A spirulina-based preparation for treating chicken intestinal inflammation and a preparation method thereof

Abstract

The patent technology scheme firstly applies the spirulina carrier technology to the field of veterinary drugs, realizes intestinal targeted drug delivery in combination with the characteristics of nalfurafine, and solves the problems of high dose and low utilization rate of traditional preparations. The preparation process is simple, the cost is low, and the industrial production is suitable; the drug residues are reduced, the drug cost is reduced, and the environmental protection and food safety requirements are met.

Description

Technical Field

[0001] This invention belongs to the field of veterinary drug formulation technology, specifically relating to an intestinal targeted drug delivery system that uses spirulina as a drug carrier to load nasitide, for treating intestinal inflammation in chickens and reducing drug dosage. Background Technology

[0002] Noscetide is a sulfur-containing polypeptide antibiotic produced by the fermentation of actinomycetes. It has broad-spectrum antibacterial activity, especially effective against Gram-positive bacteria such as Staphylococcus aureus and Streptococcus. Its mechanism of action is to inhibit bacterial protein synthesis and block peptide chain elongation, thereby achieving bacteriostatic and bactericidal effects. Because noscetide is almost not absorbed in the intestine, it has high safety and leaves no residue, and is widely used as a feed additive for poultry such as chickens and pigs to prevent and treat intestinal inflammation while promoting animal growth.

[0003] However, nasidopeptide has poor water solubility, and traditional administration methods typically involve adding it to feed as a premix. This method has certain limitations: First, nasidopeptide is not absorbed in the body; the drug moves through the intestines with the intestinal contents and is excreted along with them, resulting in low drug utilization. Higher doses and continuous administration are required to achieve a therapeutic effect (for example, in broiler feed, 40-80g of nasidopeptide needs to be added per 1000kg of feed for 7 consecutive days to treat necrotic enteritis). This not only increases farming costs but may also lead to bacterial resistance. Second, the traditional premix form of nasidopeptide has a short residence time in the intestines, failing to achieve sustained and targeted release, resulting in a shorter duration of action and requiring relatively high therapeutic doses.

[0004] Spirulina is a natural microalga belonging to the phylum Cyanobacteria. It possesses a spiral structure and is rich in nutrients such as proteins, polysaccharides, vitamins, and minerals. In recent years, due to its unique physicochemical properties and biocompatibility, spirulina has been widely used in the field of drug delivery. The surface of spirulina carries a negative charge, enabling it to load positively charged drug molecules through electrostatic adsorption. Simultaneously, its spiral structure can embed itself into the intervillous spaces of the intestinal tract, prolonging the drug's retention time and improving its bioavailability.

[0005] Spirulina offers several advantages as a drug carrier: High biocompatibility: Spirulina is a natural, non-toxic, and harmless substance with excellent biocompatibility, making it suitable for drug delivery systems in both animals and humans. High loading capacity: The porous structure and negative surface charge of spirulina enable it to efficiently load various drug molecules, including the poorly water-soluble nascetin. Targeted release: Spirulina maintains its structural integrity in the acidic environment of the stomach. Once in the intestines, its spiral structure allows it to embed itself in the intervillous spaces, achieving slow release and targeted drug delivery. Synergistic effect: Spirulina itself possesses anti-inflammatory, antioxidant, and immunomodulatory properties, which can enhance the therapeutic effects of drugs. For example, the polysaccharide components in spirulina can inhibit the expression of pro-inflammatory factors (such as TNF-α and IL-6), thereby enhancing the therapeutic effect of nascetin on intestinal inflammation.

[0006] Currently, studies have shown that spirulina can be used to load drugs such as curcumin and doxorubicin for the treatment of diseases such as colitis and colon cancer. However, there are no reports on its use in the veterinary drug field, particularly regarding the loading and application of nasitide. Therefore, developing an intestinal targeted drug delivery system based on spirulina loaded with nasitide has significant application value and market potential. Summary of the Invention

[0007] Preparation method of Spirulina loaded with narcissin:

[0008] Spirulina pretreatment: The dried spirulina is pulverized to a particle size of 100-200 mesh and ultrasonically treated to make its surface charge distribution uniform and increase its loading capacity.

[0009] Drug loading: Narcissin was added to a chitosan solution (pH 8.0-9.0), homogenized, and then mixed with spirulina at a mass ratio of 1:10 to 1:50. High-efficiency loading was achieved using electrostatic adsorption and microalgal water channels. The loading rate and release rate of the drug can be controlled by adjusting the pH and drug concentration of the solution.

[0010] Drying and Formulation: The loaded spirulina is freeze-dried to maintain its structure and drug stability. The dried complex is then mixed with excipients (such as starch and silica) to form granular or powdered premixes that can be directly added to feed.

[0011] This invention offers the following technical advantages: Targeted Release: Spirulina maintains its structural integrity in the acidic environment of the stomach. After entering the intestines, its spiral structure allows it to embed itself in the intervillous spaces, achieving slow release and targeted drug action. This targeted release mechanism prolongs the drug's duration of action in the intestines, improving therapeutic efficacy. Reduced Dosage: Due to the loading effect of spirulina, the utilization rate of nasitide is significantly improved, and the effective dose can be reduced to 10%-20% of the traditional dosage (e.g., only 10-20g per 1000kg of feed). This not only reduces breeding costs but also decreases the risk of drug residues and drug resistance. Synergistic Effect: Spirulina itself has anti-inflammatory, antioxidant, and immunomodulatory effects, which can enhance the therapeutic effect of nasitide. For example, the polysaccharide components in spirulina can inhibit the expression of pro-inflammatory factors (such as TNF-α and IL-6), thereby enhancing the therapeutic effect of nasitide on intestinal inflammation.

[0012] This patented technology is the first to apply spirulina carrier technology to the veterinary drug field, combining it with the properties of nasitide to achieve intestinal targeted drug delivery, solving the problems of high dosage and low utilization rate in traditional formulations. The formulation preparation process is simple and low-cost, suitable for industrial production; it reduces drug residues, lowers medication costs, and meets environmental protection and food safety requirements. Detailed Implementation

[0013] Example 1: Loading Process Example 100 mg of 80% nasitide was added to 50 ml of chitosan solution (pH 8.0-9.0, 10% WT), homogenized, and then 5 g of spirulina powder was added and mixed. The mixture was sonicated for 30 minutes, centrifuged, and then freeze-dried to obtain the complex. The content of nasitide in the complex was 1.36% WT.

[0014] Example 2: Loading Process Example 200 mg of 80% nasitide was added to 100 ml of chitosan solution (pH 8.0-9.0, 10% WT), homogenized, and then 5 g of spirulina powder was added and mixed. The mixture was sonicated for 30 minutes, centrifuged, and then freeze-dried to obtain the complex. The content of nasitide in the complex was 2.98% WT.

[0015] Example 3: Formulation Preparation Example The nasitide complex preparation prepared in Example 2 was added to the carrier starch according to the specified content and mixed evenly to obtain a nasitide complex preparation premix with an effective content of 1% WT.

[0016] Example 4: Animal Experiment Verification Case Eighty 10-day-old yellow-feathered broiler chickens of similar weight were randomly divided into four groups: a control group, a nasitide group, and two groups (group 1 and group 2) containing spirulina-loaded nasitide complex, with 20 chickens in each group. Except for the control group, the other three groups were challenged with wild-type Clostridium perfringens virus to establish an artificial infection model of Clostridium perfringens disease in chickens. The nasitide group was administered the virus at a dose of 40 mg / kg via feed for 7 consecutive days. Spirulina-loaded nasitide complex groups 1 and 2 were administered the virus at doses of 10 mg / kg and 20 mg / kg, respectively, via feed for 7 consecutive days. Clinical symptoms and fecal condition were observed daily before administration, at the end of administration, and on day 7 after administration. A comprehensive score was calculated, and the cure rate was determined. Fasting weight was measured in the morning before administration and on day 7 after administration to evaluate weight gain and feed conversion ratio. On day 7 after administration, 10 chickens were randomly selected from each of the four experimental groups and euthanized. Small intestinal sections were collected for histological analysis to calculate villus height and crypt depth. The results showed that both the ordinary nasitide group and the spirulina-loaded nasitide complex group could treat artificially induced Clostridium perfringens infection in chickens. Compared with the ordinary nasitide group, the spirulina-loaded nasitide complex group showed better results in terms of cure rate, feed conversion ratio, and intestinal health indicators. This indicates that, with a reduced dosage of nasitide, spirulina-loaded nasitide can not only achieve the same therapeutic effect as ordinary nasitide but also has advantages in improving feed conversion ratio and intestinal health.

[0017] Table 1. Clinical observation scores of spirulina-loaded nasitin complex in the treatment of artificially infected chickens with Clostridium perfringens. Group Dosage (mg / kg) Clinical observation score (x±STD) Infection without medication group — <![CDATA[4.02±2.12 a ]]> Nasiside group 40 <![CDATA[1.98±2.39 b ]]> Spirulina loaded with nascitide complex group 1 10 <![CDATA[1.23±4.58 b ]]> Spirulina loaded with nascitide complex group 2 20 <![CDATA[1.16±2.85 b ]]> Note: No identical letters in the same series of subheadings indicates a significant difference (p < 0.05), while identical letters indicate no significant difference (p > 0.05). Table 2. Cure rate of Spirulina-loaded nasitin complex in treating artificially infected chickens with Clostridium perfringens. Group Dosage (mg / kg) Cure rate (%) Infection without medication group — 36.15% Narciside group 40 72.36% Spirulina loaded with nascitide complex group 1 10 89.75% Spirulina loaded with nascitide complex group 2 20 92.83% Table 3 Weight gain and feed conversion ratio Group Dosage (mg / kg) Average weight gain (g) Meat-to-fat ratio Infection without medication group — <![CDATA[43.68±28.96 a ]]> 3.01 Nasiside group 40 <![CDATA[79.85±20.38 b ]]> 2.11 Spirulina loaded with nascitide complex group 1 10 <![CDATA[93.73±25.46 c ]]> 1.88 Spirulina loaded with nascitide complex group 2 20 <![CDATA[106.59±23.30 c ]]> 1.68 Note: No identical letters in the same series of subheadings indicates a significant difference (p < 0.05), while identical letters indicate no significant difference (p > 0.05). Table 4 Indicators of Gut Health Group Intestinal villus height (μm) crypt depth (μm) Plum height / crypt depth (V / C) Infection without medication group <![CDATA[239.18±12.62 a ]]> <![CDATA[124.83±7.81 a ]]> <![CDATA[1.92±1.62 a ]]> Nasiside group <![CDATA[254.67±13.77 a ]]> <![CDATA[116.57±7.63 a ]]> <![CDATA[2.18±1.80 a ]]> Spirulina loaded with nascitide complex group 1 <![CDATA[273.66±17.80 b ]]> <![CDATA[101.63±3.93 b ]]> <![CDATA[2.69±4.53 b ]]> Spirulina loaded with nascitide complex group 2 <![CDATA[316.72±7.63 b ]]> <![CDATA[93.86±3.80 b ]]> <![CDATA[3.37±2.01 b ]]> Note: No identical letters in the same series of shoulder labels indicate a significant difference (p < 0.05), while identical letters indicate no significant difference (p > 0.05).

Claims

1. A complex of spirulina loaded with nascetin, characterized in that: The complex is composed of a spirulina carrier and nascitide bound by electrostatic adsorption; the loading rate of nascitide is 10%-20%; the particle size of the spirulina carrier is 100-200 mesh, and the surface charge distribution is uniform. The complex enables sustained and targeted drug release within the intestine.

2. The Spirulina-loaded nascetin complex according to claim 1, characterized in that: The spirulina carrier is pretreated, including crushing and sieving to a particle size of 100-200 mesh, and ultrasonic treatment to make its surface charge distribution uniform; the nasitide is added to a chitosan solution (pH 8.0-9.0), treated with a homogenizer, and then mixed with spirulina at a mass ratio of 1:10 to 1:50, achieving high-efficiency loading through electrostatic adsorption and microalgal water channels.

3. The Spirulina-loaded nascetin complex according to claim 1 or 2, characterized in that: The complex is prepared by freeze-drying to maintain the structure of spirulina and the stability of the drug; the complex is mixed with pharmaceutically acceptable excipients (such as starch and silica) to form a granular or powdered premix.

4. A premix for treating chicken intestinal inflammation, characterized in that: A complex comprising Spirulina loaded with nascitide according to any one of claims 1-3; the dosage of nascitide in the premix is ​​10-20 g / 1000 kg feed; the premix can be directly added to feed for the treatment of intestinal inflammation in chickens.

5. The chicken intestinal inflammation treatment premix according to claim 4, characterized in that: The premixed agent reduces the dosage of nasitide and improves efficacy by prolonging the drug's residence time in the intestine and through a targeted release mechanism; the premixed agent increases the intestinal inflammation suppression rate by more than 20% when the dosage is reduced to 10%-20% of the original dosage.

6. A method for preparing spirulina-loaded nascetin, characterized in that... The process includes the following steps: Step 1: Pulverize dried spirulina to a particle size of 100-200 mesh, and treat it chemically and ultrasonically to ensure uniform surface charge distribution; Step 2: Add nasitide to a chitosan solution (pH 8.0-9.0), homogenize it using a homogenizer, and mix it with spirulina at a mass ratio of 1:10 to 1:50, utilizing electrostatic adsorption and microalgal water channels to achieve high-efficiency loading; Step 3: Freeze-dry the loaded spirulina to obtain a complex with a loading rate of 10%-20%; Step 4: Mix the dried complex with pharmaceutically acceptable excipients (such as starch and silica) to prepare a granular or powdered premix.

7. The preparation method according to claim 6, characterized in that: The ultrasonic treatment time is 30 minutes; the chitosan solution concentration is 5% WT; the pH value of the chitosan solution is 8.0-9.0; the freeze-drying process is used to maintain the stability of the spirulina structure and the drug.

8. The application of a Spirulina-loaded nascetin complex in the preparation of a drug for treating chicken intestinal inflammation, characterized in that: The complex reduces the dosage of nasitide and improves efficacy by prolonging the drug's residence time in the intestine and through a targeted release mechanism; when the dosage is reduced to 10%-20% of the original dosage, the complex increases the intestinal inflammation suppression rate by more than 20%.

9. The application according to claim 8, characterized in that: The complex is used to prepare a premix for treating chicken intestinal inflammation, which can be directly added to feed; the dosage of nasitide in the premix is ​​10.0-20.0g / 1000kg feed.

10. A chicken intestinal inflammation treatment system based on spirulina-loaded nascetin, characterized in that: The system comprises a complex of spirulina loaded with nascetide according to any one of claims 1-3; the system achieves sustained and targeted release of the drug by embedding the spiral structure of spirulina into the intervillous space of the intestine; the system can reduce the dosage of nascetide and improve the therapeutic effect, while reducing the risk of drug residues and drug resistance.