A curcumin-loaded fish scale gelatin-fatty acid conjugate and its preparation method

Abstract

The present invention provides a curcumin-loaded fish scale gelatin-fatty acid conjugate and a preparation method thereof, specifically including fatty acid dissolution, activation of fatty acids, preparation of fish scale gelatin-fatty acid conjugate, and preparation of curcumin-loaded fish scale gelatin-fatty acid conjugate; wherein, an amide bond is formed between the -NH2 group in fish scale gelatin and the C=O in fatty acids, and the conjugate prepared by modifying fish scale gelatin with fatty acids is an irregular spherical particle structure with uniform distribution, which is beneficial to improving the hydrophobicity of fish scale gelatin; by changing the alkyl chain length of fatty acids, it is found that with the increase of the alkyl chain length, the curcumin loading capacity and its stability of the fish scale gelatin-fatty acid conjugate are correspondingly improved, and the delivery efficiency of nutrients is improved in the nano-delivery system.

Description

Technical Field

[0001] The present invention belongs to the technical field of gelatin modification, and particularly relates to a curcumin-loaded fish scale gelatin-fatty acid conjugate and a preparation method thereof. Background Art

[0002] Fish scale gelatin is a natural hydrophilic polymer, well-known for its safety, biodegradability and biocompatibility, and is also low-cost and easy to produce. However, its gel hardness is weak, making fish scale gelatin unsuitable for traditional applications of gelatin. Therefore, it is worth further exploring to make full use of waste resources and find fields suitable for the characteristics of fish scale gelatin. In recent years, amphiphilic fish scale gelatin has received increasing attention due to its good biocompatibility and biodegradability, and is considered a promising nutrient delivery carrier due to its adjustable physicochemical and biopharmaceutical properties. Grafting hydrophobic groups onto gelatin has the ability to form self-assembled nanoparticles with a hydrophobic core for loading various hydrophobic nutrients (such as curcumin (Cur)), providing a more stable environment for hydrophobic nutrients, while the hydrophilic gelatin layer faces the external aqueous environment for circulation extension and biocompatibility. Controlling the amount of grafted hydrophobic groups can change the natural properties of the grafted hydrophobic groups, such as hydrophobicity and emulsion stability, as well as conformational flexibility from β-turn to random coil and β-sheet. However, there is still a lack of literature reports on the influence of structural characteristics such as the chain length and configuration of hydrophobic groups on the natural properties of hydrophobic group-grafted gelatin.

[0003] Fatty acids are widely used in the food industry and are considered safe agents for imparting hydrophobicity to biological macromolecules. Most importantly, fatty acids with different amounts of alkyl chains are ideal materials for studying the relationship between the chain length of hydrophobic groups and the natural properties of hydrophobic group-grafted gelatin. In addition, gelatin contains multifunctional groups such as -NH2, which can be used for conjugation with various suitable organic molecules. Considering that fish scale gelatin has amino groups and cannot directly bind to fatty acids in water, the fatty acids must be activated to react. Therefore, the generally recognized safe EDC / NHS (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide / N-hydroxysuccinimide) activator can be selected to activate fatty acids and bind to the hydrophilic segment of gelatin in water as an intermediate substance. However, few literatures have focused on the influence of the length of hydrophobic groups on the interaction and stability between modified gelatin and hydrophobic molecules. Summary of the Invention

[0004] Technical problem to be solved: The objective of the present invention is to provide a curcumin-loaded fish scale gelatin-fatty acid conjugate and its preparation method, which specifically includes fatty acid dissolution, fatty acid activation, preparation of fish scale gelatin-fatty acid conjugate (GFC), and preparation of curcumin-loaded fish scale gelatin-fatty acid conjugate, which is conducive to improving the hydrophobicity of fish scale gelatin, enhancing the loading capacity and stability of curcumin, and improving the delivery efficiency of nutrients in the nano-delivery system.

[0005] Technical solution: A preparation method of a curcumin-loaded fish scale gelatin-fatty acid conjugate includes the following steps: S1. Fatty acid dissolution: Disperse the fatty acid in 60% ethanol and 1M NaOH solution and completely dissolve it to obtain a fatty acid solution; S2. Fatty acid activation: Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide to the fatty acid solution, vortex, and shake at 100-300 rpm for 30-45 min at 30 °C to obtain a mixed solution; continue to add 2-mercaptoethanol and shake at 100-300 rpm for 15-20 min to obtain an activated fatty acid solution;

[0006] S3. Preparation of fish scale gelatin-fatty acid conjugate: Completely dissolve fish scale gelatin in 60% ethanol, add 1M NaOH, then pour it into the activated fatty acid solution, incubate, dialyze for 48 h, and freeze-dry at -60 °C for 2 days to obtain fish scale gelatin-fatty acid conjugate;

[0007] S4. Preparation of curcumin-loaded fish scale gelatin-fatty acid conjugate: Dissolve the freeze-dried fish scale gelatin-fatty acid conjugate in distilled water, heat it to the boiling state for 1-3 min, completely dissolve it, cool the solution to 15 ± 1 °C, and increase the volume to the original volume with distilled water; then add curcumin powder, homogenize at 8000-12000 rpm for 2-5 min, place it in a water bath at 30 ± 1 °C, continuously stir at a speed of 300-600 rpm for 24-36 h, centrifuge at 5000-6000×g for 10-15 min, take the supernatant for testing.

[0008] Further, in the step S1, the fatty acid is a fatty acid with 8, 12, and 18 carbon atoms in the carbon chain.

[0009] Further, the fatty acid includes caprylic acid, lauric acid, and oleic acid.

[0010] Further, in the step S1, the concentration of the fatty acid solution is 5-6%.

[0011] Further, in the step S2, the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to N-hydroxysuccinimide is 1:(1 - 3); the volume ratio of 2-mercaptoethanol to the mixed solution is (140 - 142):1.

[0012] Further, in the step S3, the concentration of fish scale gelatin is 20 - 25 mg / mL; the volume ratio of fish scale gelatin to the activated fatty acid solution is 1:(1 - 2).

[0013] Further, in the step S3, the incubation condition is to place it in a 30°C water bath machine and incubate for 12 - 18 h at an oscillation speed of 300 - 600 rpm.

[0014] Further, in the step S4, the concentration of the freeze-dried fish scale gelatin-fatty acid conjugate is 2 - 4 mg / mL.

[0015] Further, in the step S4, the mass-to-volume ratio of curcumin powder to the fish scale gelatin-fatty acid conjugate is 1:(4 - 6).

[0016] Beneficial effects:

[0017] 1. In the present invention, fish scale gelatin is modified with fatty acids (caprylic acid, lauric acid, and oleic acid), and an amide bond is formed between the -NH2 group in fish scale gelatin and the C=O in the fatty acid. The prepared fish scale gelatin-fatty acid conjugate is an irregular spherical particle structure with uniform distribution, while the untreated fish scale gelatin shows a loose, layer-like stacked coarse network. In addition, the modification of fish scale gelatin with fatty acids in the present invention is beneficial to improving the hydrophobicity of fish scale gelatin;

[0018] 2. In the present invention, by grafting fish scale gelatin with a hydrophobic group of fatty acid, it has the ability to form self-assembled nanoparticles. The nanoparticles have a hydrophobic core for loading curcumin-like hydrophobic nutrients, providing a more stable environment for hydrophobic nutrients, while the hydrophilic gelatin layer faces the external water environment for circulation extension and biocompatibility; by adjusting the alkyl chain length of the fatty acid, the natural properties of the grafted hydrophobic group can be changed, such as conformational changes, hydrophobicity, curcumin loading efficiency, and its stability; 3. In the present invention, the curcumin loading capacity of the fish scale gelatin-fatty acid conjugate loaded with curcumin was studied. By changing the chain length and configuration of the fatty acid, it was found that with the increase of the alkyl chain length, the curcumin loading capacity of the fish scale gelatin-fatty acid conjugate showed an increasing trend, which was beneficial to improving the delivery efficiency of nutrients. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the preparation process of the fish scale gelatin-fatty acid conjugate;

[0020] Figure 21H NMR spectra of unmodified fish scale gelatin, fish scale gelatin-fatty acid conjugates, and fish scale gelatin-fatty acid conjugates loaded with curcumin. Among them, a represents unmodified fish scale gelatin (FSG), fish scale gelatin-caprylic acid conjugate (GCC), fish scale gelatin-lauric acid conjugate (GLC), and fish scale gelatin-oleic acid conjugate (GOC); b represents curcumin-loaded unmodified fish scale gelatin (CL-FSG), curcumin-loaded fish scale gelatin-caprylic acid conjugate (CL-GCC), curcumin-loaded fish scale gelatin-lauric acid conjugate (CL-GLC), and curcumin-loaded fish scale gelatin-oleic acid conjugate (CL-GOC).

[0021] Figure 3 FTIR spectra of unmodified fish scale gelatin, fish scale gelatin-fatty acid conjugates, and fish scale gelatin-fatty acid conjugates loaded with curcumin. Among them, a represents FSG, GCC, GLC, and GOC; b represents Cur, CL-FSG, CL-GCC, CL-GLC, and CL-GOC.

[0022] Figure 4 Scanning electron microscopy images of unmodified fish scale gelatin and fish scale gelatin-fatty acid conjugates. Among them, a represents FSG; b represents GCC; c represents GLC; d represents GOC.

[0023] Figure 5 Stability profiles of curcumin, curcumin-loaded unmodified fish scale gelatin, and curcumin-loaded fish scale gelatin-fatty acid conjugates. Among them, a represents the photo-stability profile; b represents the thermal-stability profile. Detailed implementation manners

[0024] The present invention will be further described below in conjunction with the accompanying drawings and embodiments. The following embodiments are explanations of the present invention, and the present invention is not limited to the following embodiments:

[0025] Example 1

[0026] A preparation method of a curcumin-loaded fish scale gelatin-caprylic acid conjugate, comprising the following steps:

[0027] S1. Fatty acid dissolution: Disperse 1 mL of caprylic acid in 20 mL of 60% ethanol and 200 μL of 1 M NaOH solution, and completely dissolve to obtain a caprylic acid solution;

[0028] S2. Activation of fatty acid: 38.34 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 57.54 mg of N-hydroxysuccinimide (NHS) were added to the octanoic acid solution, vortexed, and the resulting solution was placed in a 30 °C water bath. The octanoic acid activation reaction was carried out at a shaking speed of 100 rpm for 45 min. At the end of the reaction, 150 μL of 2-mercaptoethanol was added, and the resulting solution was shaken for 15 min under the same conditions to inactivate the unreacted EDC, thus obtaining the activated octanoic acid solution;

[0029] S3. Preparation of fish scale gelatin-octanoic acid conjugate: 500 mg of fish scale gelatin was completely dissolved in 20 mL of 60% ethanol, 200 μL of 1 M NaOH was added, and then the solution was poured into the activated octanoic acid solution. The mixture was incubated in a water bath at 30 °C and an oscillation speed of 300 rpm for 18 h. After dialysis for 48 h to purify the conjugate, it was freeze-dried at -60 °C for 2 days, thus obtaining the fish scale gelatin-octanoic acid conjugate (GCC); S4. Preparation of curcumin-loaded fish scale gelatin-octanoic acid conjugate: 40 mg of the freeze-dried fish scale gelatin-octanoic acid conjugate was dissolved in 20 mL of distilled water and heated to the boiling state for 1 min. After complete dissolution, the solution was cooled to 15 ± 1 °C and the volume was increased to 20 mL with distilled water. 10 mg of curcumin powder was added to 20 mL of the above solution, and the solution was homogenized at 12000 rpm for 2 min using a homogenizer. To ensure sufficient contact between curcumin and the fish scale gelatin-octanoic acid conjugate, the homogenized suspension was continuously stirred at 300 rpm in a 30 ± 1 °C water bath for 36 h. Finally, the solution was centrifuged at 5600×g for 10 min, and the supernatant was collected for further analysis.

[0030] Example 2

[0031] A preparation method of curcumin-loaded fish scale gelatin-lauric acid conjugate, comprising the following steps:

[0032] S1. Dissolution of fatty acid: 1 mL of lauric acid was dispersed in 20 mL of 60% ethanol and 200 μL of 1 M NaOH solution and completely dissolved to obtain the lauric acid solution;

[0033] S2. Activation of fatty acid: 38.34 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 57.54 mg of N-hydroxysuccinimide (NHS) were added to the lauric acid solution, vortexed, and the resulting solution was placed in a 30 °C water bath. The lauric acid activation reaction was carried out at a shaking speed of 200 rpm for 40 min. At the end of the reaction, 150 μL of 2-mercaptoethanol was added, and the resulting solution was shaken for 18 min under the same conditions to inactivate the unreacted EDC, thus obtaining the activated lauric acid solution;

[0034] S3. Preparation of fish scale gelatin - lauric acid conjugate: Dissolve 500 mg of fish scale gelatin completely in 20 mL of 60% ethanol, add 200 μL of 1 M NaOH, then pour it into the activated lauric acid solution, incubate in a water bath at 30 °C with an oscillation speed of 400 rpm for 14 h. After purifying the conjugate by dialysis for 48 h, freeze - dry it at - 60 °C for 2 days to obtain fish scale gelatin - lauric acid conjugate (GLC).

[0035] S4. Preparation of curcumin - loaded fish scale gelatin - lauric acid conjugate: Dissolve 40 mg of freeze - dried fish scale gelatin - lauric acid conjugate in 20 mL of distilled water, heat it to the boiling state for 2 min. After complete dissolution, cool the solution to 15 ± 1 °C and adjust the volume to 20 mL with distilled water. Add 10 mg of curcumin powder to 20 mL of the above - mentioned solution, homogenize the solution at 8000 rpm for 5 min using a homogenizer. To ensure sufficient contact between curcumin and fish scale gelatin - lauric acid conjugate, continuously stir the homogenized suspension in a water bath at 30 ± 1 °C at a rotation speed of 400 rpm for 30 h. Finally, centrifuge the solution at 5000×g for 15 min, collect the supernatant for further analysis.

[0036] Example 3

[0037] A preparation method of curcumin - loaded fish scale gelatin - oleic acid conjugate, comprising the following steps:

[0038] S1. Fatty acid dissolution: Disperse 1 mL of oleic acid in 20 mL of 60% ethanol and 200 μL of 1 M NaOH solution, and dissolve it completely to obtain an oleic acid solution.

[0039] S2. Activation of fatty acid: Add 38.34 mg of 1 - (3 - dimethylaminopropyl) - 3 - ethylcarbodiimide (EDC) and 57.54 mg of N - hydroxysuccinimide (NHS) to the oleic acid solution, vortex, and place the resulting solution in a 30 °C water bath. The oleic acid activation reaction is carried out at a shaking speed of 300 rpm for 30 min. At the end of the reaction, add 150 μL of 2 - mercaptoethanol and shake the resulting solution under the same conditions for 20 min to inactivate the unreacted EDC, thus obtaining the activated oleic acid solution.

[0040] S3. Preparation of fish scale gelatin-oleic acid conjugate: Dissolve 500 mg of fish scale gelatin completely in 20 mL of 60% ethanol, add 200 μL of 1 M NaOH, then pour it into the activated oleic acid solution, incubate in a water bath at 30 °C with an oscillation speed of 600 rpm for 12 h, after purifying the conjugate by dialysis for 48 h, freeze-dry it at -60 °C for 2 days to obtain fish scale gelatin-oleic acid conjugate (GOC); S4. Preparation of curcumin-loaded fish scale gelatin-oleic acid conjugate: Dissolve 40 mg of freeze-dried fish scale gelatin-oleic acid conjugate in 20 mL of distilled water, heat it to the boiling state for 3 min, after complete dissolution, cool the solution to 15 ± 1 °C, and increase the volume to 20 mL with distilled water; Add 10 mg of curcumin powder to 20 mL of the above solution, homogenize the solution at 10000 rpm for 4 min using a homogenizer, in order to make curcumin fully contact with fish scale gelatin-oleic acid conjugate, continuously stir the homogenized suspension in a water bath at 30 ± 1 °C at a rotation speed of 600 rpm for 24 h; Finally, centrifuge the solution at 6000×g for 10 min, collect the supernatant for further analysis.

[0041] Comparative Example 1

[0042] The difference between this comparative example and Example 1 lies in the preparation of unmodified fish scale gelatin loaded with curcumin, specifically as follows:

[0043] A preparation method of unmodified fish scale gelatin loaded with curcumin includes the following steps:

[0044] S1. Preparation of unmodified fish scale gelatin: Dissolve 500 mg of fish scale gelatin completely in 20 mL of 60% ethanol, add 200 μL of 1 M NaOH, freeze-dry it at -60 °C for 2 days to obtain unmodified fish scale gelatin (FSG);

[0045] S2. Preparation of unmodified fish scale gelatin loaded with curcumin: Dissolve 40 mg of freeze-dried fish scale gelatin in 20 mL of distilled water, heat it to the boiling state for 1 min, after complete dissolution, cool the solution to 15 ± 1 °C, and increase the volume to 20 mL with distilled water; Add 10 mg of curcumin powder to 20 mL of the above solution, homogenize the solution at 12000 rpm for 2 min using a homogenizer, in order to make curcumin fully contact with unmodified fish scale gelatin, continuously stir the homogenized suspension in a water bath at 30 ± 1 °C at a rotation speed of 300 rpm for 36 h; Finally, centrifuge the solution at 5600×g for 10 min, collect the supernatant for further analysis.

[0046] The NMR spectra of unmodified fish scale gelatin, fish scale gelatin-fatty acid conjugate and curcumin-loaded fish scale gelatin-fatty acid conjugate are as Figure 2As shown. The peak observed at a chemical shift of 2.00 - 2.30 ppm is attributed to the acetyl group, and the proton resonance on the acetyl CH3 can also be easily identified as being close to 2.50 ppm. It is worth noting that the NMR spectrum confirmed the successful reaction between FSG and fatty acids. The disappearance of the characteristic peak of the - carboxyl group (-COOH) of fatty acids at 12 ppm in the GFC spectrum ( Figure 2 as indicated by the black arrow in a) indicates the formation of amide bonds through the reaction of the -COOH group of fatty acids and the amino group (-NHR) of FSG. On the other hand, Figure 2 the characteristic peak of the olefinic bond (-CH=CH-) of oleic acid at 5.32 ppm in b) confirmed the successful grafting of oleic acid onto FSG. The grafting of hydrophobic groups onto FSG may lead to the formation of random coil assemblies, which helps it resist the action of the environment on gelatin molecules compared to unfolded gelatin molecules, thereby improving the loading efficiency of curcumin.

[0047] The FTIR spectra of unmodified fish scale gelatin, fish scale gelatin-fatty acid conjugate, and fish scale gelatin-fatty acid conjugate loaded with curcumin are as shown in Figure 3 as follows. Characteristic peaks were observed in the FTIR spectrum of FSG at 3421 cm -1 , corresponding to the O-H stretching vibration coupled with hydrogen bonds. The formation of hydrogen bonds is an indispensable factor affecting the microstructure and properties of these nanoparticles. Figure 3 After the formation of the GFC nanocomposite in a, the O-H vibration absorption band in the range of 3500 - 3100 cm -1 shifted from 3421 cm -1 (FSG) to 3425 cm -1 , 3429 cm -1 and 3432 cm -1 corresponding to GCC, GLC, and GOC respectively, indicating that hydrogen bonds were also formed between the amide groups in FSG and the hydroxyl groups in fatty acids. At the same time, after loading curcumin, the amide A bands of FSG and GFC both shifted to 3395 cm -1 ( Figure 3 b), indicating that the participation of curcumin may lead to the formation of hydrogen bonds between the ester groups and hydroxyl groups in curcumin, resulting in a change in the wave number. In addition, compared with CL-FSG, the intensity of the amide A band of CL-GFC increased, indicating that there are stronger hydrogen bonds in the structure of GFC, which can effectively encapsulate curcumin in the hydrophobic cavity. On the other hand, in the FTIR spectra of the two nanocomposites CL-FSG and CL-GFC, the peak positions related to the methyl C-H stretching in the range of the amide B band (2800 - 3000 cm -1 ) shifted from 2921 cm -1 to 2924 cm -1, indicating that hydrophobic interactions were formed between FSG, fatty acids, and curcumin during the self-assembly process of the sample, which may be attributed to the interaction between the hydrophobic amino acids of GFC and the hydrophobic groups of curcumin. Notably, after loading curcumin onto GFC, the amide B band was simultaneously enhanced, and this result may indicate that the hydrophobic CH2 chains in GFC participated in the non-covalent interaction with curcumin. In addition, the intensity of the CH2 stretching band before and after loading curcumin increased significantly with the increase in the carbon chain length. These results suggest that introducing longer GFC carbon chains generates more hydrophobic groups, which is more conducive to the formation of intermolecular hydrophobic regions, and it is inferred that GOC has the best hydrophobic interaction before and after loading curcumin compared with GCC and GLC. Meanwhile, the FTIR spectrum of FSG showed characteristic bands of amide-I (1600 - 1700 cm -1 ), amide-II (1500 - 1600 cm -1 ), and amide-III (1220 - 1330 cm -1 ). Specifically, the C=O bond frequency of FSG in the FTIR spectrum was 1641 cm -1 . When conjugated with fatty acids of different carbon chain lengths, the peak of the C=O bond shifted to 1602 cm -1 , and the peak intensity increased, which confirmed the successful conjugation between FSG and fatty acids, and an amide bond was formed between the NH2 group of FSG and the C=O of fatty acids with different carbon chain lengths. The peak at 1547 cm -1 of FSG was related to amide-II, mainly originating from the in-plane N-H bending mode coupled with C-N stretching vibration and C-C stretching vibration. Notably, during the fatty acid modification process, the amide-II band of GFC disappeared, indicating the existence of electrostatic interaction forces between FSG and fatty acids. The peak at 1242 cm -1 of GFC was stronger than that of the FSG group and was related to amide-III, mainly attributed to the C-N stretching vibration of the amide bond, the N-H deformation of the amide bond, and the wagging vibration of the CH2 groups of glycine and proline, indicating that fatty acid modification changed the C-N or N-H bonds of FSG. After loading curcumin, curcumin may have promoted the change of N-H bond or C-N stretching vibration during the fatty acid modification process due to the hydrophobic interaction with non-polar amino acids, shielding the protein chain, and thus destroying the internal structure of FSG. It can be seen that during the fatty acid modification process, GFC aggregated, its hydrophobic residues were exposed, and the surface hydrophobicity increased. In addition, new absorption peaks were found at 1348 cm -1 and 1385 cm -1 in GFC, which indicated the appearance of in-phase combination of C-H stretching in GFC. The results showed that the secondary structure of the gelatin structure changed during the fatty acid modification process.

[0048] To further explore the changes in the functional groups of nanoparticles after loading curcumin, the infrared spectra of curcumin were compared with those of other samples. As Figure 3 shown in -1 b, multiple characteristic peaks were observed in curcumin. Notably, there were no characteristic peaks in the range of 1800 - 1650 cm -1 , indicating that curcumin exists in the keto - enol tautomeric form. Among them, the characteristic peaks at 961 cm -1 , 812 cm -1 represent monosubstituted, trisubstituted alkenes and phenyl groups respectively. The peaks at 1628 and 1602 cm -1 correspond to C=O and aromatic C=C stretching respectively. The alkene C=C stretching was detected by the 1509 cm -1 peak. The peaks of curcumin at 1427 cm -1 and 1151 cm -1 correspond to the presence of δ(C - C - C) and δ(C - O - C) of the aromatic ring and the inter - ring chain of pure curcumin. The peaks at 1276, 1230, 1207 cm -1 correspond to CCC, CCH and the aromatic ketone moiety. The peak at 1151 cm -1 is attributed to O - CH3 and CCH of the curcumin aromatic ring. However, it should be noted that the binding of Cur and GFC also caused the disappearance of two characteristic peaks of curcumin at 3506 and 1628 cm -1 , which refer to phenolic - OH and C=O in the curcumin curve respectively, further proving the disappearance of the characteristic peaks of curcumin, thus confirming that curcumin has been successfully encapsulated in GFC. In addition, the characteristic absorption peaks of pure curcumin at 1276 and 1151 cm -1 disappeared in both CL - FSG and CL - GFC, indicating that the characteristic peaks of curcumin merged or overlapped with the absorption bands of the polymer. At the same time, no obvious differences were detected in the spectra between CL - GFC, meaning that no new chemical bonds were formed and curcumin was encapsulated in GFC only through intermolecular interactions (such as hydrogen bonding or hydrophobic interactions).

[0049] As Figure 4 shown, it was found by scanning electron microscopy that the untreated fish scale gelatin presented a relatively loose, layered - stacked coarse network ( Figure 4 a), while the fish scale gelatin - octanoic acid conjugate (GCC), fish scale gelatin - lauric acid conjugate (GLC) and fish scale gelatin - oleic acid conjugate (GOC) prepared by modifying fish scale gelatin with fatty acids ( Figure 4b, c, d), there are huge morphological changes, changing from a grid structure to an irregular spherical particle structure. More specifically, compared with GOC, the particle size distribution of GCC and GLC is wider. When the fatty acid alkyl chain length is 18 carbon atoms (GOC), the aggregation of this polymer is closer and the structure is more regular.

[0050] Performance determination:

[0051] (1) Calculation of curcumin loading capacity

[0052] Mix the curcumin-loaded fish scale gelatin-fatty acid conjugate with 80% ethanol and dilute it to an appropriate concentration. Measure the absorbance value at a wavelength of 425 nm using an A560 ultraviolet-visible spectrophotometer and plot a standard curve; calculate the curcumin concentration by substituting the absorbance value into the standard curve. The curcumin loading capacity (CLC) of the fish scale gelatin-fatty acid conjugate (in 1 mL of the curcumin-loaded fish scale gelatin-fatty acid conjugate solution) is calculated according to the following formula:

[0053]

[0054] The experiment measured the effect of different alkyl chain lengths of the fish scale gelatin-fatty acid conjugate on the curcumin loading capacity. The results showed that when the internal structure of fish scale gelatin unfolds, only a very small amount of curcumin is encapsulated in its neutral hydrophobic region. The measured curcumin loading amount of curcumin-loaded unmodified fish scale gelatin is only 1.51 ± 0.26 μg / mg. With the increase of the alkyl chain length, the curcumin loading efficacy of the fish scale gelatin-fatty acid conjugate shows an increasing trend, increasing from 8.20 ± 0.54 μg / mg to 31.18 ± 1.41 μg / mg.

[0055] (2) Stability

[0056] Compared with free curcumin, curcumin encapsulated by FSG and GFC has better stability. As Figure 5As shown in a, the percentage of free curcumin that was not degraded after exposure to ultraviolet light (for 24 hours) was 66.12%, while those of CL-FSG, CL-GCC, CL-GLC, and CL-GOC were 83.34%, 87.25%, 92.83%, and 95.82%, respectively. This result indicates that, compared with natural FSG, GFC nanoparticles greatly improve the photostability of curcumin. The formation of the shell during the self-assembly of GFC may provide a physical barrier to prevent curcumin from contacting ultraviolet light. In addition, CL-GOC has a better photoprotective effect than other nanoparticles, which may be due to the fact that the fatty acids encapsulated on the surface of FSG molecules increase the thickness of the molecules, thus endowing them with better ultraviolet shielding ability. Compared with GCC and GLC, GOC has a higher height, which creates a favorable structural advantage for avoiding the loss of ultraviolet light. In addition, CL-GFC has excellent heat treatment protection ability, which may be attributed to the fact that the core-shell structure of GFC nanoparticles provides an opportunity for encapsulating curcumin, avoiding the contact between curcumin and the high-temperature environment.

[0057] As Figure 5 shown in b, as the temperature increases, the curcumin retention rate of CL-GFC also decreases, but compared with free curcumin, its degradation rate is significantly much lower. In particular, CL-GOC has excellent thermal stability compared with other nanoparticles. After treatment at 75 °C, 85 °C, and 95 °C, the curcumin retention rates at different temperatures are significantly increased by 1.27 times, 1.34 times, and 1.51 times, respectively, compared with free curcumin. This result indicates that the encapsulation of curcumin by GOC significantly improves the thermal stability of curcumin, and the increase in the carbon chain length enhances the interaction between curcumin and GFC, resulting in higher thermal stability. In summary, the photostability and thermal stability can be controlled by adjusting the carbon chain length of GFC nanoparticles.

Claims

1. A preparation method of a curcumin-loaded fish scale gelatin-fatty acid conjugate, characterized in that, It includes the following steps: S1. Fatty acid dissolution: Disperse the fatty acid in 60% ethanol and 1M NaOH solution and completely dissolve it to obtain a fatty acid solution; the fatty acid includes caprylic acid or lauric acid; S2. Activation of fatty acid: Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide to the fatty acid solution, vortex, and shake at 100 - 300 rpm for 30 - 45 min at 30 °C to obtain a mixed solution; continue to add 2-mercaptoethanol and shake at 100 - 300 rpm for 15 - 20 min to obtain an activated fatty acid solution; S3. Preparation of fish scale gelatin-fatty acid conjugate: Completely dissolve fish scale gelatin in 60% ethanol, add 1M NaOH, then pour it into the activated fatty acid solution, incubate, dialyze for 48 h, and freeze-dry at -60 °C for 2 days to obtain fish scale gelatin-fatty acid conjugate; S4. Preparation of curcumin-loaded fish scale gelatin-fatty acid conjugate: Dissolve the freeze-dried fish scale gelatin-fatty acid conjugate in distilled water, heat it to the boiling state for 1 - 3 min, after complete dissolution, cool the solution to 15 ± 1 °C and increase the volume to the original volume with distilled water; then add curcumin powder, homogenize at 8000 - 12000 rpm for 2 - 5 min, place it in a water bath at 30 ± 1 °C, continuously stir at a speed of 300 - 600 rpm for 24 - 36 h, centrifuge at 5000 - 6000×g for 10 - 15 min, take the supernatant for testing; the mass-volume ratio of curcumin powder to fish scale gelatin-fatty acid conjugate in step S4 is 1:(4 - 6).

2. The preparation method of a curcumin-loaded fish scale gelatin-fatty acid conjugate according to claim 1, characterized in that: In step S1, the fatty acid is a fatty acid with 8, 12, and 18 carbon atoms in the carbon chain.

3. The preparation method of a curcumin-loaded fish scale gelatin-fatty acid conjugate according to claim 1, characterized in that: In step S1, the concentration of the fatty acid solution is 5 - 6%.

4. The preparation method of a curcumin-loaded fish scale gelatin-fatty acid conjugate according to claim 1, characterized in that: In step S2, the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to N-hydroxysuccinimide is 1:(1 - 3); the volume ratio of 2-mercaptoethanol to the mixed solution is (140 - 142):

1.

5. The preparation method of a curcumin-loaded fish scale gelatin-fatty acid conjugate according to claim 1, characterized in that: In step S3, the concentration of fish scale gelatin is 20 - 25 mg / mL; the volume ratio of fish scale gelatin to the activated fatty acid solution is 1:(1 - 2).

6. The preparation method of a curcumin-loaded fish scale gelatin-fatty acid conjugate according to claim 1, characterized in that: In step S3, the incubation condition is to place it in a 30 °C water bath machine and incubate at an oscillation speed of 300 - 600 rpm for 12 - 18 h.

7. The preparation method of a curcumin-loaded fish scale gelatin-fatty acid conjugate according to claim 1, characterized in that: In step S4, the concentration of the freeze-dried fish scale gelatin-fatty acid conjugate is 2 - 4 mg / mL.

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