A pH-sensitive intestinal-targeting gel particle based on prickly pear SOD and tannins and its preparation method

Abstract

This invention relates to the field of targeted release gel technology, and discloses a pH-sensitive intestinal-targeting gel particle based on prickly pear SOD and tannins, and its preparation method, comprising the following steps: extracting SOD and tannins from prickly pear; mixing the SOD, tannins, and sodium alginate powder, and then dispersing them in a shellac solution to obtain a gel; forming a solution from the gel using an injection method, and then dripping the solution into a gelling solution to form a gel, and stirring to obtain the pH-sensitive intestinal-targeting gel particle. This invention utilizes the combined use of SOD and tannins, which not only enhances the individual encapsulation effect but also enhances the overall antioxidant efficacy. The alginate-shellac composite gel particle achieves the specific release of SOD and tannins in the colon.

Description

Technical Field

[0001] This invention relates to the field of targeted release gel technology, and more specifically, to a pH-sensitive intestinal-targeting gel particle based on prickly pear SOD and tannins and its preparation method. Background Technology

[0002] Superoxide dismutase (SOD) is an important antioxidant enzyme in living organisms, capable of efficiently scavenging superoxide anion free radicals and playing a crucial role in the body's oxidation-antioxidant balance. It has therapeutic and preventative effects on various diseases, showing potential in the treatment and prevention of ulcerative colitis, diabetic complications, and cancer. It can also be used as a nutritional supplement and as a functional food ingredient. The SOD content in fresh prickly pear fruit far exceeds that of most plants, earning it the title of "King of SOD in the Plant Kingdom." This high activity gives it a significant advantage in antioxidant applications. Furthermore, compared to animal-derived SOD (such as bovine blood SOD), prickly pear SOD is derived from plants, possessing characteristics such as being natural, safe, and without allergy risks, better meeting the modern food and pharmaceutical industries' demand for "green raw materials."

[0003] In addition to superoxide dismutase (SOD), prickly pear is rich in polyphenols and other antioxidants. These components work synergistically with SOD to enhance antioxidant effects. After juicing, the remaining prickly pear pomace is rich in polyphenolic compounds, with tannins being the most abundant (accounting for 78.20% of the total phenolic acids in the fruit). Tannins are natural water-soluble plant polyphenolic compounds with a multi-ortho- and ortho-hydroxyl structure, a characteristic that endows them with various physiological activities such as antioxidant, antibacterial, and protein coagulation. The synergistic ability of SOD and tannins to scavenge free radicals is significantly higher than that of either component alone. Extracting tannins from the pomace not only utilizes waste and reduces environmental pollution but also meets the needs of a circular economy and sustainable development.

[0004] However, when SOD and tannins are taken orally directly, they are easily degraded by gastric acid and pepsin in the upper digestive tract, resulting in low bioavailability. Studies have shown that SOD aqueous solutions are completely inactivated in simulated gastric juice within just 20 minutes, therefore, encapsulation technology is necessary to protect their activity. The intestine (especially the colon) is an important site for SOD to exert its effects, such as repairing damaged intestinal mucosal barriers and treating inflammatory bowel disease (IBD). The colon is characterized by long transit times and low proteolytic enzyme activity, making it suitable for drug accumulation, but a carrier is needed to achieve targeted release and avoid premature release in the stomach and small intestine.

[0005] Existing liposome preparation methods are complex and costly, and have low encapsulation efficiency for hydrophilic substances; the biocompatibility and safety of nanoparticles need further verification; although protein carriers can bind SOD, they may cause gluten allergy, which limits their application.

[0006] Therefore, providing a pH-sensitive intestinal-targeting gel particle with excellent stability based on prickly pear SOD and tannins and its preparation method is of great practical significance. Summary of the Invention

[0007] In view of this, the present invention proposes a pH-sensitive intestinal-targeting gel particle based on prickly pear SOD and tannin and its preparation method. The present invention uses the combined use of SOD and tannin, which not only enhances the single encapsulation effect, but also enhances the overall antioxidant effect. The alginate-shellac composite gel particle realizes the specific release of SOD and tannin in the colon.

[0008] This invention proposes a method for preparing pH-sensitive intestinal-targeting gel particles based on prickly pear SOD and tannins, characterized by the following steps:

[0009] SOD and tannins were extracted from prickly pear.

[0010] The SOD, tannin, and sodium alginate powder were mixed and then dispersed in a shellac solution to obtain a gel.

[0011] The gel was injected to form a solution, which was then dropped into the gelation solution to form a gel. After stirring, the pH-sensitive intestinal-targeting gel particles were obtained.

[0012] Preferably, the concentration of SOD is 2.00 mg / mL, and the activity is 6 × 10⁻⁶. 4 U / g.

[0013] Preferably, the tannin has a concentration of 40.96 mg / g and a purity of 89%.

[0014] Preferably, when SOD, tannin, and sodium alginate powder are mixed, the mass ratio of SOD to tannin is 1:6 to 3:1.

[0015] Preferably, the amount of sodium alginate powder added is 2%.

[0016] Preferably, the shellac solution has a mass fraction concentration of 5-10%.

[0017] Preferably, the gelation solution comprises a solution of 0.50‰ chitosan and 2.00% CaCl2.

[0018] Preferably, the injection height in the injection method is 8 to 10 centimeters.

[0019] The present invention also provides pH-sensitive intestinal-targeting gel particles obtained by the preparation method of pH-sensitive intestinal-targeting gel particles based on prickly pear SOD and tannins as described in the above technical solution.

[0020] This invention also provides the application of the pH-sensitive intestinal-targeting gel particles described above in the treatment and prevention of ulcerative colitis, diabetic complications, and cancer drugs.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] (1) The present invention uses the combined use of SOD and tannin, which not only enhances the single encapsulation effect, but also enhances the overall antioxidant effect; the present invention extracts tannin from prickly pear pomace, realizing the high-value utilization of waste, reducing pollution, and conforming to the circular economy and sustainable development.

[0023] (2) The alginate-shellac composite gel particles in this invention achieve the specific release of SOD and tannins in the colon; in addition, all materials used in this invention are natural materials, and the cost is low, and the preparation process is simple, easy to scale up production, and has high commercialization potential.

[0024] (3) The non-polymerized carboxyl group of the shellac described in this invention has a pKa between 5.60 and 7.00 and is only soluble in aqueous solutions with pH > 7.00. Therefore, it cannot be dissolved in gastric juice (pH 2.00) or intestinal juice (pH 7.00) but can be dissolved in colonic juice (pH 7.40). This invention releases SOD and tannins in colonic juice by adding shellac, which is crucial for the treatment of inflammatory bowel disease (such as ulcerative colitis). Attached Figure Description

[0025] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Furthermore, the same reference numerals denote the same parts throughout the drawings. In the drawings:

[0026] Figure 1 This is a schematic diagram of the inactivation process of SOD aqueous solution in simulated gastric juice.

[0027] Figure 2 This is a schematic diagram showing the encapsulation efficiency results of the gel particles obtained in Examples 1-8 of the present invention;

[0028] Figure 3 This is a schematic diagram comparing the antioxidant activity of Example 7 of the present invention with that of the control group;

[0029] Figure 4 This is a schematic diagram showing the effect of shellac on the release of SOD and tannins according to the present invention. Detailed Implementation

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0031] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0033] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0034] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0035] This invention provides a method for preparing pH-sensitive intestinal-targeting gel particles based on prickly pear SOD and tannins, characterized by comprising the following steps:

[0036] SOD and tannins were extracted from prickly pear.

[0037] The SOD, tannin, and sodium alginate powder were mixed and then dispersed in a shellac solution to obtain a gel.

[0038] The gel was injected to form a solution, which was then dropped into the gelation solution to form a gel. After stirring, the pH-sensitive intestinal-targeting gel particles were obtained.

[0039] SOD and tannins were extracted from prickly pear.

[0040] The preferred specific procedure for extracting SOD and tannins from prickly pear in this invention is as follows: After removing the pit and pulverizing the prickly pear, add 3% NaCl solution at a ratio of 1g:5mL, and extract with ultrasonic assistance for 30 minutes, followed by filtration. The filtrate is then subjected to a water bath at 55-60℃ for 30 minutes, the pH is adjusted to 5 with 10% NaHCO3, and then purified by precipitation with 10% ethanol. Finally, NaCl powder is added to a concentration of 20-30%, the precipitate is washed, and then freeze-dried to obtain an activity of 6×10⁻⁶. 4 The present invention uses the pyrogallol method to detect the activity of SOD at U / g;

[0041] The fruit pomace after SOD extraction was freeze-dried and ground into powder. After defatting with n-hexane, it was added to a 70% ethanol solution containing 0.1% HCl at a ratio of 1g:10mL, and extracted with ultrasound-assisted extraction for 30 minutes, followed by filtration. Purification was performed using a D101 macroporous resin column. Separation was achieved by high-speed countercurrent chromatography (HSCCC), followed by freeze-drying, and purity determination and identification by liquid chromatography-mass spectrometry (LC / MS). Tannin with a purity of 40.96 mg / g and 89% was obtained.

[0042] The SOD, tannin, and sodium alginate powder were mixed and then dispersed in shellac solution to obtain a gel. The gel was then injected to form a solution, which was then dropped into the gelation solution to form a gel. After stirring, the pH-sensitive intestinal-targeting gel particles were obtained.

[0043] Specifically, in this invention, the concentration of SOD is 2.00 mg / mL. SOD and tannins are mixed in a specific ratio, then mixed with sodium alginate powder. The pH of the mixed solution is adjusted to 4-7 using 4M NaOH. The solution is then dispersed in shellac solutions of different concentrations and diluted with deionized water. Using an injection method, the gel-forming solution is dripped into the gelation solution through a 32-gauge needle. The injection height is approximately 8-10 cm. The gel particles form a gel in the gelation solution over 30 minutes, during which time a magnetic stirrer is used for stirring. Finally, the gel particles are rinsed with deionized water to obtain pH-sensitive intestinal-targeting gel particles.

[0044] In this invention, the concentration of the tannin is 40.96 mg / g and the purity is 89%.

[0045] In this invention, when SOD, tannin and sodium alginate powder are mixed, the mass ratio of SOD to tannin is preferably 1:6 to 3:1, and more preferably 3:1, 1:1, 1:3 and 1:6.

[0046] In this invention, the amount of sodium alginate powder added is preferably 2%.

[0047] The 2% addition of sodium alginate powder refers to the composition of sodium alginate.

[0048] In this invention, the mass fraction concentration of the shellac solution is preferably 5-10%, and more preferably 5% and 10%.

[0049] In this invention, the gelation solution preferably comprises 0.50‰ chitosan and 2.00% CaCl2.

[0050] In this invention, the injection height in the injection method is preferably 8 to 10 centimeters.

[0051] The present invention also provides pH-sensitive intestinal-targeting gel particles obtained by the preparation method of pH-sensitive intestinal-targeting gel particles based on prickly pear SOD and tannins as described in the above technical solution.

[0052] This invention also provides the application of the pH-sensitive intestinal-targeting gel particles described above in the treatment and prevention of ulcerative colitis, diabetic complications, and cancer drugs.

[0053] Example 1

[0054] (1) After removing the core and crushing the prickly pear, add 3% NaCl solution at a ratio of 1g:5mL, and extract with ultrasonic assistance for 30 minutes, then filter. The filtrate is then subjected to a water bath at 55-60℃ for 30 minutes, the pH is adjusted to 5 with 10% NaHCO3, and then purified by precipitation with 10% ethanol. Finally, NaCl powder is added to a concentration of 20-30%, the precipitate is washed, and then freeze-dried to obtain an activity of 6×10⁻⁶. 4 U / g SOD; the fruit pomace after SOD extraction was freeze-dried and ground into powder, defatted with n-hexane, and then added to a 70% ethanol solution containing 0.1% HCl at a ratio of 1g:10mL. Extraction was performed under ultrasonic assistance for 30 minutes, followed by filtration. Purification was carried out using a D101 macroporous resin column. Separation was performed by high-speed countercurrent chromatography (HSCCC), followed by freeze-drying. Purity was determined and identified by liquid chromatography-mass spectrometry (LC / MS), yielding tannins with a purity of 89% and a concentration of 40.96 mg / g.

[0055] (2) SOD at a concentration of 2 mg / mL and tannin at a mass ratio of 40.96 mg / g were mixed at a ratio of 3:1, and then mixed with 2% sodium alginate powder. The pH of the mixed solution was adjusted to 5 using 4M NaOH. The solution was then dispersed in a 5% shellac solution to obtain a gel. The gel was injected to a height of 8 cm, and then the solution was dripped into the gelation solution through a 35-gauge needle. The gelation solution contained 0.50‰ chitosan and 2.00% CaCl2. The gel particles formed a gel in the gelation solution over 30 minutes, during which time a magnetic stirrer was used for stirring. Finally, the gel particles were rinsed with deionized water to obtain pH-sensitive intestinal-targeting gel particles.

[0056] Example 2

[0057] (1) After removing the core and crushing the prickly pear, add 3% NaCl solution at a ratio of 1g:5mL, and extract with ultrasonic assistance for 30 minutes, then filter. The filtrate is then subjected to a water bath at 55-60℃ for 30 minutes, the pH is adjusted to 5 with 10% NaHCO3, and then purified by precipitation with 10% ethanol. Finally, NaCl powder is added to a concentration of 20-30%, the precipitate is washed, and then freeze-dried to obtain an activity of 6×10⁻⁶. 4 U / g SOD; the fruit pomace after SOD extraction was freeze-dried and ground into powder, defatted with n-hexane, and then added to a 70% ethanol solution containing 0.1% HCl at a ratio of 1g:10mL. Extraction was performed under ultrasonic assistance for 30 minutes, followed by filtration. Purification was carried out using a D101 macroporous resin column. Separation was performed by high-speed countercurrent chromatography (HSCCC), followed by freeze-drying. Purity was determined and identified by liquid chromatography-mass spectrometry (LC / MS), yielding tannins with a purity of 89% and a concentration of 40.96 mg / g.

[0058] (2) Mix SOD with a concentration of 2 mg / mL and tannin with a concentration of 40.96 mg / g at a mass ratio of 1:1, then mix with 2% sodium alginate powder, adjust the pH of the mixed solution with 4M NaOH to make the pH value 5, and then disperse it in a 10% shellac solution to obtain a gel.

[0059] The gel was injected to form a solution at a height of 10 cm. The solution was then dripped into a gelation solution containing 0.50‰ chitosan and 2.00% CaCl2 through a 35-gauge needle. The gel particles formed a gel in the gelation solution over 30 minutes, stirred with a magnetic stirrer. Finally, the gel particles were rinsed with deionized water to obtain pH-sensitive intestinal-targeting gel particles.

[0060] Example 3

[0061] (1) After removing the core and crushing the prickly pear, add 3% NaCl solution at a ratio of 1g:5mL, and extract with ultrasonic assistance for 30 minutes, then filter. The filtrate is then subjected to a water bath at 55-60℃ for 30 minutes, the pH is adjusted to 5 with 10% NaHCO3, and then purified by precipitation with 10% ethanol. Finally, NaCl powder is added to a concentration of 20-30%, the precipitate is washed, and then freeze-dried to obtain an activity of 6×10⁻⁶. 4 U / g SOD; the fruit pomace after SOD extraction was freeze-dried and ground into powder, defatted with n-hexane, and then added to a 70% ethanol solution containing 0.1% HCl at a ratio of 1g:10mL. Extraction was performed under ultrasonic assistance for 30 minutes, followed by filtration. Purification was carried out using a D101 macroporous resin column. Separation was performed by high-speed countercurrent chromatography (HSCCC), followed by freeze-drying. Purity was determined and identified by liquid chromatography-mass spectrometry (LC / MS), yielding tannins with a purity of 89% and a concentration of 40.96 mg / g.

[0062] (2) Mix SOD with a concentration of 2 mg / mL and tannin with a concentration of 40.96 mg / g at a mass ratio of 1:3, then mix with 2% sodium alginate powder, adjust the pH of the mixed solution with 4M NaOH to make the pH value 5, and then disperse it in a 5% shellac solution to obtain a gel.

[0063] The gel was injected to form a solution at a height of 8 cm. The solution was then dripped into a gelation solution containing 0.50‰ chitosan and 2.00% CaCl2 through a 35-gauge needle. The gel particles formed a gel in the gelation solution over 30 minutes, stirred with a magnetic stirrer. Finally, the gel particles were rinsed with deionized water to obtain pH-sensitive intestinal-targeting gel particles.

[0064] Example 4

[0065] The steps are the same as in Example 1, except that the mass ratio of SOD to tannin is 1:6.

[0066] Examples 5-8

[0067] The steps are the same as in Example 3, except that the pH environment during gel preparation in step (2) is 4, 5, 6 and 7 respectively.

[0068] Performance and Testing

[0069] 1. The encapsulation efficiency of the gel particles obtained in Examples 1-4 and Examples 5-8 was tested, and the results are as follows: Figure 2 As shown, Figure 2 (a) is a schematic diagram showing the encapsulation efficiency of the gel particles obtained in Examples 1-4. Figure 2 (b) is a schematic diagram showing the encapsulation efficiency of the gel particles obtained in Examples 5-8; based on Figure 2 It was found that the best overall effect was achieved when the ratio of SOD to tannic acid was 1:3 and the pH was 5, with encapsulation rates of SOD and tannic acid of 66.55±1.27% and 79.67±2.96%, respectively. However, the encapsulation rates of gel particles prepared by using SOD and tannic acid alone were 51.12±1.96% and 76.89±2.25%, respectively, indicating that the present invention improves the encapsulation rate by using a combination of these components.

[0070] 2. The antioxidant activity of the gel particles obtained in Example 7 was tested. Specifically, the gel particles obtained in Example 7 were used as the experimental group, and three control groups were set up: a blank gel particle group, a gel particle group encapsulated with SOD alone, and a gel particle group encapsulated with tannin alone. The antioxidant activity of these control groups was tested, and the results are as follows: Figure 3 As shown, based on Figure 3It was found that gel particles co-encapsulated with SOD and tannins exhibited a significant synergistic antioxidant effect in scavenging DPPH and hydroxyl radicals. This synergistic effect resulted in gel particles co-encapsulating SOD and tannins having superior antioxidant capacity compared to gel particles encapsulating SOD or tannins alone. This indicates that liposomes can not only effectively encapsulate these active ingredients but also enhance their antioxidant capacity, demonstrating potential application value.

[0071] 3. The gel particles obtained in Example 7 were subjected to an in vitro digestion simulation experiment; specifically: the gel particles were mixed with simulated gastric juice (pH 2.0, containing 50 mM NaCl and 3.2 mg / mL pepsin), and gastric digestion was carried out at 37°C for 2 hours. Small intestinal digestion stage: the sample after gastric digestion was adjusted to pH 7.0, bile salt solution (15.57 mg / mL bile salt) and pancreatic enzyme solution (48 mg / mL pancreatic enzyme) were added, and small intestinal digestion was carried out at 37°C for 6 hours. Colonic juice digestion stage: the sample after small intestinal digestion was placed in phosphate buffer (PBS, 50 mM, pH 7.4) for 2 hours.

[0072] 4. The effects of different concentrations of shellac solution on gel particles were determined. The stability of SOD and tannin in the gastrointestinal tract by increasing the shellac concentration is shown in Table 1.

[0073] Table 1. Effects of shellac concentration on SOD and tannin sequestration efficiency and retention rate after gastric digestion.

[0074]

[0075] As shown in Table 1, with the increase of shellac concentration, the retention rates of SOD and tannins after 2 hours of gastric digestion were both improved.

[0076] SOD and tannin gel particles without shellac and SOD and tannin gel particles with 8% shellac were set as control groups. Intestinal digestion was simulated for 6 hours and colonic fluid digestion for 2 hours, respectively. The results are as follows: Figure 4 As shown, based on Figure 4 It was found that SOD and tannin gel particles without shellac were essentially released in the intestine, while those with 8% shellac were delivered to the colon. This is due to the unique physical properties of shellac. When the pH of the environment is greater than 7.0, the carboxyl groups of shellac acid begin to dissociate (-COOH→-COO-), causing the shellac to gradually dissolve. Through these processes, shellac significantly improves the bioavailability of SOD, enabling it to better exert its antioxidant and therapeutic effects in the colon.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the protection scope of the claims of the present invention.

Claims

1. A method for preparing pH-sensitive intestinal-targeting gel particles based on prickly pear SOD and tannins, characterized in that, Includes the following steps: SOD and tannins were extracted from prickly pear. The SOD, tannin, and sodium alginate powder were mixed and then dispersed in a shellac solution to obtain a gel. The gel was injected to form a solution, which was then dropped into the gelation solution to form a gel. After stirring, the pH-sensitive intestinal-targeting gel particles were obtained.

2. The method for preparing pH-sensitive intestinal-targeting gel particles based on prickly pear SOD and tannins according to claim 1, characterized in that, The concentration of SOD was 2.00 mg / mL, and the activity was 6 × 10⁻⁶. 4 U / g.

3. The method for preparing pH-sensitive intestinal-targeting gel particles based on prickly pear SOD and tannins according to claim 1, characterized in that, The concentration of the tannin was 40.96 mg / g, and the purity was 89%.

4. The method for preparing pH-sensitive intestinal-targeting gel particles based on prickly pear SOD and tannins according to claim 1, characterized in that, When SOD, tannin, and sodium alginate powder are mixed, the mass ratio of SOD to tannin is 1:6 to 3:

1.

5. The method for preparing pH-sensitive intestinal-targeting gel particles based on prickly pear SOD and tannins according to claim 1, characterized in that, The amount of sodium alginate powder added is 2%.

6. The method for preparing pH-sensitive intestinal-targeting gel particles based on prickly pear SOD and tannins according to claim 1, characterized in that, The mass fraction concentration of the shellac solution is 5-10%.

7. The method for preparing pH-sensitive intestinal-targeting gel particles based on prickly pear SOD and tannins according to claim 1, characterized in that, The gelation solution comprises a solution of 0.50‰ chitosan and 2.00% CaCl2.

8. The method for preparing pH-sensitive intestinal-targeting gel particles based on prickly pear SOD and tannins according to claim 1, characterized in that, The injection height in the aforementioned injection method is 8-10 cm.

9. A pH-sensitive intestinal-targeting gel particle obtained by the preparation method of pH-sensitive intestinal-targeting gel particles based on prickly pear SOD and tannins as described in any one of claims 1 to 8.

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