A composition of euglena for improving gout and a method of preparing the same

Abstract

This invention belongs to the food field, specifically health food, and discloses a Euglena composition for improving gout and its preparation method. The composition is prepared from the following raw materials in parts by weight: 3-8 parts of Coix seed, 3-8 parts of Poria cocos, 0.1-6 parts of Gardenia jasminoides, 0.1-6 parts of Imperata cylindrica root, 2-5 parts of corn silk, 2-5 parts of Pueraria lobata root, 0.1-10 parts of Euglena galanga powder, 0.1-5 parts of Lactobacillus plantarum powder, 0.1-4 parts of mangiferin, 0.1-6 parts of celery seed extract, and 0.1-5 parts of sour cherry extract. The preparation process is efficient and controllable, and the application is safe and effective.

Description

Technical Field

[0001] This invention relates to the food industry, and in particular to a Euglena composition for improving gout and its preparation method. Background Technology

[0002] Gout is a common metabolic disorder characterized by the excessive deposition of uric acid in the joints, leading to the formation of tophi (gouty nodules). Symptoms include severe joint pain, inflammation, and swelling, significantly impacting patients' quality of life. A study based on data from the Global Burden of Disease, Injury, and Risk Factors Study (GBD) systematically analyzed the epidemiological trends of gout globally and in China from 1990 to 2021. Data showed that from 1990, the number of gout cases in China was 1,182,498, with an incidence rate of 122.522 per 100,000 people. By 2021, the number of gout cases in China had increased to 3,079,836, and the incidence rate had risen to 151.612 per 100,000 people. Furthermore, the average annual percentage change (AAPC) of gout incidence rate in China was 0.7014, significantly higher than the global average of 0.5166, indicating a more rapid increase in gout incidence in China.

[0003] The onset of gout is influenced by various factors, including external environmental factors, internal dietary habits, and even racial and familial genetics. Essentially, the pathogenesis of gout stems from an imbalance in the metabolic pathways of purines, leading to excessive production and / or reduced excretion of uric acid metabolites. Currently, there are generally three types of drugs used to lower uric acid: xanthine oxidase inhibitors, drugs that promote uric acid excretion, and uricase inhibitors. While these drugs are widely used due to their significant effects, their severe side effects often deter patients. A safe and effective medication is urgently needed. Summary of the Invention

[0004] The purpose of this invention is to provide a safe and effective composition for improving gout, wherein *Lactobacillus plantarum* powder can increase the beneficial bacteria in the gut of gout patients. A method for preparing this composition is also provided, solving problems such as the poor solubility of mangiferin and the inactivation of *Lactobacillus plantarum* powder upon contact with acid, and ensuring a stable preparation process.

[0005] To address the aforementioned problems, this invention provides a Euglena composition for improving gout, prepared from the following raw materials in parts by weight: 3-8 parts of Coix seed, 3-8 parts of Poria cocos, 0.1-6 parts of Gardenia jasminoides, 0.1-6 parts of Imperata cylindrica root, 2-5 parts of corn silk, 2-5 parts of Pueraria lobata root, 0.1-10 parts of Euglena powder, 0.1-5 parts of Lactobacillus plantarum powder, 0.1-4 parts of mangiferin, 0.1-6 parts of celery seed extract, and 0.1-5 parts of sour cherry extract.

[0006] The composition is prepared from the following raw materials in parts by weight: 2-6 parts of Coix seed, 2-6 parts of Poria cocos, 0.1-3 parts of Imperata cylindrica root, 0.1-3 parts of Gardenia jasminoides, 3-5 parts of Pueraria lobata root, 3-5 parts of corn silk, 0.1-5 parts of Euglena gracilis powder, 0.1-3 parts of Lactobacillus plantarum powder, 0.1-2 parts of mangiferin, 0.1-3 parts of celery seed extract, and 0.1-3 parts of sour cherry extract.

[0007] The composition is prepared from the following raw materials in parts by weight: 5 parts of Coix seed, 5 parts of Poria cocos, 0.7 parts of Imperata cylindrica root, 0.7 parts of Gardenia jasminoides, 4 parts of Pueraria lobata root, 3 parts of corn silk, 1.5 parts of Euglena argyi powder, 0.3 parts of Lactobacillus plantarum powder, 0.2 parts of mangiferin, 0.6 parts of celery seed extract, and 0.2 parts of sour cherry extract.

[0008] The composition further comprises a food additive, which is one or more of the following: sodium alginate, micronized silica gel, cross-linked polyvinylpyrrolidone, 50% ethanol, calcium chloride, hydroxypropyl-β-cyclodextrin (HP-β-CD), microcrystalline cellulose, and magnesium stearate.

[0009] The composition is prepared by the following method:

[0010] Step 1: Inoculate Lactobacillus plantarum powder into MRS liquid medium and culture until the viable count reaches 10. 9 After centrifugation, the supernatant was discarded and the collected bacterial cells were made into a bacterial suspension. The bacterial suspension and 20 g / L sodium alginate aqueous solution were mixed evenly at a volume ratio of 1:3 and poured into a pressure-resistant bottle. The mixed liquid was added dropwise to 10 g / L CaCl2 solution by high-frequency oscillation using a microcapsule granulator. The parameters were adjusted to make the droplets free of agglomeration and uniform. After granulation, the mixture was solidified for 40 min and centrifuged to obtain the sample. The CaCl2 residue on the surface of the microcapsules was washed away with physiological saline to obtain the finished microcapsules, which were stored at -20℃.

[0011] Step 2: Weigh an appropriate amount of ultra-finely pulverized coix seed, poria cocos, corn silk, gardenia, kudzu root, and imperata root, add 8 times the weight of water, extract in a 100℃ water bath for 2 hours, filter, repeat twice, combine the filtrates, concentrate under reduced pressure, and spray dry to obtain a mixed extract of traditional Chinese medicine.

[0012] Step 3: Weigh out appropriate amounts of celery seed extract, sour cherry extract, and mixed extract of traditional Chinese medicine, and premix with appropriate amount of micronized silica gel for 5 min. Finally, add microcapsules of Lactobacillus plantarum powder, mix at 100 r / min, granulate by dry method, adjust granulation pressure, granulate through 14 mesh sieve, add magnesium stearate (0.5%) and mix for 5 min to obtain the upper granules.

[0013] Step 4: Prepare mangiferin-HP-β-CD inclusion complex at a 1:1 ratio, dry and pulverize through a 100-mesh sieve; premix Euglena powder with an appropriate amount of micronized silica gel for 10 min, and pass through an 80-mesh sieve; mix mangiferin inclusion complex, pretreated Euglena powder, an appropriate amount of microcrystalline cellulose, and an appropriate amount of cross-linked polyvinylpyrrolidone (PVPP) evenly, add 50% ethanol aqueous solution as a binder to prepare a soft material, granulate, dry, add 0.5% magnesium stearate and mix for 10 min to obtain the lower layer particles; finally, load the upper and lower layer particles into the two feeding hoppers of a multi-functional rotary tablet press and compress them into tablets to obtain Euglena compound tablets.

[0014] In the preparation method of the composition, the pressure is controlled at 7.013 kN and the rotation speed at 37 r / min throughout the tableting process, and the amount of feed is adjusted according to the filling ratio of 1.996:1 to ensure uniform layering.

[0015] The solid dosage form of the composition is a bilayer tablet.

[0016] The application of the composition in the preparation of foods that improve gout.

[0017] The application of the composition in the preparation of health food products for improving gout.

[0018] Job's tears (Coix lacryma-jobi) is the dried, mature seed of the grass Coix lacryma-jobi L. var. mayuen (Roman.) Stapf. It is sweet, bland, and cool in nature. It enters the spleen, stomach, and lung meridians. Its main functions are to promote diuresis and eliminate dampness, strengthen the spleen and stop diarrhea, relieve numbness, drain pus, and detoxify and dissipate nodules. It is rich in esters and fatty acids, polysaccharides, proteins, as well as various active ingredients such as phenolic acids, sterols, flavonoids, lactams, triterpenes, alkaloids, and adenosine. It possesses various pharmacological effects, including anti-inflammatory, anti-tumor, metabolic and immune regulation, anti-aging and antioxidant effects, hypotensive effects, bone repair, and intestinal flora regulation. Studies have found that coix seed exhibits significant uric acid-lowering effects in adenosine-induced HK-2 cells and in mouse models of hyperuricemia. The mechanisms involve inhibiting xanthine oxidase activity, regulating urate transporter expression, and alleviating kidney damage caused by hyperuricemia by downregulating the IL-6 / Janus kinase 2 / signal transducer and activator of transcription 3 signaling pathways. Furthermore, coix seed can also alleviate systemic diseases associated with hyperuricemia by regulating gut microbiota.

[0019] Poria cocos is the dried sclerotium of the fungus *Poria cocos* (Schw.) Wolf (family Polyporaceae). It is sweet, bland, and neutral in nature. It enters the heart, lung, spleen, and kidney meridians. Its main functions are to promote diuresis and eliminate dampness, strengthen the spleen, and calm the mind. It is used for edema with oliguria, phlegm retention with dizziness and palpitations, spleen deficiency with poor appetite, loose stools and diarrhea, restlessness, palpitations, and insomnia. Modern research shows that Poria cocos can reduce serum uric acid levels in animal models of hyperuricemia by inhibiting the expression of urate transporters. Studies have found that Poria cocos extract can significantly reduce serum uric acid, creatinine, and urea nitrogen levels, inhibit xanthine oxidase activity, and alleviate kidney damage. Further research based on metabolomics and network pharmacology has identified xanthine dehydrogenase and fatty acid synthase as key targets, with purine metabolism, fatty acid biosynthesis, and primary bile acid biosynthesis as the main pathways of action.

[0020] Corn silk, the style and stigma of *Zea mays* L., a plant of the Poaceae family, is sweet and bland in taste and neutral in nature. It enters the bladder, liver, and gallbladder meridians, and has diuretic, swelling-reducing, liver-clearing, and gallbladder-benefiting effects. Lu Jing et al., through pharmacological experiments, found that total flavonoids from corn silk (TFCS) significantly reduced kidney damage in rats with uric acid nephropathy. The mechanism may be related to TFCS reducing the expression levels of URAT1 and GLUT9 proteins and increasing the expression level of ABCG2 protein in kidney tissue.

[0021] Gardenia is the dried, ripe fruit of *Gardenia jasminoides* Ellis, a plant in the Rubiaceae family. It is bitter and cold in nature. It enters the heart, lung, and triple burner meridians. It has the effects of purging fire and relieving irritability, clearing heat and promoting diuresis, cooling blood and detoxifying; externally, it reduces swelling and relieves pain. Studies by Zhu Jixiao et al. have found that gardenia extract can lower serum uric acid in hyperuricemic mice, and inhibiting XOD activity is one of the mechanisms by which it lowers serum uric acid levels in hyperuricemic mice. Meng Zhaoqing et al. found that geniposide can lower serum uric acid levels in hyperuricemic mice by promoting uric acid excretion.

[0022] Kudzu root is the dried root of *Pueraria lobata* (Willd.) Ohwi, a plant in the legume family. It has a sweet and pungent taste, and is cool in nature; it enters the spleen, stomach, and lung meridians. It has the effects of relieving muscle tension and reducing fever, promoting body fluid production and quenching thirst, promoting rash eruption, raising yang and stopping diarrhea, unblocking meridians and activating collaterals, and detoxifying alcohol. Studies by Xiong Yichen et al. have found that the mechanism of action of puerarin in treating acute gouty arthritis in rats may be related to puerarin inhibiting the TLR4 / NF-κB signaling pathway and suppressing the production of downstream inflammatory factors IL-1β and TNF-α, thereby reducing the degree of inflammation in acute gouty arthritis and playing a therapeutic role.

[0023] Imperata cylindrica root is the dried rhizome of *Imperata cylindrica* Beauv. var. major (Nees) CE Hubb., a plant belonging to the Poaceae family. It is sweet and cold in nature, and enters the lung, stomach, and bladder meridians. It has the effects of cooling the blood and stopping bleeding, clearing heat and promoting diuresis. It is used for hematemesis due to blood heat, epistaxis, hematuria, thirst due to febrile diseases, jaundice due to damp-heat, edema with oliguria, and painful urination due to heat. It has a wide range of pharmacological effects; its main components include triterpenes, phenylpropanoids, organic acids, flavonoids, steroids, volatile substances, polysaccharides, and other chemical components, possessing hemostatic, antitumor, renal function-improving, anti-inflammatory, anthelmintic, antibacterial, antioxidant, hypoglycemic, and hypotensive effects.

[0024] Euglena, also known as Euglena algae, is a type of single-celled eukaryotic microalgae that can perform strong photosynthesis. Moreover, Euglena cells contain a great deal of vitamins, chlorophyll, antioxidants, and other essential nutrients that the body needs every day.

[0025] Celery seed, the dried, mature fruit of the umbelliferous plant *Apium spp.*, is a classic food and medicine ingredient, primarily containing apigenin, volatile oils, flavonoids, and coumarins. Traditional Chinese medicine considers it to be cool in nature, pungent and bitter in taste, and associated with the liver and heart meridians. It is believed to have the effects of clearing liver heat, extinguishing wind, and promoting diuresis, and is often used to treat dizziness and edema. Modern pharmacological research has found that celery seed has anti-inflammatory and diuretic effects, and can help regulate uric acid metabolism and blood pressure, often appearing in dietary supplements related to high uric acid and gout.

[0026] Mango leaves are the leaves of *Mangiferaindica* L., a plant in the Anacardiaceae family. Records of mango leaves as a traditional Chinese medicine date back to the 1977 edition of the *Dictionary of Traditional Chinese Medicine*. Mango leaves are rich in mangiferin, a single component of the herb. This medicinal material has wide applications and is a major ingredient in many traditional Chinese medicine preparations, such as honeysuckle mango granules and mango cough tablets. Mangiferin, also known as anemarrhena asphodeloides or mangiferin, is a natural uric acid-lowering flavonoid compound. Studies have shown that mangiferin is highly effective and low in toxicity in lowering blood uric acid levels. Its uric acid-lowering effect is comparable to, and even superior to, that of allopurinol, a first-line clinical uric acid-lowering drug, while its toxicity is far less than that of allopurinol. It is a typical poorly water-soluble compound.

[0027] Sour cherries are a common edible fruit belonging to the genus *Prunus* in the Rosaceae family. They are rich in anthocyanins, proanthocyanidins, polyphenols, vitamin C, potassium, and other nutrients. Their core activity lies in their strong antioxidant and anti-inflammatory capabilities. Modern research has confirmed that they can help relieve joint inflammation and reduce joint discomfort.

[0028] The gut microbiota of gout patients differs significantly from that of healthy individuals, mainly manifested as an imbalance in microbiota structure and abnormal function. In particular, the abundance of beneficial bacteria that produce short-chain fatty acids (SCFAs), such as Bifidobacterium, Lactobacillus plantarum powder, Faecalibacterium, and Faecalibacterium, is reduced in the gut of gout patients.

[0029] This invention (Euglena compound tablets) has the following advantages:

[0030] 1. Explanation of Traditional Chinese Medicine Theory

[0031] Although ancient Chinese medical texts do not explicitly record gout as a disease, it can be categorized under "Bi syndrome" or "arthralgia" based on its clinical manifestations. It is often caused by factors such as damp-heat, phlegm, and blood stasis, and is triggered by consuming high-purine foods or excessive alcohol consumption. Gout is characterized by a deficiency in the root and an excess in the branch. The deficiency is attributed to dysfunction of the liver, spleen, and kidneys, while the excess arises from the internal accumulation of phlegm, blood stasis, dampness, turbidity, and heat toxins due to these dysfunctions. Excessive consumption of rich and fatty foods can lead to the internal generation of phlegm and dampness, which, over time, transforms into heat; or phlegm, dampness, and blood stasis can combine to cause the disease. Rich, fatty foods and improper dietary control are contributing factors.

[0032] According to the "Standards for Diagnosis and Efficacy of Internal Medicine Diseases in Traditional Chinese Medicine," gout can be divided into four syndrome types. Among them, damp-heat accumulation syndrome is the most common and the main syndrome type during the acute attack of gout. Clinical manifestations include sudden redness, swelling, heat, and pain in the small joints of the lower limbs, tenderness upon touch, local burning sensation, and relief upon cooling. Accompanying symptoms include fever, thirst, restlessness, and dark yellow urine. The tongue is red with a yellow, greasy coating, and the pulse is slippery and rapid. Damp-heat accumulation is mainly caused by external pathogenic factors, excessive consumption of rich and fatty foods, etc., leading to the internal generation of damp-heat. Starting with dampness, it interferes with the normal transportation and transformation of the internal organs, eventually transforming into heat and leading to the accumulation of pathological products. As recorded in "Gezhi Yulun," "Generally, it is caused by the blood being heated, already boiling, and then wading in cold water or standing on damp ground... therefore, pain occurs, which is worse at night, and it travels through the yin." This indicates that in the pathogenesis of gout, dampness and excessive heat in the blood interact, causing damp-heat to accumulate in the body, thus triggering gout. Treatment should focus on clearing heat and dampness, unblocking the meridians, and relieving pain. This invention is based on the TCM theory of "damp-heat accumulation type gout," establishing a classic formula with Poria cocos and Coix seed as the principal herbs to invigorate the spleen and eliminate dampness; Gardenia jasminoides and Imperata cylindrica as the assistant herbs to clear heat and cool the blood; and Corn silk and Pueraria lobata as adjuvant herbs to unblock the channels and eliminate turbidity, achieving the effects of clearing heat and eliminating dampness, unblocking the channels and relieving pain. Building upon this, it innovatively introduces Euglena gracilis powder, sour cherry extract, and celery seed extract. Euglena gracilis is rich in polysaccharides and various active substances, which synergistically enhance metabolic regulation with the TCM components; sour cherry and celery seed provide strong antioxidant support. The entire formula not only follows the TCM principle of "invigorating the spleen and eliminating dampness, clearing heat and unblocking the channels" to treat the root cause, but also combines modern pharmacological uric acid-lowering and anti-inflammatory components to treat the symptoms, achieving multi-target synergistic intervention.

[0033] 2. Three core processes ensure activity and stability

[0034] Microencapsulation protection of Lactobacillus plantarum powder activity: The Lactobacillus plantarum powder in the composition is encapsulated using microencapsulation technology, which effectively isolates it from the erosion of gastric acid and bile salts, significantly improves the intestinal survival rate after oral administration, thereby effectively regulating the imbalanced intestinal flora in gout patients, promoting the production of short-chain fatty acids, and improving intestinal barrier function.

[0035] Improved bioavailability of poorly soluble components: For the water-poorly soluble active ingredient mangiferin, hydroxypropyl-β-cyclodextrin (HP-β-CD) inclusion technology is used to embed its molecules into the hydrophobic cavity of cyclodextrin, which significantly improves its water solubility and dissolution rate, overcomes the absorption bottleneck, and ensures that the uric acid-lowering effect is fully exerted.

[0036] Double-layer tablet preparation process: A double-layer tablet structure is adopted, with the upper layer rich in probiotics and antioxidants, and the lower layer containing Euglena powder and inclusion complexes. By optimizing the tableting parameters (pressure 7.013 kN, rotation speed 37 r / min, filling ratio 1.996:1), physical isolation of functional components is achieved to avoid mutual interference. Simultaneously, the tablets are endowed with excellent hygroscopic resistance and mechanical strength, ensuring storage stability and fractionated release in vivo.

[0037] 3. The drug has significant and dose-dependent effects.

[0038] Pharmacological experiments have confirmed that the composition of this invention performs excellently in a hyperuricemia-gout model. Compared with the model group, this invention can significantly reduce serum uric acid levels (by more than 25%), effectively inhibit xanthine oxidase activity, and reduce uric acid production at its source; at the same time, it significantly reduces the levels of inflammatory factors (IL-1β, IL-6, TNF-α), alleviates joint swelling index and gait abnormalities, improves pathological changes in synovial tissue, and its efficacy is dose-dependent, which is superior to single-component formulations.

[0039] 4. High safety, suitable for long-term conditioning.

[0040] Although the active ingredient mangiferin in the formula has a uric acid-lowering effect comparable to allopurinol, its toxicity is far lower than that of first-line clinical drugs. The other ingredients are mostly food and medicine ingredients (such as coix seed, poria cocos, kudzu root, etc.), which are highly safe and have no obvious toxic side effects. They are suitable for daily long-term conditioning and adjuvant treatment for people with gout. Attached Figure Description

[0041] Figure 1 This is a standard curve for mangiferin concentration.

[0042] Figure 2-1 , Figure 2-2 The images show 3D curves and contour plots illustrating the interaction between tableting pressure and filler ratio.

[0043] Figure 3-1 , Figure 3-2 The images show 3D curves and contour plots illustrating the interaction between tableting pressure and tableting speed.

[0044] Figure 4-1 , Figure 4-2 The figures are a 3D curve and a contour plot showing the interaction between filler ratio and tableting speed, respectively.

[0045] Figure 5 The results of Euglena compound tablets on the 24-hour ankle swelling index in gouty rats (x±s, n=10).

[0046] Figure 6 The results of Euglena compound tablets on ankle swelling index in gouty rats at 48 h (x±s, n=10).

[0047] Figure 7 The results of 24-hour gait score of ankle joint in gout rats using Euglena compound tablets (x±s, n=10).

[0048] Figure 8 The results of Euglena compound tablets on the gait score of ankle joint in gout rats at 48 h (x±s, n=10).

[0049] Figure 9 The results of serum uric acid detection in gouty rats were obtained using Euglena compound tablets (x±s, n=10).

[0050] Figure 10 The results of the detection of xanthine oxidase activity in gouty rats by Euglena compound tablets (x±s, n=10).

[0051] Figure 11 The results of the detection of IL-1β in gouty rats by Euglena compound tablets (x±s, n=10).

[0052] Figure 12 The results of IL-6 detection in gouty rats using Euglena compound tablets (x±s, n=10).

[0053] Figure 13 The results of TNF-α detection in gouty rats using Euglena compound tablets (x±s, n=10) are shown.

[0054] Figure 14 HE staining images of the right ankle joint of rats in each group (left image × 50, right image × 200);

[0055] Figure 15 The results of pathological scoring of Euglena compound tablets in gout rats (x±s, n=3). Detailed Implementation

[0056] In the following examples: Sour cherry extract (5% anthocyanins (UV method)), Xi'an Longze Bioengineering Co., Ltd.; Celery seed extract (20:1, i.e., 20 parts by weight of celery seed extract yielded 1 part by weight of celery seed extract), Fufeng Sinote Biotechnology Co., Ltd.; Mangiferin (97%), Shanghai Maclean Biochemical Technology Co., Ltd.; Euglena powder (Euglena polysaccharide 50-70%), Shanghai Guangyu Biotechnology Co., Ltd.; Lactobacillus plantarum powder, Guangzhou Haoxiang Fine Chemical Co., Ltd.; Coix seed, Poria cocos, Gardenia jasminoides, Imperata cylindrica root, corn silk, and Pueraria lobata were all purchased from Bozhou Chuangxin Technology Consulting Service Co., Ltd., and met the standards of the Chinese Pharmacopoeia (2025 edition).

[0057] Example 1: Process Optimization of Double-Layer Tablets (Compressed Candy)

[0058] 1. Preparation and process optimization of upper layer particles

[0059] 1.1 Optimization of the microencapsulation preparation process of Lactobacillus plantarum powder

[0060] 1.1.1 Preparation of capsule core material

[0061] 3g of *Lactobacillus plantarum* powder was inoculated into MRS liquid medium and anaerobically cultured at 37°C in a constant temperature biochemical incubator until the liquid became turbid and the viable count reached approximately 10⁻⁶. 9 CFU / mL, after centrifuging the lactic acid bacteria in the early stage of the stationary phase obtained from the culture, discard the supernatant, make a bacterial suspension from the collected bacteria, add 3.5%-9% of the total weight of the bacterial suspension as a bacterial strain protectant, let it stand for a period of time, and that is the core material of the lactic acid bacteria liquid capsule.

[0062] 1.1.2 Preparation of Lactobacillus microcapsules

[0063] The collected bacterial sludge and sodium alginate solution were mixed evenly at a volume ratio of 1:3 and poured into a pressure-resistant bottle. Using a microencapsulation apparatus, the mixture of sodium alginate and bacterial suspension was added dropwise to a 10 g / L CaCl2 solution via high-frequency oscillation. Parameters were adjusted to ensure the droplets were non-agglomerated and uniform. After granulation, the mixture was allowed to solidify for 40 min. The sample was obtained by centrifugation, and the residual CaCl2 solution on the surface of the microcapsules was washed away with physiological saline to obtain the finished microcapsules, which were then stored at -20℃.

[0064] 1.1.3 Count of viable bacteria

[0065] Dissolve 1g of microcapsules in 50mL of sterile physiological saline → shake at 37℃ and 180 r / min for 1 h until the powder is completely dissolved → according to GB4789.35-2023 "Food Microbiology Examination - Lactic Acid Bacteria Examination", perform serial dilution and plate the sample to obtain the viable count.

[0066] 1.1.4 Orthogonal Experiment

[0067] Four factors that significantly influence the preparation of lactic acid bacteria microcapsules were selected: sodium alginate concentration, volume ratio of bacterial culture to sodium alginate, chitosan concentration, and CaCl2 concentration. A four-factor, three-level L9(3) model was used. 4 An orthogonal experimental design was used, with encapsulation rate and viable cell count as evaluation indicators, to screen the preparation process of lactic acid bacteria microcapsules. See Table 1.

[0068] Table 1 Microcapsule Formulation L9(3) 4 Orthogonal experimental design table

[0069]

[0070] 1.1.5 Determination of the encapsulation efficiency of Lactobacillus plantarum microcapsules

[0071] Add 1g of Lactobacillus microcapsules to 9 mL of 0.06 mol / L sodium citrate solution, place on a shaker, and incubate at 37℃ and 180 r / min for 3 h before counting viable bacteria.

[0072]

[0073] In the formula: N1 is the total number of live bacteria in the microcapsules after probiotic encapsulation (the product of the bacterial content per gram of microcapsule and the total mass of collected microcapsules); N0 is the total number of live bacteria before encapsulation (the product of the bacterial content per milliliter of concentrated bacterial solution and the volume of concentrated bacterial solution).

[0074] 1.1.6 Analysis of Experimental Results

[0075] Table 2 Orthogonal Experiment Table for Microencapsulation

[0076]

[0077] Table 2 shows that the effects of various factors on the encapsulation rate of *Lactobacillus plantarum* powder are as follows: sodium alginate concentration > ratio of bacterial solution to sodium alginate > calcium chloride concentration > chitosan concentration; the effects of various factors on the viable count of *Lactobacillus plantarum* powder are as follows: sodium alginate concentration > calcium chloride concentration > ratio of bacterial solution to sodium alginate > chitosan concentration. Under optimal conditions, the following were selected: sodium alginate concentration 20 g / L, bacterial solution to sodium alginate ratio 1:3, calcium chloride concentration 10 g / L, and chitosan concentration 5 g / L. The microcapsule encapsulation rate was measured to be 85.33%, and the viable count was 1.71 × 10⁻⁶. 9 cfu / g.

[0078] 1.1.7 Results of the tolerance test of microcapsules to simulated gastric juice

[0079] Weigh 1 g of microcapsules and dissolve them in 9 ml of simulated gastric fluid. Incubate at 37°C and 175 r / min on a shaker. After 2 h, take a sample, filter the microcapsules, wash with physiological saline until neutral, and dry. Treat the dried microcapsules with decapsulation solution and then perform viable counts using a serial dilution method. After 2 h in simulated gastric fluid, the viable count of the lactic acid bacteria microcapsules reached 10-1. 8 The CFU / g of the microcapsules resulted in a cell survival rate of 42% (as shown in Table 3); while the number of viable bacteria in the unencapsulated bacterial solution decreased by two orders of magnitude, with a cell survival rate of 1.95%, indicating that the lactic acid bacteria microcapsules have good acid resistance.

[0080] Table 3. Changes in viable bacterial count of microcapsules in artificial gastric fluid

[0081]

[0082] 1.2 Preparation of Traditional Chinese Medicine Extracts

[0083] 1.2.1. Raw material pretreatment

[0084] Weigh out 50g of coix seed, 50g of poria cocos, 30g of corn silk, 7g of gardenia, 40g of kudzu root, and 7g of imperata root, and then grind them into ultrafine powder to increase the contact area.

[0085] 1.2.2 Extraction Process

[0086] Place the pulverized medicinal powder into an extraction vessel, add 8 times its weight of purified water, and soak for 60 minutes. Heat to boiling, then maintain a gentle boil for 2 hours. Filter and collect the extract. Add 6 times its weight of purified water to the residue and repeat the extraction twice, 1 hour each time. Combine all filtrates and pass through a 100-mesh sieve to remove fine impurities.

[0087] 1.2.3 Concentration and Drying

[0088] The combined extracts were concentrated under reduced pressure at 70°C until a paste with a relative density of 1.10-1.20 was obtained. The paste was then further spray-dried (inlet air temperature 160°C, outlet air temperature 90°C) and pulverized through an 80-mesh sieve to obtain a fine powder of the mixed herbal extract.

[0089] 1.3 Preparation of upper layer particles

[0090] 6g of celery seed extract and 2g of sour cherry extract were vacuum dried at 40℃ until the moisture content was ≤3%, pulverized and passed through a 100-mesh sieve, and premixed with an appropriate amount of micronized silica gel for 5 min. Then, Lactobacillus plantarum powder microcapsules were added, and dry granulation was performed by adjusting the granulation pressure and sizing through a 14-mesh sieve. Magnesium stearate (0.5%) was added and mixed for 5 min.

[0091] 2. Preparation and Process Optimization of Lower Layer Particles

[0092] 2.1 Preparation and optimization of mangiferin inclusion complex

[0093] Weigh 2g of HP-β-CD and 2g of mangiferin into a beaker, add an appropriate amount of 75% ethanol, stir magnetically for the corresponding time, recover the ethanol under reduced pressure at 45℃, dissolve the concentrated product in 30 mL of water, filter, collect the filtrate and precipitate separately, and dry in an oven at 65℃.

[0094] 2.1.1 Drawing the Standard Curve

[0095] Mangiferin reference standard was accurately weighed and prepared into solutions with concentrations of 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 μg / mL. The absorbance was measured at 240 nm. A linear regression was performed on the absorbance (y) against the mass concentration (x), yielding the regression equation: y = 0.0691x + 0.0028, R². 2 =0.9988. See Figure 1 This indicates that the absorbance of mangiferin exhibits a good linear relationship within the mass concentration range of 1.0-10.0 μg / mL.

[0096] 2.1.2 Determination of solubility

[0097] Take mangiferin-HP-β-CD inclusion complexes, add water to prepare supersaturated solutions, shake in a water bath at 25℃ for 1 day to equilibrate, filter with a microporous membrane, take the filtrate, dilute with water to an appropriate factor, and measure the absorbance at 240 nm. Calculate the solubility of each mangiferin in water according to the standard curve equation.

[0098] 2.1.3 Determination of inclusion rate

[0099] Accurately weigh the total mass of mangiferin used in the preparation of the inclusion complex, and simultaneously weigh the total mass of the final dried inclusion complex. Take a certain mass of the dried inclusion complex, add an appropriate amount of 70% ethanol solution, and extract ultrasonically for 30 min. Centrifuge the extract in a high-speed centrifuge (3000 r / min, 4℃) for 5 min, take the supernatant, and measure the absorbance at 240 nm. Substitute the absorbance into the regression equation to calculate the concentration of mangiferin in the supernatant.

[0100] 2.1.4 Single-factor experiment

[0101] Using inclusion rate and solubility as evaluation indicators, single-factor experiments were conducted to analyze the ratio of mangiferin to HP-β-CD, solution pH, inclusion temperature, and inclusion time.

[0102] (1) Ratio of mangiferin to HP-β-CD: The solution pH was fixed at 7.5, the inclusion temperature was 40℃, and the inclusion time was 2 h. The effects of the ratio of mangiferin to HP-β-CD (1:0.5, 1:1, 1:1.5, 1:2, 1:2.5) on the inclusion rate and solubility of mangiferin were investigated.

[0103] (2) Solution pH: The ratio of mangiferin to HP-β-CD was fixed at 1:1, the inclusion temperature was 40℃, and the inclusion time was 2 h. The effect of solution pH (6.5, 7.0, 7.5, 8.0, 8.5) on the inclusion rate and solubility of mangiferin was investigated.

[0104] (3) Inclusion temperature: The ratio of mangiferin to HP-β-CD was fixed at 1:1, the solution pH was 7.5, and the inclusion time was 2 h. The effect of inclusion temperature (10, 20, 30, 40, 50℃) on the inclusion rate and solubility of mangiferin was investigated.

[0105] (4) Inclusion time: The ratio of mangiferin to HP-β-CD was fixed at 1:1, the solution pH was 7.5, and the inclusion temperature was 40℃. The effects of inclusion time (1, 2, 3, 4, 5 h) on the inclusion rate and solubility of mangiferin were investigated.

[0106] 2.1.5 Orthogonal Experiment

[0107] To optimize the inclusion conditions, based on the results of single-factor experiments, the L9(3) method was adopted. 4 An orthogonal experiment was conducted. See Table 4.

[0108] Table 4. Factor Level Table for Orthogonal Experiments

[0109]

[0110] 2.1.6 Analysis of Experimental Results

[0111] Table 5. Results of the orthogonal experiment

[0112]

[0113] Table 5 shows that the main factors affecting the inclusion rate of mangiferin, in order of importance, are the ratio of mangiferin to HP-β-CD, inclusion temperature, solution pH, and inclusion time. Similarly, the main factors affecting the solubility of mangiferin, in order of importance, are the ratio of mangiferin to HP-β-CD, inclusion temperature, solution pH, and inclusion time. Under optimal conditions, a 1:1 ratio of mangiferin to HP-β-CD, an inclusion temperature of 40℃, an inclusion time of 2 h, and a solution pH of 8.0 were selected. The resulting inclusion rate of mangiferin was 82.33%, and the solubility was 30.17 mg / mL.

[0114] 2.2 Preparation of lower layer particles

[0115] Premix 15g of Euglena gracilis powder with micronized silica gel for 10 min, then pass through an 80-mesh sieve. Next, mix mangiferin inclusion complex, pretreated Euglena gracilis powder, microcrystalline cellulose, and PVPP evenly, add 50% ethanol aqueous solution as a binder to prepare a soft mass, and granulate using a 14-mesh sieve. Dry the wet granules in a 40℃ vacuum drying oven until the moisture content is ≤5%, then granulate using a 12-mesh sieve, add magnesium stearate, and mix for 10 min.

[0116] 3. Optimization of the double-layer tableting process

[0117] The upper and lower layers of granules were respectively loaded into the two hoppers of a multi-functional rotary tablet press for tableting. Based on the preliminary experimental results, tableting pressure, double-layer filling ratio, and tableting speed were selected as influencing factors, and the comprehensive score was used as the evaluation index. A Box-Behnken design was conducted, and the experimental factors and levels are shown in Table 6. The total weighting coefficients for granule yield, appearance, disintegration time, hardness, and friability were 13.36%, 11.24%, 29.17%, 31.78%, and 14.45%, respectively.

[0118] Table 6 Orthogonal Experimental Design Table for Double-Layer Tablets

[0119]

[0120] 3.1 Box-Behnken Design

[0121] Table 7 Box-Behnken Design Results

[0122]

[0123] Table 8 Results of Analysis of Variance

[0124]

[0125] Using Design-Expert 13 software, a quadratic multiple regression analysis was conducted. The comprehensive score of the regression equation between the response value and each factor was 62.824 - 6.88875A + 1.2625B + 0.98375C + 2.5325AB + 2.26AC + 5.7575BC + 5.423A² + 6.9855B² + 11.823C². The model's F-value was 16.85, and the P-value was <0.001, indicating that the model was highly significant. The lack-of-fit term had a P-value of 0.0621, indicating that the influence of non-experimental factors was not significant, and the model was valid. Table 8 shows that the linear term M of the model was highly significant (P < 0.01), while B and C were not significant (P > 0.05). The quadratic term L... 2 N 2 For highly significant differences (P<0.01), M 2The difference was statistically significant (P<0.05). The order of influence of each factor was M>L>N, and the response surface and contour plot of the interaction are shown in Figure 2-4.

[0126] 3.2 Process Validation Experiment

[0127] Using Design Expert 13 software, the optimal conditions obtained through regression model prediction analysis were a tableting pressure of 7.013 kN, a double-layer filling ratio of 1.996:1, and a tableting speed of 37 r / min. The comprehensive scores of the three validation experiments were 84.29, 86.47, and 89.13, respectively, with an average score of 86.63 and an RSD of 1.86%, indicating that the optimized process is stable and feasible.

[0128] Example 2: Take 500g of coix seed, 500g of poria cocos, 70g of Imperata cylindrica root, 70g of gardenia, 400g of kudzu root, 300g of corn silk, 150g of Euglena gracilistylus powder, 30g of Lactobacillus plantarum powder, 20g of mangiferin, 60g of celery seed extract, 20g of sour cherry extract, and appropriate amounts of food additives, and prepare them into double-layer tablets.

[0129] Step 1: Inoculate Lactobacillus plantarum powder into MRS liquid medium and culture until the viable count reaches approximately 10. 9 After centrifugation, the supernatant was discarded and the collected bacterial cells were made into a bacterial suspension. The bacterial suspension and 20 g / L sodium alginate aqueous solution were mixed evenly at a volume ratio of 1:3 and poured into a pressure-resistant bottle. The mixed liquid was added dropwise to 10 g / L CaCl2 solution by high-frequency oscillation using a microcapsule granulator. The parameters were adjusted to make the droplets free of agglomeration and uniform. After granulation, the mixture was solidified for 40 min and centrifuged to obtain the sample. The CaCl2 residue on the surface of the microcapsules was washed away with physiological saline to obtain the finished microcapsules, which were stored at -20℃.

[0130] Step 2: Add 8 times the weight of water to the ultra-finely pulverized coix seed, poria cocos, corn silk, gardenia, kudzu root, and imperata root, extract in a 100℃ water bath for 2 hours, filter, repeat twice, combine the filtrates, concentrate under reduced pressure, and spray dry to obtain a mixed extract of traditional Chinese medicine.

[0131] Step 3: Premix celery seed extract, sour cherry extract and mixed Chinese medicine extract with micronized silica gel for 5 minutes. Finally, add Lactobacillus plantarum powder microcapsules, mix at 100 r / min, granulate by dry method, adjust granulation pressure, granulate through 14 mesh sieve, add magnesium stearate (0.5%) and mix for 5 minutes to obtain the upper granules.

[0132] Step 4: Prepare mangiferin-HP-β-CD inclusion complex at a 1:1 ratio, dry, and pulverize through a 100-mesh sieve; premix Euglena powder with an appropriate amount of micronized silica gel for 10 min, and pass through an 80-mesh sieve; mix the mangiferin inclusion complex, pretreated Euglena powder, an appropriate amount of microcrystalline cellulose, and an appropriate amount of cross-linked polyvinylpyrrolidone (PVPP) evenly, add 50% ethanol aqueous solution as a binder to prepare a soft mass, granulate, dry, add 0.5% magnesium stearate and mix for 10 min to obtain the lower layer particles; finally, load the upper and lower layer particles into the two feeding hoppers of a multi-functional rotary tablet press for tableting. Throughout the tableting process, control the pressure at 7.013 kN and the rotation speed at 37 r / min, adjusting the feeding amount according to a 1.996:1 filling ratio to ensure uniform layering. (The mixed upper and lower layer particles are used as the test substance in animal experiments.)

[0133] Example 3: Take 600g of coix seed, 600g of poria cocos, 90g of Imperata cylindrica root, 100g of gardenia, 450g of kudzu root, 500g of corn silk, 160g of Euglena gracilistylus powder, 160g of Lactobacillus plantarum powder, 40g of mangiferin, 70g of celery seed extract, 40g of sour cherry extract, and appropriate amounts of food additives, and prepare them into double-layer tablets.

[0134] Step 1: Inoculate Lactobacillus plantarum powder into MRS liquid medium and culture until the viable count reaches approximately 10. 9 After centrifugation, the supernatant was discarded and the collected bacterial cells were made into a bacterial suspension. The bacterial suspension and 20 g / L sodium alginate aqueous solution were mixed evenly at a volume ratio of 1:3 and poured into a pressure-resistant bottle. The mixed liquid was added dropwise to 10 g / L CaCl2 solution by high-frequency oscillation using a microcapsule granulator. The parameters were adjusted to make the droplets free of agglomeration and uniform. After granulation, the mixture was solidified for 40 min and centrifuged to obtain the sample. The CaCl2 residue on the surface of the microcapsules was washed away with physiological saline to obtain the finished microcapsules, which were stored at -20℃.

[0135] Step 2: Add 8 times the weight of water to the ultra-finely pulverized coix seed, poria cocos, corn silk, gardenia, kudzu root, and imperata root, extract in a 100℃ water bath for 2 hours, filter, repeat twice, combine the filtrates, concentrate under reduced pressure, and spray dry to obtain a mixed extract of traditional Chinese medicine.

[0136] Step 3: Premix celery seed extract, sour cherry extract and mixed Chinese medicine extract with micronized silica gel for 5 minutes. Finally, add Lactobacillus plantarum powder microcapsules, mix at 100 r / min, granulate by dry method, adjust granulation pressure, granulate through 14 mesh sieve, add magnesium stearate (0.5%) and mix for 5 minutes to obtain the upper granules.

[0137] Step 4: Prepare mangiferin-HP-β-CD inclusion complex at a 1:1 ratio, dry it, and then pulverize it through a 100-mesh sieve. Premix Euglena powder with an appropriate amount of micronized silica gel for 10 min, and then pass it through an 80-mesh sieve. Mix the mangiferin inclusion complex, pretreated Euglena powder, an appropriate amount of microcrystalline cellulose, and an appropriate amount of cross-linked polyvinylpyrrolidone (PVPP) evenly. Add 50% ethanol aqueous solution as a binder to prepare a soft material, granulate it, dry it, add 0.5% magnesium stearate, and mix for 10 min to obtain the lower layer granules. Finally, load the upper and lower layer granules into the two feeding hoppers of a multi-functional rotary tablet press for tableting. Control the pressure at 7.013 kN and the rotation speed at 37 r / min throughout the tableting process, and adjust the feeding amount according to a 1.996:1 filling ratio to ensure uniform layering.

[0138] Example 4: Take 800g of coix seed, 800g of poria cocos, 100g of Imperata cylindrica root, 100g of gardenia, 500g of kudzu root, 500g of corn silk, 200g of Euglena gracilis powder, 150g of Lactobacillus plantarum powder, 40g of mangiferin, 80g of celery seed extract, 30g of sour cherry extract, and appropriate amounts of food additives, and prepare them into double-layer tablets.

[0139] Step 1: Inoculate Lactobacillus plantarum powder into MRS liquid medium and culture until the viable count reaches approximately 10. 9 After centrifugation, the supernatant was discarded and the collected bacterial cells were made into a bacterial suspension. The bacterial suspension and 20 g / L sodium alginate aqueous solution were mixed evenly at a volume ratio of 1:3 and poured into a pressure-resistant bottle. The mixed liquid was added dropwise to 10 g / L CaCl2 solution by high-frequency oscillation using a microcapsule granulator. The parameters were adjusted to make the droplets free of agglomeration and uniform. After granulation, the mixture was solidified for 40 min and centrifuged to obtain the sample. The CaCl2 residue on the surface of the microcapsules was washed away with physiological saline to obtain the finished microcapsules, which were stored at -20℃.

[0140] Step 2: Add 8 times the weight of water to the ultra-finely pulverized coix seed, poria cocos, corn silk, gardenia, kudzu root, and imperata root, extract in a 100℃ water bath for 2 hours, filter, repeat twice, combine the filtrates, concentrate under reduced pressure, and spray dry to obtain a mixed extract of traditional Chinese medicine.

[0141] Step 3: Premix celery seed extract, sour cherry extract and mixed Chinese medicine extract with micronized silica gel for 5 minutes. Finally, add Lactobacillus plantarum powder microcapsules, mix at 100 r / min, granulate by dry method, adjust granulation pressure, granulate through 14 mesh sieve, add magnesium stearate (0.5%) and mix for 5 minutes to obtain the upper granules.

[0142] Step 4: Prepare mangiferin-HP-β-CD inclusion complex at a ratio of 1:1, dry it, and then crush it through a 100-mesh sieve; premix the euglena powder with an appropriate amount of microcrystalline silica gel for 10 min, and then sieve it through an 80-mesh sieve. Mix the mangiferin inclusion complex, pretreated euglena powder, an appropriate amount of microcrystalline cellulose, and an appropriate amount of cross-linked polyvinylpyrrolidone (PVPP) evenly, add 50% ethanol aqueous solution as a binder to prepare soft materials, granulate, dry, add 0.5% magnesium stearate and mix for 10 min to obtain the lower-layer granules; finally, load the upper-layer granules and the lower-layer granules into two feed hoppers of a multi-functional rotary tablet press respectively for tabletting. Control the pressure at 7.013 kN and the rotation speed at 37 r / min throughout the tabletting process, and adjust the feeding amount according to a filling ratio of 1.996:1 to ensure uniform layering.

[0143] Example 5: Influence of the present invention on gout model mice

[0144] 1 Experimental materials

[0145] 1.1 Drugs and reagents

[0146] Euglena compound tablets: Prepared according to the method of Example 2. Allopurinol tablets, Hefei Jiulian Pharmaceutical Co., Ltd., batch number 20221006. Colchicine tablets, Yunnan Botanic Pharmaceutical Co., Ltd., batch number 20230621. Potassium oxonate, Shanghai Macklin Biochemical Co., Ltd., batch number C15784654. Sodium urate, Shanghai Macklin Biochemical Co., Ltd., batch number C16381356. Uric acid kit (product number 70925), Shandong Becton Dickinson Biotechnology Co., Ltd., batch number 10330025Y. Rat xanthine oxidase (product number AF3600-A), TNF-α (product number AF3056-A), IL-1β (product number AF2923-A), and IL-6 (product number AF3066-A) ELISA kits, Hunan Aifang Biotechnology Co., Ltd., batch numbers 20240420, 20240416, 20240423, 20240415 respectively. Tween-80, Beijing Solarbio Science & Technology Co., Ltd., batch number 1215E019. Paraformaldehyde fixative, Wuhan Sevier Biotechnology Co., Ltd., batch number CR2206002.

[0147] 1.2 Experimental animals

[0148] 60 male SD rats, weighing 180 - 220 g, were purchased from Hangzhou Medical College, and the animal certificate number is SCXK (Zhe) 2024-0002. This experiment has passed the review of the Animal Ethics Committee of Nanjing University of Chinese Medicine, and the ethics number is 202402A050.

[0149] 1.3 Experimental instruments

[0150] Synergy HT microplate reader, Bio-Tek, USA. BK-280 fully automated biochemical analyzer, Shandong Boke Biotechnology Co., Ltd. 85-2A dual digital display thermostatic magnetic stirrer, Changzhou Surui Instrument Co., Ltd. FA1004 electronic balance, Lichen Technology Co., Ltd.

[0151] 2. Experimental Methods

[0152] 2.1 Preparation of modeling reagents

[0153] Following the method described in the reference, 3 g of potassium oxonate was weighed and added to 97 mL of 0.9% physiological saline. The mixture was stirred and mixed at 70°C to prepare a 3% potassium oxonate suspension, which was then cooled and stored for later use. 250 mg of sodium urate crystals were weighed and added to 9 mL of physiological saline and 1 mL of Tween 80. The mixture was heated and stirred until thoroughly mixed to prepare a sodium urate suspension with a mass concentration of 25 mg / mL. This suspension was stored at 4°C for later use.

[0154] 2.2 Grouping and Dosing

[0155] Male SD rats were randomly divided into a blank control group, a model group, a positive control group, and low-, medium-, and high-dose Euglena compound tablet groups, with 10 rats in each group. The blank control group and the model group were administered an equal volume of physiological saline by gavage. The low-dose Euglena compound tablets were calculated based on a daily human dose of 3.5 g of the finished product, with a rat equivalent dose of 0.3 g / kg. The medium and high doses were 0.6 g / kg and 1.2 g / kg, respectively. The positive control group was initially administered allopurinol 0.1 g / kg by gavage for 4 consecutive days, and from the 5th day onwards, it was switched to colchicine 0.35 × 10⁻⁶. -3 The dose was 100 mg / kg for 3 consecutive days. The other groups were given the corresponding drugs by gavage once a day for 7 consecutive days. The control group and the model group were given an equal volume of distilled water by gavage.

[0156] 2.3 Replication of a rat model of hyperuricemia combined with gout

[0157] Except for the control group, other rats received intraperitoneal injections of 10 μL / g of 3% potassium oxonate suspension twice daily for 7 consecutive days. On day 5, the rats were anesthetized, and a No. 5 sterile syringe was inserted into the joint cavity from the posterior side of the right ankle joint along the medial side of the Achilles tendon at a 30°–40° angle, injecting 200 μL of 25 mg / mL sodium urate suspension. This injection was repeated for 3 consecutive days. The control group received intraperitoneal injections or combined intra-articular injections of an equal volume of physiological saline at the corresponding time points.

[0158] 2.3. Indicator Testing:

[0159] 2.3.1 Joint swelling index and gait score

[0160] Ankle swelling index: The circumference of the same area of ​​the right ankle was measured using the suture method before injection of sodium urate, and 24 and 48 hours after injection. Ankle swelling index = [(circumference of right ankle after modeling / before modeling)] × 100%.

[0161] Gait scoring: The joint swelling index and gait score of rats were assessed. Gait scoring: The gait and behavior of rats were observed before injection of sodium urate, and at 24 h and 48 h after injection to determine the gait score. 0 points: normal walking on both feet; 1 point: mild lameness with the right hind limb able to touch the ground but slightly bent; 2 points: moderate lameness with the right hind limb able to touch the ground but immediately retracted; 3 points: severe lameness with the right hind limb unable to touch the ground.

[0162] 2.3.2 Serum biochemical detection

[0163] On the evening of the 6th day after drug administration, rats were fasted but allowed free access to water for 8 hours. One hour after the last administration, blood was collected from the abdominal aorta under 3% isoflurane anesthesia. After the blood coagulated, it was centrifuged (2000 r / min) for 10 min, and serum was collected. Serum uric acid content was detected by biochemical methods, and serum xanthine oxidase, TNF-α, IL-1β and IL-6 content were determined by ELISA.

[0164] 2.3.3 Histopathological examination of rat ankle synovial tissue

[0165] Rats were euthanized by cervical dislocation after anesthesia with 3% isoflurane and blood collection. Tissue from the right ankle joint was harvested, periarticular tissue was removed while preserving the joint cavity, and the tissue was fixed with 4% paraformaldehyde, dehydrated with ethanol at graded concentrations, and embedded in paraffin. The tissue blocks were cut into 5 μm thick sections, stained with hematoxylin and eosin, dehydrated with ethanol, cleared with xylene, topped with resin, mounted, and the pathological changes of the synovial tissue were observed under a light microscope. Pathological scoring was performed according to the following criteria.

[0166] (1) Swelling of synovial tissue: None (0 points), mild (1 point), moderate (2 points), severe (3 points);

[0167] (2) Inflammatory cell infiltration: None (0 points), mild (1 point), moderate (2 points), severe (3 points);

[0168] (3) Small blood vessel and fibroblast proliferation: None (0 points), mild (1 point), moderate (2 points), severe (3 points).

[0169] 2.4 Statistical Methods

[0170] Data analysis and plotting were performed using Graphpad Prism 8, and results are expressed as mean ± standard deviation (x ± s). One-way ANOVA was used to analyze data from multiple groups, and a p-value < 0.05 was considered statistically significant.

[0171] 3. Experimental Results

[0172] 3.1 Joint swelling index and gait score results

[0173] Compared with the control group, the swelling index of the right ankle joint in the model group was significantly increased 24 h and 48 h after injection of sodium urate (P < 0.01), reaching a peak at 48 h. The swelling index of the affected ankle joint was significantly decreased in the medium and high dose groups of Euglena compound tablets (P < 0.05). Furthermore, gait analysis at 24 h and 48 h showed that the normal group exhibited a normal gait, while the gait score of the model group was significantly higher than that of the control group (P < 0.01), and the rats exhibited a tripedal gait. Similar to the swelling index, the gait score was significantly decreased in the medium and high dose groups of Euglena compound tablets (P < 0.05). The results are shown in […]. Figure 5-8 As shown.

[0174] Table 9. Swelling index of rats at 24h and 48h

[0175]

[0176] 3.2 Serum biochemical test results

[0177] Compared with the control group, the serum levels of uric acid, xanthine oxidase, IL-1β, IL-6, and TNF-α in the model group rats were significantly increased (P < 0.01). Compared with the model group, the low, medium, and high dose groups of Euglena compound tablets all reduced uric acid and xanthine oxidase levels (P < 0.05, 0.01), the medium and high dose groups reduced IL-6 and TNF-α levels (P < 0.05, 0.01), and the high dose group reduced IL-1β levels (P < 0.05). These results suggest that Euglena compound tablets can reduce serum uric acid levels in gout rats, improve abnormal uric acid metabolism, and alleviate the inflammatory response caused by hyperuricemia and gout. (See attached figures). Figure 9-13 As shown.

[0178] Table 10 Serum Biochemical Index Detection Data

[0179]

[0180] 3.3 Histopathological examination results of rat ankle joint synovial tissue

[0181] In the control group, the synovial tissue structure of the right ankle joint was clear, with normal synovial cell arrangement, intact and smooth structure, and visible joint cavity. There was virtually no inflammatory cell infiltration or capillary proliferation. In the model group, significant inflammatory cell infiltration was observed in the synovial tissue, with synovial tissue proliferation further filling the joint cavity. In the positive control group, mild swelling of the synovial tissue and slight cell proliferation were observed, along with a small amount of inflammatory cell infiltration and capillary proliferation. In the low- and medium-dose Euglena compound tablet groups, synovial tissue proliferation was reduced, joint cavity space increased, and inflammatory cell infiltration and capillary proliferation decreased. In the high-dose Euglena compound tablet group, the synovial tissue structure was even clearer, and synovial cell proliferation and inflammatory cell infiltration were further significantly improved. Results are shown below. Figure 14 .

[0182] In addition, pathological scoring was performed based on the degree of synovial tissue swelling, inflammatory cell infiltration, and the degree of small blood vessel and fibroblast proliferation. The results showed that the pathological score of the ankle joint synovial tissue in the model group was significantly higher than that in the normal group (P < 0.01), and a significant decrease in score was observed in the positive group (P < 0.01). The three dosage groups of Euglena compound tablets also showed a decrease in pathological score, exhibiting a good dose-response relationship (P < 0.05, 0.01). The results are shown in […]. Figure 15 As shown.

Claims

1. A Euglena composition for improving gout, characterized in that, It is prepared from the following raw materials in parts by weight: 3-8 parts of coix seed, 3-8 parts of poria cocos, 0.1-6 parts of gardenia, 0.1-6 parts of imperata root, 2-5 parts of corn silk, 2-5 parts of kudzu root, 0.1-10 parts of Euglena gracilis powder, 0.1-5 parts of Lactobacillus plantarum powder, 0.1-4 parts of mangiferin, 0.1-6 parts of celery seed extract, and 0.1-5 parts of sour cherry extract.

2. The composition according to claim 1, characterized in that, It is prepared from the following raw materials in parts by weight: 2-6 parts of Coix seed, 2-6 parts of Poria cocos, 0.1-3 parts of Imperata cylindrica root, 0.1-3 parts of Gardenia jasminoides, 3-5 parts of Pueraria lobata root, 3-5 parts of corn silk, 0.1-5 parts of Euglena gracilis powder, 0.1-3 parts of Lactobacillus plantarum powder, 0.1-2 parts of mangiferin, 0.1-3 parts of celery seed extract, and 0.1-3 parts of sour cherry extract.

3. The composition according to claim 2, characterized in that, It is prepared from the following raw materials in parts by weight: 5 parts of coix seed, 5 parts of poria cocos, 0.7 parts of imperata root, 0.7 parts of gardenia, 4 parts of kudzu root, 3 parts of corn silk, 1.5 parts of Euglena gracilis powder, 0.3 parts of Lactobacillus plantarum powder, 0.2 parts of mangiferin, 0.6 parts of celery seed extract, and 0.2 parts of sour cherry extract.

4. The composition according to any one of claims 1-3, characterized in that, It also contains food additives, which are one or more of the following: 20 g / L sodium alginate aqueous solution, micronized silica gel, croscarmellose, 50% ethanol, 10 g / L calcium chloride aqueous solution, hydroxypropyl-β-cyclodextrin HP-β-CD, microcrystalline cellulose, and magnesium stearate.

5. The composition according to claim 4, characterized in that, The preparation methods include the following: Step 1: Inoculate Lactobacillus plantarum powder into MRS liquid medium and culture until the viable count reaches 10. 9 After centrifugation, the supernatant was discarded and the collected bacterial cells were made into a bacterial suspension. The bacterial suspension and 20 g / L sodium alginate aqueous solution were mixed evenly at a volume ratio of 1:3 and poured into a pressure-resistant bottle. The mixed liquid was added dropwise to 10 g / L CaCl2 solution by high-frequency oscillation using a microcapsule granulator. The parameters were adjusted to make the droplets free of agglomeration and uniform. After granulation, the mixture was solidified for 40 min and centrifuged to obtain the sample. The CaCl2 residue on the surface of the microcapsules was washed away with physiological saline to obtain the finished microcapsules, which were stored at -20℃. Step 2: Weigh an appropriate amount of ultra-finely pulverized coix seed, poria cocos, corn silk, gardenia, kudzu root, and imperata root, add 8 times the weight of water, extract in a 100℃ water bath for 2 hours, filter, repeat twice, combine the filtrates, concentrate under reduced pressure, and spray dry to obtain a mixed extract of traditional Chinese medicine. Step 3: Weigh an appropriate amount of celery seed extract, sour cherry extract and mixed extract of traditional Chinese medicine and premix with micronized silica gel for 5 min. Finally, add microcapsules of Lactobacillus plantarum powder, mix at 100 r / min, granulate by dry method, adjust the granulation pressure, granulate through a 14-mesh sieve, add 0.5% magnesium stearate and mix for 5 min to obtain the upper granules. Step 4: Prepare mangiferin-HP-β-CD inclusion complex at a 1:1 ratio, dry and pulverize through a 100-mesh sieve; premix Euglena powder with an appropriate amount of micronized silica gel for 10 min, and pass through an 80-mesh sieve; mix mangiferin inclusion complex, pretreated Euglena powder, an appropriate amount of microcrystalline cellulose, and an appropriate amount of cross-linked polyvinylpyrrolidone (PVPP) evenly, add 50% ethanol aqueous solution as a binder to prepare a soft material, granulate, dry, add 0.5% magnesium stearate and mix for 10 min to obtain the lower layer particles; finally, load the upper and lower layer particles into the two hoppers of a multi-functional rotary tablet press for tableting.

6. The composition according to claim 5, characterized in that, In the preparation method, the pressure is controlled at 7.013 kN and the rotation speed at 37 r / min throughout the tableting process. The amount of feed is adjusted according to the filling ratio of 1.996:1 to ensure uniform layering.

7. The composition according to claim 4, characterized in that, The solid dosage form is a double-layered tablet.

8. The use of the composition according to claim 1 in the preparation of gout-relieving foods.

9. The use of the composition according to claim 1 in the preparation of a health food for improving gout.

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