Garlic powder medicine capsule as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and discloses a garlic powder medicine capsule as well as a preparation method and application thereof. The garlic powder medicine capsule is prepared from garlic powder, a gelatin solution, a chitosan hydrochloride solution, an alginate solution and a calcium chloride solution through 3D printing. The encapsulation efficiency of the 3D printing garlic powder medicine capsule on the garlic powder is remarkably improved. The generation and development of hepatic fibrosis are improved by adjusting the intestinal-hepatic axis to influence the stability of the micro-ecology of the liver, and the pharmaceutical composition has huge commercial development and clinical treatment prospects.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to a garlic powder medicine capsule and a preparation method and application thereof. Background Art

[0002] Non-alcoholic steatohepatitis (liver fibrosis) is a chronic liver disease and a severe subtype of non-alcoholic fatty liver disease (NAFLD). The salient features of this disease are inflammation and liver cell damage inside the liver, even though the patient has no history of long-term heavy drinking. Liver fibrosis may worsen into more serious liver diseases, including liver fibrosis, cirrhosis, and even liver cancer. According to studies, about one-third of the world's population is affected by NAFLD, of which about 20% develop liver fibrosis, and they face a higher risk of developing cirrhosis and hepatocellular carcinoma.

[0003] Liver fibrosis is a liver disease caused by multiple factors, and its development process involves complex and multi-faceted dynamic changes in the liver's internal environment. First, liver fibrosis is closely related to metabolic syndrome, and patients are usually accompanied by obesity, insulin resistance, hypertension, and abnormal blood lipid levels. These metabolic disorders can cause excessive accumulation of fat in the liver, resulting in lipid accumulation, which is toxic to liver cells and triggers an inflammatory response. As inflammation persists, the level of oxidative stress in the liver increases, producing a large amount of reactive oxygen species, which are highly reactive and can damage cell structures, leading to impaired cell function and, in severe cases, cell death. At the same time, inflammatory mediators released during the inflammation process, such as tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β), further aggravate damage to liver cells. In addition, the inflammatory response also triggers the activation of hepatic stellate cells, which are responsible for maintaining the stability of liver structure under normal conditions. However, in liver fibrosis, they are activated and transformed into collagen-producing cells, which promotes the occurrence and development of liver fibrosis. If this inflammatory and fibrotic process is not effectively controlled, it may gradually develop into cirrhosis and severely weaken the function of the liver. The pathophysiological mechanism of liver fibrosis also involves complex interactions of genetics, dietary habits and environmental factors, all of which may affect an individual's susceptibility to the disease and the speed of disease progression. As the core absorption and metabolic organ of the human body, the balance of the intestinal microenvironment has a significant impact on the body's metabolic level. Among the many pathological factors, the connection between the intestine and the liver is particularly critical. The so-called gut-liver axis refers to the interaction between the intestine and its microbiota and the liver, and the intestinal mucosa and vascular barriers play a vital role in this process. They not only limit the systemic spread of microorganisms and toxins, but also allow nutrients to enter the blood circulation and eventually reach the liver, playing a key role in the occurrence and development of liver fibrosis. The balance of intestinal flora is crucial to maintaining the homeostasis of the gut-liver axis. Under normal conditions, microorganisms in the intestine coexist in a certain proportion, jointly maintaining the balance of the intestinal microecology, and participating in physiological processes such as metabolism and immunity of the body.

[0004] The intestinal flora affects the occurrence and development of liver fibrosis through multiple mechanisms. For example, the integrity of the intestinal barrier is affected by intestinal flora imbalance, which may lead to impaired intestinal mucosal barrier function and increased intestinal permeability. This allows bacterial metabolites and bacterial components to enter the blood circulation and affect the liver through the portal vein system, triggering inflammatory responses, oxidative stress, and lipid accumulation, ultimately leading to liver damage. In addition, the intestinal flora may also affect liver lipid metabolism and inflammatory responses by affecting bile acid metabolism and related receptor signaling pathways, such as farnesoid X receptor (FXR) and G protein-coupled bile acid receptor (TGR5). Changes in the composition and ratio of bile acids, especially the increase in secondary bile acids such as deoxycholic acid (DCA), may aggravate lipid accumulation and inflammation in the liver.

[0005] Studies have confirmed that the composition and diversity of intestinal flora in NAFLD patients are significantly different from those in healthy people, indicating that the disorder of intestinal flora may be involved in the pathogenesis of NAFLD. The formation of fatty liver can be induced by transplanting the intestinal flora of patients with fatty liver into normal germ-free mice, while no significant changes were observed after transplanting the intestinal flora of obese children who had undergone diet to lose weight. These findings provide strong evidence for the treatment of NAFLD by regulating the intestinal flora. Therefore, regulating the species and abundance of intestinal flora, improving the intestinal mucosal barrier function, and regulating the signal transduction of intestinal flora metabolites are expected to become a new strategy for the treatment of liver fibrosis. The intestinal environment has great variability, and the pH value of different parts ranges from 1 (extremely acidic) to 7 (neutral). Natural changes in diet time and content lead to frequent changes in lipid abundance, high bacterial load, 100% humidity, 37°C temperature and a wide variety of proteases, lipases and other enzymes, all of which are not conducive to the integrity of biomaterials and the long-term stability of drugs.

[0006] At present, the development of oral or rectal drug sustained-release delivery systems has great clinical application prospects and market demand. Sodium alginate / chitosan microcapsules are an excellent sustained-release drug delivery system with good biocompatibility and mechanical strength, and play an important role in the field of drug controlled release. Although some progress has been made in the preparation of the capsules, and the encapsulation rate of oil-soluble and water-soluble drugs has been greatly improved, the encapsulation effect of powdered drugs is not ideal, whether it is a one-step method, a two-step method or a composite method. Therefore, it is necessary to expand the scope of application of microcapsules by continuously improving the preparation method. Summary of the invention

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a garlic powder medicine capsule and a preparation method and application thereof.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] In a first aspect, the present invention provides a garlic powder medicine capsule, which is prepared by 3D printing from garlic powder, gelatin solution, chitosan hydrochloride solution, alginate solution and calcium chloride solution.

[0010] Compared with traditional garlic powder microcapsules, the 3D printed garlic powder medicinal capsules of the present invention can greatly improve the encapsulation rate of garlic powder.

[0011] As a preferred embodiment of the garlic powder medicinal capsules described in the present invention, the concentration of the garlic powder is 5g / L-200g / L; the mass concentration of the chitosan hydrochloride solution is 0.4%-3%; the mass concentration of the alginate solution is 1%-5%; the mass concentration of the calcium chloride solution is 0.5%-5%; and the mass concentration of the gelatin solution is 1%-30%.

[0012] As a preferred embodiment of the garlic powder medicine capsule of the present invention, the garlic powder medicine capsule is a core-shell structure, and the diameter of the garlic powder medicine capsule is 3mm-4mm.

[0013] In a second aspect, the present invention provides a method for preparing a garlic powder medicament capsule, comprising the following steps:

[0014] (1) dissolving garlic powder in a mixed solution of gelatin, calcium chloride and chitosan hydrochloride, and placing the mixture at 4° C. to 10° C. to obtain a gel;

[0015] Preferably, the temperature is any one or both of 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, and 10°C;

[0016] (2) Using a 3D printing device, the gel is dripped into an alginate solution in the form of droplets to form hydrogel microspheres.

[0017] In the preparation process of the preparation method of the present invention, a mixed solution of gelatin, calcium chloride and chitosan hydrochloride is placed under low temperature conditions to form an intermediate phase between liquid and solid; a water droplet-shaped gel is accurately printed under specific conditions using low-temperature pneumatic extrusion 3D printing technology, and the gel is dripped into a pre-configured alginate solution in the form of droplets to form hydrogel microspheres. In the process of contact between the gel mixed solution and the alginate solution, calcium ions and carboxyl groups in the alginate molecules undergo ionic crosslinking, and the three-dimensional network structure of the hydrogel is quickly constructed. At the same time, gelatin molecules may interact with other molecules through physical entanglement or hydrogen bonds, thereby enhancing the mechanical stability of the hydrogel; chitosan hydrochloride, due to its high charge density, can more effectively synergistically combine with drug molecules, which not only helps to regulate the distribution of drugs inside the hydrogel, but also effectively controls the release rate of the drugs.

[0018] As a preferred embodiment of the method for preparing the garlic powder medicinal capsules of the present invention, in the mixed solution of step (1), the concentration of the garlic powder is 5 g / L-200 g / L.

[0019] Preferably, the concentration of the garlic powder is 5g / L, 6g / L, 7g / L, 8g / L, 9g / L, 10g / L, 15g / L, 20g / L, 25g / L, 30g / L, 35g / L, 40g / L, 45g / L, 50g / L, 55g / L, 60g / L, 65g / L, 70g / L, 75g / L, 80g / L, 85g / L, 90g / L, 95g / L, 100g / L, 110g / L, 120g / L, 130g / L, 140g / L, 150g / L, 160g / L, 170g / L, 180g / L, 190g / L, and 200g / L, or any one or both of the range values.

[0020] As a preferred embodiment of the method for preparing the garlic powder medicinal capsules of the present invention, in step (1), the gelatin, calcium chloride and chitosan hydrochloride are pre-made into solutions, the mass concentration of the prepared gelatin solution is 1%-30%, the mass concentration of the calcium chloride solution is 0.5%-5%, and the mass concentration of the chitosan hydrochloride solution is 0.4%-3%.

[0021] As a preferred embodiment of the method for preparing the garlic powder medicinal capsules of the present invention, in step (2), the 3D printing adopts a low-temperature pneumatic extrusion 3D printing device; the temperature is 8°C-10°C, the pressure is 5kPa-15kPa; and the mass concentration of the alginate solution is 1%-5%.

[0022] Preferably, the temperature is a range value of any one or both of 8°C, 8.5°C, 9°C, 9.5°C, and 10°C; the pressure is a range value of any one or both of 5kPa, 6kPa, 7kPa, 8kPa, 9kPa, 10kPa, 11kPa, 12kPa, 13kPa, 14kPa, and 15kPa.

[0023] In a third aspect, the present invention provides a garlic powder medicine capsule prepared by the preparation method of the above-mentioned garlic powder medicine capsule.

[0024] In a fourth aspect, the present invention uses the above-mentioned garlic powder medicine capsule in the preparation of medicine for treating or preventing liver fibrosis.

[0025] In a fifth aspect, the present invention uses the above-mentioned garlic powder medicine capsule in the preparation of a medicine for treating or preventing intestinal flora disorder caused by liver fibrosis.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention prepares a hydrogel microsphere based on sodium alginate / chitosan natural biopolymer, which exhibits excellent biocompatibility and biodegradability, and the encapsulation rate of powdered drugs is much higher than that of traditional sodium alginate / chitosan microcapsules. The 3D printed garlic powder drug capsule of the present invention improves the occurrence and development of liver fibrosis by regulating the intestinal-liver axis to affect the stability of liver microecology, and has a significant improvement effect on inflammation and fibrosis in the liver fibrosis mouse model induced by CDHF (methionine-deficient high-fat diet), and has a significant regulatory effect on the intestinal flora disorder of CDHF-induced liver fibrosis mice. It has great prospects for commercial development and clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the encapsulation efficiency of garlic powder drug capsule.

[0029] Figure 2 Drug characterization for 3D printed garlic powder drug capsules; Figure 2 In the figure, A is the measured size of the drug capsule; B is the optical microscopic image of the drug capsule (×40, scale bar = 100 μm); C is the SEM image of the drug capsule (scale bar on the left = 100 μm, scale bar on the right = 1 μm); D is the confocal microscopic imaging of the drug capsule (scale bar = 100 μm).

[0030] Figure 3 In the acute liver fibrosis mouse model, 3D printed garlic powder drug capsules significantly restored the body weight and liver mass of mice; Figure 3 In the figure, A is the percentage change of body weight of mice; B is the change of liver-to-body weight ratio; n=5 mice / group.

[0031] Figure 4 In the acute liver fibrosis mouse model, 3D printed garlic powder drug capsules significantly improved inflammation and fibrosis; Figure 4 In the figure, A is a representative anatomical image of liver tissue, B is a representative image of H&E staining (×100, scale bar=100 μm), and C is a representative image of Masson staining (×100, scale bar=100 μm); n=5 mice / group.

[0032] Figure 5 In the acute liver fibrosis mouse model, 3D printed garlic powder drug capsules significantly reduced the expression of liver injury and fibrosis-related factors; Figure 5 In the figure, A is the serum ALT level; B is the serum AST level; C is the liver triglyceride level; D is the liver collagen level; n=5 / group.

[0033] Figure 6 In the acute liver fibrosis mouse model, garlic powder capsules significantly reduced the expression of pro-inflammatory and pro-fibrosis related genes; Figure 6In the figure, (A) Tnf-α, (B) IL-1β, (C) Ccl2 and (D) Ccl5 are the hepatic mRNA expressions of pro-inflammatory markers; (E) Col1a1, (F) Col3a1, (G) Acta2 and (H) Fn are the hepatic mRNA expressions of pro-fibrotic markers; n = 5 mice / group.

[0034] FIG7 is an analysis of the diversity composition spectrum of the microbial community in mouse feces to study the regulatory effect of 3D printed garlic powder drug capsules on the intestinal flora of mice with liver fibrosis; Fig. 7A is a graph of relative abundance of species analyzed at the phylum level; Figure 7B This is the taxonomic cladogram of species analyzed using LEfSe; Figure 7C It is the predicted KEGG secondary functional pathway map. DETAILED DESCRIPTION

[0035] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. It should be understood by those skilled in the art that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0036] Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0037] The raw materials of the 3D printed garlic powder medicine capsules involved in the embodiment include: gelatin (Gelatin), alginate (Alginate, Alg), chitosan hydrochloride (Chitosan Hydrochloride, CS) and calcium chloride (CalciumChloride, CaCl2).

[0038] The garlic powder involved in the examples was purchased from Beijing Wusheng Biotechnology Co., Ltd.

[0039] The statistical analysis involved in the examples was performed using GraphPad Prism 9.5 software. The measurement data that conformed to the normal distribution were represented by xs, and the comparison between multiple groups was performed by one-way analysis of variance, and the multiple comparison between groups was performed by LSD-t test. P < 0.05 was considered statistically significant.

[0040] Example 1: Preparation of drug capsules

[0041] 1. Preparation of 3D printed garlic powder capsules

[0042] Garlic powder (5g / L, 10g / L, 20g / L, 40g / L, 80g / L, 160g / L, 200g / L) was dissolved in a mixed solution of 1% Gelatin, 1% CaCl2 and 1% chitosan sodium hydrochloride (the solute was sterile water) and placed at 4°C to form a solid-liquid interphase; a low-temperature pneumatic extrusion 3D printing device was used to drip the obtained solid-liquid interphase into a pre-configured 1% sodium alginate solution (the solute was sterile water) in the form of droplets by setting the temperature to 9°C and the pressure to 10kPa, thereby forming hydrogel microspheres to obtain 3D printed garlic powder drug capsules.

[0043] During the preparation process, the mixed solution of Gelatin, CaCl2 and chitosan sodium hydrochloride is placed under low temperature conditions to form an intermediate phase between liquid and solid; the low-temperature pneumatic extrusion 3D printing technology is used to accurately print water droplet-shaped gel under specific conditions, and the gel is dripped into the pre-configured sodium alginate solution in the form of droplets to form hydrogel microspheres. In the process of contact between the gel mixed solution and the sodium alginate solution, calcium ions and carboxyl groups in the sodium alginate molecules undergo ionic crosslinking, and the three-dimensional network structure of the hydrogel is quickly constructed. At the same time, gelatin molecules may interact with other molecules through physical entanglement or hydrogen bonds, thereby enhancing the mechanical stability of the hydrogel; chitosan sodium hydrochloride can more effectively bind to drug molecules due to its high charge density, which not only helps to regulate the distribution of drugs inside the hydrogel, but also effectively controls the release rate of drugs.

[0044] 2. Traditional one-step preparation method of microcapsules

[0045] Dissolve garlic powder (5g / L, 10g / L, 20g / L, 40g / L) in 2% sodium alginate solution to obtain a mixed solution; slowly drop a 3% chitosan sodium hydrochloride and 25mmol / L CaCl2 mixed solution into the mixed solution with a needle, and stir at a speed of 200r / min to form microcapsules, add 1% glutaraldehyde solution and stir rapidly, and place it in a 4°C environment for fixation for 10 hours; obtain traditional garlic powder microcapsules.

[0046] 3. Characterization of garlic powder capsules

[0047] (1) The 3D printed garlic powder drug capsules and traditional garlic powder microcapsules were placed on a horizontal table and their sizes were measured from a macroscopic perspective. The core-shell structure of the capsules was observed under an inverted fluorescence microscope (Nikon) and a confocal microscope (Leica). The capsules were then freeze-dried and observed using a field emission scanning electron microscope.

[0048] Results Figure 1:When the concentration of garlic powder reaches 20g / L, the traditional garlic powder microcapsule has drug loss phenomenon, and when it reaches 40g / L, it is impossible to prepare microcapsules. However, the 3D printed garlic powder drug capsule can encapsulate garlic powder up to 160g / L. When it reaches 200g / L, the solution fluidity is poor due to the high drug concentration, which no longer meets the conditions of 3D printing. Compared with traditional garlic powder microcapsules, 3D printed garlic powder drug capsules can greatly improve the encapsulation rate of garlic powder.

[0049] (2) The physical properties of 3D printed garlic powder drug capsules, including the size and core-shell structure of the drug capsules, were comprehensively analyzed by using naked eye observation, SEM, optical imaging, and confocal microscopy techniques.

[0050] Results Figure 2 : It can be seen with the naked eye that the diameter of the 3D printed garlic powder drug capsule is approximately between 3mm and 4mm. When magnified and observed using an optical microscope, the core-shell structure characteristics of the 3D printed garlic powder drug capsule can be clearly identified. The 3D printed garlic powder drug capsule was observed at the microscopic level using a scanning electron microscope, revealing the porous three-dimensional network structure inside the capsule. This structure is essential for maintaining the stability of the drug capsule and controlling the drug release rate. At the same time, the outer shell of the 3D printed garlic powder drug capsule shows a denser and smoother texture, which helps to effectively prevent the unexpected loss of internal drug ingredients.

[0051] In order to further verify the composition of the 3D printed garlic powder drug capsule shell, the capsule was prepared using sodium alginate containing green fluorescent markers. Under confocal microscopy, it can be clearly seen that the 3D printed garlic powder drug capsule shell exhibits green fluorescence, which not only verifies the composition of sodium alginate in the capsule shell, but also shows that sodium alginate can form a uniform covering layer on the capsule surface during the preparation process.

[0052] It can be seen that the 3D printed garlic powder drug capsule has a core-shell structure, which provides a guarantee for the precise delivery of garlic powder drugs and ensures the continuity and stability of drug treatment.

[0053] Example 2: Effects of 3D printed garlic powder drug capsules on changes in body weight and liver mass in mice with liver fibrosis induced by CDHF (methionine-deficient high-fat diet)

[0054] 1. Construction of CDHF-induced mouse liver fibrosis model

[0055] C57BL / 6N mice aged between 6 and 8 weeks were selected, and the breeding temperature was maintained at 23℃-26℃, the humidity was controlled at 40%-70%, and the light and dark cycles were 12 hours each. Before the experiment, the mice had a one-week adaptation period, during which time there was sufficient food and water supply.

[0056] After the adaptation period, the mice were randomly divided into three groups. The mice in the control group (NC) were fed a standard diet; the mice in the methionine and choline deficient diet group (CDHF) received a specially formulated diet (feed product number A06071301B) for the next 4 weeks; the mice in the treatment group (CDHF+TGPC) received traditional garlic powder drug microcapsules (500 mg / kg / time) in addition to the CDHF diet; the mice in the treatment group (CDHF+3DGPC) received 3D printed garlic powder drug capsules (500 mg / kg / time) in addition to the CDHF diet. During the four-week experiment, the mice were dosed three times a week, and the weight changes of the mice in each group were monitored and recorded weekly.

[0057] After the experiment, blood samples were collected by eye bleeding, and the mice were dissected and liver tissue was collected. The body lobe of the liver was fixed in 4% paraformaldehyde (product number BL539A, Biosharp) for subsequent tissue paraffin embedding; the remaining liver lobe was stored in a refrigerator at -80°C for molecular biological analysis. The blood samples were centrifuged at 12000g and 4°C for 10 minutes, and the serum was separated and stored at -80°C.

[0058] 2. Effects on body weight and liver quality of model mice

[0059] Results Figure 3 : CDHF-induced liver fibrosis mice showed a significant decrease in body weight and an increase in liver-to-body weight ratio compared to the control group fed a normal diet. Both were improved after treatment with 3D printed garlic powder drug capsules, and the effect of 3DGPC was significantly higher than that of TGPC.

[0060] It can be seen that 3D printed garlic powder drug capsules have a significant improvement effect on the changes in body weight and liver weight of mice with CDHF-induced liver fibrosis.

[0061] 3. Effects on liver pathological status of model mice

[0062] The collected mouse livers were photographed, and the mouse liver sections were stained with HE and Masson's trichrome to observe the effect of the drug capsule on the pathological state of the liver in mice with liver fibrosis. The body lobe of the mouse liver was fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 6 μm thick sections. Subsequently, hematoxylin and eosin (HE) staining and Masson's trichrome (MT) staining were performed according to the manufacturer's instructions.

[0063] The specific steps of hematoxylin-eosin staining (G1120, Solarbio) are as follows:

[0064] Dewaxing and rehydration: The liver sections were placed on a staining rack and first dewaxed in xylene for 10 minutes, which was repeated twice. Gradient alcohol rehydration: Subsequently, the sections were rehydrated through a series of gradient alcohols, including anhydrous ethanol for 3 minutes, 95% ethanol for 3 minutes, 85% ethanol for 3 minutes, and 75% ethanol for 3 minutes, and finally soaked in distilled water for 2 minutes to prepare for staining. Staining: The sections were stained in hematoxylin stain for 5 minutes and then rinsed with distilled water to remove floating color. Differentiation: The sections were placed in differentiation solution for 30 seconds and rinsed in tap water for 3 minutes, which was repeated twice. Eosin staining: The sections were stained in eosin stain for 40 seconds, and the excess staining solution was removed and then quickly dehydrated. Dehydration and transparency: The sections were dehydrated in 75%, 85%, 95% ethanol and anhydrous ethanol in sequence, and then transparentized in xylene. Mounting: The sections were mounted with neutral gum and observed using an inverted microscope (Nikon, Ti2) after drying overnight.

[0065] The specific steps of Masson's trichrome staining (G1346, Solarbio) are as follows:

[0066] Dewaxing and rehydration: Same as HE staining, the sections were dewaxed in xylene for 10 minutes, repeated twice, and then rehydrated through gradient alcohol. Mordant: The sections were immersed in the mordant solution, placed in a 60°C incubator for 1 hour for mordant, and then rinsed with running water for 10 minutes. Drop staining: Use lapis lazuli blue, Mayer hematoxylin, and Ponceau fuchsin staining solution for drop staining for 2 minutes in sequence, and rinse with water twice after each drop staining, each for 10 seconds. Differentiation and water washing: Use acidic differentiation solution for differentiation for 5 seconds, then terminate differentiation with distilled water and rinse for 10 minutes. Staining: Ponceau fuchsin staining solution was dripped for 10 minutes, washed twice with distilled water, each for 10 seconds; the sections were immersed in phosphomolybdic acid solution for 10 minutes, the upper liquid was poured off, and then stained with aniline blue solution for 5 minutes. Weak acid treatment: Use weak acid solution to wash off the staining, and then cover the sections for 2 minutes. Dehydration and transparentization: Dehydration and transparentization were performed with 95% ethanol, anhydrous ethanol and xylene. Sealing and observation: Finally, use neutral gum to seal the slides, let them dry overnight, and then observe them using an inverted microscope.

[0067] Results Figure 4 :The liver of mice in the control group was dark brown, with clear cell structure, intact nuclei and cytoplasm, and no inflammation and fibrosis. Compared with the control group, the liver tissue of mice in the CDHF group was orange due to lipid accumulation, and a large number of hepatocytes showed fatty degeneration. Round vacuoles of lipid droplets of varying sizes were visible in the cytoplasm; a large amount of blue collagen deposition was shown around the cortex and in the interstitial area (the purple in the figure is a positive field of view of blue collagen fibers combined with a red background). Compared with the CDHF group, the degree of inflammation and fibrosis in the liver tissue of mice in the drug capsule group was significantly alleviated, and the effect of 3DGPC was significantly higher than that of TGPC.

[0068] It can be seen that 3D printed garlic powder drug capsules have a significant improvement effect on inflammation and fibrosis in the CDHF-induced liver fibrosis mouse model.

[0069] 4. Effects on liver injury and fibrosis-related factors in model mice

[0070] The levels of liver injury factors (ALT, AST) and fibrosis factors (triglyceride, collagen) in the collected serum and liver samples were detected.

[0071] Colorimetric assay: ALT / GPT test kit (C009-2-1, Nanjing Jiancheng) and AST / GOT kit (C010-2-1, Nanjing Jiancheng) were used to quantitatively detect the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse serum. The triglyceride content assay kit (Shanghai Tongwei) was used to determine the level of triglycerides in mouse liver. The collagen level in mouse liver was detected using the HYP collagen assay kit (A030-2-1, Nanjing Jiancheng). All colorimetric assay experiments were carried out in strict accordance with the protocol provided by the manufacturer to ensure the accuracy and reproducibility of the results.

[0072] Results Figure 5 : Compared with the control group, mice in the CDHF group had severe liver damage, with increased ALT and AST levels; and increased levels of triglycerides and collagen, markers of fibrosis (P<0.01). Compared with the CDHF group, the CDHF+3DGPC group had attenuated liver damage and fibrosis, with decreased ALT and AST, triglycerides and collagen levels (P<0.01).

[0073] It can be seen that 3D printed garlic powder drug capsules have a significant inhibitory effect on liver damage and upregulation of fibrosis-related factors in CDHF-induced liver fibrosis mice.

[0074] 5. Effects on inflammation and fibrosis in model mice

[0075] The expression of genes related to inflammation (Tnf-α, IL-1β, Ccl2, Ccl5) ​​and fibrosis (Col1a1, Col3a1, Acta2, Fn) was detected by RT-qPCR.

[0076] The extraction of total RNA and real-time quantitative polymerase chain reaction (RT-qPCR) were performed as follows:

[0077] Total liver RNA was efficiently extracted from frozen liver tissue using the RNAeasyTM Animal RNA Isolation Kit (Beyotime) with spin columns. The extracted RNA concentration was determined using a Nanodrop instrument. RNA was converted to cDNA using the BeyoRTTMIII cDNA First-Strand Synthesis Kit (5X) (Beyotime). The expression of the target gene was quantitatively analyzed by qPCR using the SYBR Green qPCR Mix (2X) (Beyotime) technology and a three-step method (including denaturation, annealing, and extension) with 40 cycles. The expression data were normalized with the expression of the internal reference gene GAPDH mRNA. Application 2 -ΔΔCt Methods The fold changes of the expression levels of the target genes relative to the control group were calculated. All mouse-specific primers were purchased from GeneVision Biotech (Suzhou, China), and the specific sequence information of the primers is shown in Table 1.

[0078] Table 1 Primer sequence information

[0079]

[0080]

[0081] Results Figure 6 : Compared with the control group fed with a normal diet, the mRNA expression levels of pro-inflammatory factors Tnf-α, IL-1β, Ccl2, Ccl5, and pro-fibrotic factors Col1a1, Col3a1, Acta2, and Fn in the CDHF-induced liver fibrosis mouse model were significantly increased. After drug capsule treatment, the mRNA levels of pro-inflammatory factors and pro-fibrotic factors in the liver tissue of mice decreased, and the degree of reduction of 3DGPC was more significant.

[0082] It can be seen that 3D printed garlic powder drug capsules significantly reduced the upregulation of inflammation and fibrosis-related genes in CDHF-induced liver fibrosis mice.

[0083] 6. Effects on the intestinal flora of model mice

[0084] The fecal microbiota of the three groups of mice was analyzed using 16S high-throughput sequencing technology. The specific composition of each group at the phylum level was tested to understand the overall profile. The differences in species abundance composition between different sample groups were further measured by LEfSe analysis to find marker species. According to the sequencing results of 16S rRNA, 18S rRNA and ITS genes, the metabolic pathways with high abundance in the samples were found based on the KEGG database to predict the metabolic function of the sample's microbiota.

[0085] The method for 16S rRNA sequencing is as follows: extract the total DNA from the microbiome sample, quantify it using Nanodrop, and detect the quality of DNA extraction by 1.2% agarose gel electrophoresis. Use Pfu high-fidelity DNA polymerase from Quanshijin Company and strictly control the number of amplification cycles to perform PCR amplification on the rRNA target gene fragment. The amplified product is purified and recovered by magnetic beads and quantified by fluorescence. According to the fluorescence quantification results, the samples are mixed in corresponding proportions according to the sequencing amount requirements of each sample. Sequencing analysis was performed at Hangzhou Repugene Technology Co., Ltd., and the sequencing library was prepared using Illumina's TruSeq Nano DNALT Library Prep Kit.

[0086] The results are shown in Figure 7: Compared with the NC group, at the phylum level, the ratio of Firmicutes to Bacteroidetes in the CDHF group increased dramatically, and the ratio decreased significantly after the administration of garlic powder capsules. The LEfSe analysis results showed the average relative abundance of taxonomic units from phylum to genus in each group, and the rank sum test was used to screen out 36 genera with significant differences between groups. According to the KEGG analysis of the prediction results of the intestinal flora, carbohydrate metabolism and lipid metabolism are highly correlated with liver fibrosis.

[0087] It can be seen that the 3D printed garlic powder drug capsule has a significant regulatory effect on the intestinal flora disorder of CDHF-induced liver fibrosis mice. The garlic powder drug capsule of the present invention can improve liver fibrosis by regulating the intestinal-liver axis.

[0088] The present invention uses 3D printing technology to introduce the dynamic dimension of time, and prepares a hydrogel capsule with a core-shell structure. The garlic powder drug is used as the core, and the pH-responsive shell is composed of sodium alginate and calcium chloride. It can stably exist in the acidic gastric environment, and under the neutral pH conditions of the intestine, the shell will responsively expand and gradually disintegrate, thereby achieving accurate delivery of the drug. In addition, the solubility of the chitosan component inside the capsule microspheres gradually increases under neutral pH conditions, which not only enhances its adhesion, but also provides a sustained release and targeted release mechanism for the drug, controls the amount of drug released, and realizes intelligent release of the drug. pH-sensitive hydrogels are widely used in oral drug delivery because they can adjust their own properties or structures according to the different pH values ​​of the human microenvironment, form semi-solids locally in the body, have good adhesion, and thus deliver drugs in a targeted manner, thereby reducing the toxic and side effects of drugs.

[0089] The present invention establishes a mouse model of liver fibrosis, designs a 3D intelligent drug carrier to encapsulate garlic powder to target the microecology of the intestinal-liver axis, and uses proteomics, molecular biology and 16S rRNA analysis technology to find that in the process of liver fibrosis occurrence and development, 3D printed garlic powder drug capsules can improve the occurrence and development of liver fibrosis by regulating the intestinal-liver axis to affect the stability of liver microecology, and relieve liver inflammation and fibrosis. It has great prospects for commercial development and clinical treatment.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A garlic powder medicine capsule, characterized in that: It is made of garlic powder, gelatin solution, chitosan hydrochloride solution, alginate solution and calcium chloride solution through 3D printing.

2. The garlic powder medicine capsule according to claim 1, characterized in that: The concentration of the garlic powder is 5g / L-200g / L; the mass concentration of the chitosan hydrochloride solution is 0.4%-3%; the mass concentration of the alginate solution is 1%-5%; the mass concentration of the calcium chloride solution is 0.5%-5%; and the mass concentration of the gelatin solution is 1%-30%.

3. The garlic powder medicine capsule according to claim 1, characterized in that: The garlic powder medicine capsule is a core-shell structure, and the diameter of the garlic powder medicine capsule is 3mm-4mm.

4. A method for preparing garlic powder medicine capsules, characterized in that: The following steps are involved: (1) dissolving garlic powder in a mixed solution of gelatin, calcium chloride and chitosan hydrochloride, and placing the mixture at 4° C. to 10° C. to obtain a gel; (2) Using a 3D printing device, the gel is dripped into an alginate solution in the form of droplets to form hydrogel microspheres.

5. The method for preparing the garlic powder medicine capsule according to claim 4, characterized in that: In the mixed solution of step (1), the concentration of the garlic powder is 5 g / L-200 g / L.

6. The method for preparing the garlic powder medicine capsule according to claim 4, characterized in that: In step (1), the gelatin, calcium chloride and chitosan hydrochloride are prepared into solutions in advance, wherein the mass concentration of the prepared gelatin solution is 1%-30%, the mass concentration of the calcium chloride solution is 0.5%-5%, and the mass concentration of the chitosan hydrochloride solution is 0.4%-3%.

7. The method for preparing the garlic powder medicine capsule according to claim 4, characterized in that: In step (2), the 3D printing adopts a low-temperature pneumatic extrusion 3D printing device; the temperature is 8°C-10°C, the pressure is 5kPa-15kPa; and the mass concentration of the alginate solution is 1%-5%.

8. A garlic powder capsule prepared by the method for preparing the garlic powder capsule according to any one of claims 4 to 7.

9. Use of the garlic powder medicament capsule according to any one of claims 1 to 3 or claims 4 to 7 in preparing a medicament for treating or preventing liver fibrosis.

10. Use of the garlic powder medicament capsule according to any one of claims 1 to 3 or claims 4 to 7 in the preparation of a medicament for treating or preventing intestinal flora disturbance caused by liver fibrosis.

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