Abufagin-gastrodia elata colloid polysaccharide aerogel as well as preparation method and application of arenobufagin-gastrodia elata colloid polysaccharide aerogel

Abstract

The invention discloses arenobufagin-gastrodia elata colloid polysaccharide aerogel as well as a preparation method and application thereof. The arenobufagin-gastrodia elata colloid polysaccharide aerogel is formed by compounding gastrodia elata colloid aerogel and arenobufagin. According to the invention, the gastrodia elata colloid polysaccharide with specific molecular weight is prepared into aerogel, and then the aerogel is compounded with arenobufagin according to a specific ratio, so that the aerogel can be continuously released at the end of colon, and has colon targeting and slow release effects. Meanwhile, a better anti-colorectal cancer effect can be achieved under the condition that less arenobufagin is used; and when the same dosage of arenobufagin is used, the anti-tumor effect is better, the synergistic interaction effect is generated, and the arenobufagin composition has actual popularization and application values.

Description

Technical Field

[0001] The present invention specifically relates to a toad venom-Gastrodia elata colloid polysaccharide aerogel, a preparation method thereof and uses thereof. Background Art

[0002] Colorectal cancer (CRC) refers to malignant tumors that develop in the colon (large intestine) or rectum. It typically originates from adenomatous polyps in the lining of the colon or rectum. These polyps are mostly benign, but some may develop into cancer over the years. CRC is one of the most common cancers worldwide and the second leading cause of cancer-related death. Early detection and treatment of colorectal cancer improves the prognosis, while advanced disease leads to a poorer prognosis. Therefore, timely and appropriate treatment is crucial to improving survival rates and quality of life.

[0003] Gastrodia elata (Gastrodia elata Bl.), a perennial fungi-vegetative herb in the Orchidaceae family, has a dried tuber that is a valuable traditional Chinese medicine. It is sweet and neutral in nature, entering the liver meridian and possessing multiple benefits, including calming wind and relieving spasms, calming the liver and suppressing yang, and dispelling wind and unblocking the meridians. Rich in a variety of active ingredients, Gastrodia elata is commonly used clinically to treat symptoms such as epilepsy, convulsions, vertigo, headache, and rheumatic pain. It possesses analgesic, sedative, antihypertensive, and cardioprotective properties, with significant analgesic effects for trigeminal neuralgia and vascular neurosis headaches. It also increases coronary blood flow and protects the heart, making it a prominent component of Traditional Chinese Medicine. Duan Hao et al., in their study "Research Progress on Bioactive Components and Efficacy of Gastrodia elata," revealed that Gastrodia elata polysaccharides in Gastrodia elata can regulate intestinal flora and improve intestinal health.

[0004] Arenobufagin (AB), the primary active ingredient in toad venom, is a C24 steroidal compound with broad-spectrum antitumor potential, significantly inhibiting the proliferation, migration, and invasion of liver cancer cells and inducing apoptosis. While AB demonstrates significant anticancer activity, its drugability remains a concern. Arenobufagin, a cardiac glycoside, has a narrow therapeutic safety margin, with typical therapeutic doses approaching 60% of the toxic dose. This means that even the slightest misuse of the drug can lead to toxicity. Therefore, designing carriers that enhance AB cellular uptake is crucial for reducing AB dosage, improving safety, and ensuring efficacy.

[0005] There is currently no report on combining Gastrodia elata polysaccharide with fenbufantrine for the treatment of colorectal cancer to reduce the dose of fenbufantrine while improving the therapeutic efficacy. Summary of the Invention

[0006] In order to solve the above problems, the present invention provides a bufotoxin-Gastrodia elata gum polysaccharide aerogel, which is a composite of Gastrodia elata gum aerogel and bufotoxin;

[0007] The mass ratio of the gastrodia elata aerogel to bufotoxin is 50 to 150:10;

[0008] The Gastrodia elata gum aerogel is a solid substance obtained by dispersing the colloidal white solid obtained by water extraction and alcohol precipitation of Gastrodia elata in water, heating it, and then freeze-drying it.

[0009] Furthermore, the mass ratio of the Gastrodia elata aerogel to bufotoxin is 100:10.

[0010] The present invention also provides a method for preparing the aforementioned bufotoxin-Gastrodia elata colloid polysaccharide aerogel, comprising the following steps:

[0011] 1) taking Gastrodia elata, soaking and extracting it in water, concentrating the extract and adding ethanol, collecting the colloidal white solid to obtain Gastrodia elata colloid polysaccharide;

[0012] 2) dispersing the Gastrodia elata polysaccharide obtained in step 1) with water, heating and stirring to obtain a Gastrodia elata hydrogel, freezing the Gastrodia elata hydrogel at room temperature, and drying to obtain a Gastrodia elata aerogel;

[0013] 3) Gastrodia elata aerogel obtained in step 2) is ground into particles, and then a dichloromethane solution of bufotoxin is evenly added dropwise. After the addition is completed, the mixture is aged, and finally washed with dichloromethane, the dichloromethane is removed, and the mixture is dried to obtain the obtained product.

[0014] Furthermore, the amount of water added in step 1) is 15 times the amount of Gastrodia elata; the Gastrodia elata is a Gastrodia elata block with a diameter of less than 1 cm; the soaking time is 4 to 5 hours, the extraction is water bath extraction, the number of water bath extractions is 3, each temperature is 70 to 90°C, and the time is 2 to 4 hours; the concentration is reduced pressure concentration, the temperature is 40 to 60°C, and the concentration is concentrated to a relative density of 1.05; the ethanol is added to the solution to achieve an ethanol concentration of 70%.

[0015] Furthermore, in step 2), the mass volume ratio of the Gastrodia elata polysaccharide to water is 0.5-1.5 g:1 ml; the heating temperature is 40-60° C., the stirring speed is 500-1500 r / min, and the time is 2-6 h.

[0016] Furthermore, in step 2), the freezing temperature of the Gastrodia elata hydrogel is -20°C and the time is 4 hours; the drying is vacuum drying, the vacuum drying temperature is -70 to -90°C, the pressure is 0.01 to 0.02 bar, and the time is 30 to 40 hours, preferably the temperature is -80°C, the pressure is 0.018 bar, and the time is 36 hours.

[0017] Furthermore, in step 3), the mass volume ratio of the Gastrodia elata aerogel to the bufotoxin dichloromethane solution is 50-150 mg:2 ml; and the concentration of the bufotoxin dichloromethane solution is 3-7 mg / ml.

[0018] Furthermore, the aging in step 3) is performed by standing at 40-60° C. for 2-5 hours.

[0019] The present invention also provides a use of the aforementioned nervosa bufotoxin-Gastrodia elata colloid polysaccharide aerogel in preparing a drug for treating colorectal cancer.

[0020] Furthermore, the colorectal cancer includes colorectal adenocarcinoma, colon cancer, and colon adenocarcinoma.

[0021] The present invention's bufotoxin-Gastrodia elata pectin aerogel is made by forming aerogels from Gastrodia elata pectin polysaccharides of a specific molecular weight, which are then compounded with bufotoxin in a specific ratio. This aerogel allows for sustained release at the distal end of the colon, achieving colon-targeted and sustained-release effects. Furthermore, while using less bufotoxin, it achieves a better anti-colorectal cancer effect; even with the same bufotoxin dose, the anti-tumor effect is even greater. This creates a synergistic effect in terms of therapeutic efficacy, reduces the risk of bufotoxin poisoning, improves safety while ensuring efficacy, and possesses practical application value.

[0022] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0023] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Preparation process of Gastrodia elata glue loaded with bufotoxin;

[0025] Figure 2 Gastrodia elata after processing;

[0026] Figure 3 Crude polysaccharides from Gastrodia elata after water extraction and alcohol precipitation (left) and Gastrodia elata pectin polysaccharides after separation (right);

[0027] Figure 4 Standard curve of amylose of Gastrodia elata polysaccharide fragment;

[0028] Figure 5 Absolute molecular weight analysis chart;

[0029] Figure 6 Molecular configuration diagram;

[0030] Figure 7 Gastrodia elata polysaccharide fragment chain length distribution;

[0031] Figure 8 Gastrodia elata gum swelling (left) and hydrogel formation (right);

[0032] Figure 9 Freeze drying of Gastrodia elata hydrogel;

[0033] Figure 10 Preparation of AB@GGEPs-AG;

[0034] Figure 11 NMR characterization;

[0035] Figure 12 Infrared absorption spectroscopy;

[0036] Figure 13 Electron microscopy images;

[0037] Figure 14 UV-visible absorption spectrum of bufotoxin;

[0038] Figure 15 UV-visible absorption spectrum of Gastrodia elata polysaccharide;

[0039] Figure 16 Standard curve of bufotoxin;

[0040] Figure 17 In vitro release profile of AB@GGEPs-AG (SGF is gastric fluid, SDF is duodenal fluid, SIF is small intestinal fluid, and SCF is colonic fluid);

[0041] Figure 18 Caco-2 cell survival rate;

[0042] Figure 19 LS174T cell survival rate;

[0043] Figure 20 CT26 cell survival rate;

[0044] Figure 21 Cell survival rate of LS174T cells after treatment with different concentrations of drug-loaded Gastrodia elata gum

[0045] Figure 22 Cell apoptosis and necrosis. DETAILED DESCRIPTION

[0046] Example 1: Preparation of the present invention's bufotoxin-Gastrodia elata polysaccharide aerogel

[0047] 1. Preparation of Gastrodia elata polysaccharide

[0048] 2 kg of Gastrodia elata slices were weighed and crushed into small pieces with a diameter of less than 1 cm. The pieces were soaked in 15 times the volume (v / w) of water for 4-5 hours, and then extracted in an 80°C water bath for 3 hours. The extract was filtered through a 300-mesh filter cloth while still hot to separate the extract. The residue was extracted twice again using the same method. The three extracts were combined and concentrated under reduced pressure at 50°C to a relative density of 1.05. After cooling to room temperature, ethanol was added until the ethanol concentration reached 70%. The colloidal white solid was collected to obtain Gastrodia elata gelatinous polysaccharides (GGEPs).

[0049] 2. Preparation of Gastrodia elata hydrogel

[0050] Weigh 4 g of Gastrodia elata polysaccharide and 4 ml of water into a 25 ml conical flask, sonicate until the Gastrodia elata polysaccharide is evenly dispersed, heat to 50°C, add a magnetic stirrer and stir at 1000 r / min for 4 hours to obtain Gastrodia elata polysaccharide hydrogel (GGEPs-HG);

[0051] 3. Preparation of Gastrodia elata aerogel

[0052] Weigh 0.5 g of Gastrodia elata hydrogel, equilibrate at room temperature for 0.5 h, freeze at -20°C for 4 h, and finally freeze-dry under vacuum at a pressure of 0.018 bar and a temperature of -80°C for 36 h to obtain Gastrodia elata aerogel;

[0053] 4. Preparation of bufotoxin-Gastrodia elata polysaccharide aerogel

[0054] Weigh 100 mg of Gastrodia elata aerogel, grind it through a 50-mesh sieve to obtain small particles, and then evenly add 2 ml of 5 mg / ml bufotoxin dichloromethane solution. After the addition is completed, let it stand at 50°C for 3 hours, and finally wash it with dichloromethane. After removing dichloromethane, dry it to obtain bufotoxin-Gastrodia elata gum polysaccharide aerogel AB@GGEPs-AG.

[0055] The beneficial effects of the present invention are described below through test examples.

[0056] Experimental Example 1 Study on the Agrobacterium Toxoplasmosis-Gastrodia Gum Polysaccharide Aerogel

[0057] 1. Research route and content

[0058] Research roadmap see Figure 1 .

[0059] 1.1 Processing of Gastrodia elata

[0060] Gastrodia elata, a rare Chinese medicinal herb, is primarily used for its dried tubers. In Traditional Chinese Medicine, Gastrodia elata possesses multiple benefits, including calming wind and relieving spasms, calming the liver and suppressing yang, and dispelling wind and unblocking meridians. It is widely used to treat conditions such as headaches, dizziness, limb numbness, and infantile convulsions. Its mild properties allow it to be used alone or in combination with other herbs for synergistic effects. Gastrodia elata's demanding growing conditions, preferring a moist and cool environment, and its limited production, further enhances its preciousness. Its processing and preparation are complex, involving timely harvesting, meticulous cleaning, grading, soaking or steaming, slicing, and drying, all designed to enhance its shelf life, efficacy, and clinical applicability. Furthermore, specialized methods such as stir-frying, stewing, or stir-frying with ginger juice can be used to enhance its efficacy. After processing, Gastrodia elata must be stored in a cool, dry place to prevent deterioration and ensure its quality and efficacy for clinical use. The processing method of Gastrodia elata used in this study was carried out in accordance with the Chongqing local standard "Technical Regulations for Primary Processing of Gastrodia elata" (No.: DB50 / T 1498-2023). The processed Gastrodia elata slices and their morphology after cutting into small pieces were shown in Figure 2. Figure 2 .

[0061] 1.2 Extraction of Gum-like Polysaccharides from Gastrodia elata Pieces (Gastrodia elata Gum-like Polysaccharide Fragments)

[0062] Take 2kg of processed Gastrodia elata and chop it into pieces smaller than 1cm by hammering or chopping with a guillotine. Place it in a 20L vat, immerse it in water (volume ratio of 1:15 or more), and soak it for 4-5 hours. Subsequently, extract it in an 80℃ water bath for 3 hours, filter it with a 300-mesh filter cloth while it is still hot, repeat the extraction twice, and combine the three filtrates. Use a rotary evaporator to distill the extract under reduced pressure at 50℃ until it becomes slightly viscous (relative density of about 1.05) to obtain a concentrated solution. After cooling, add ethanol to a concentration of 70%. At this time, two types of polysaccharides, dark block and white colloidal, are precipitated. The white colloidal solid is colloidal Gastrodia elata polysaccharide (abbreviated as Gastrodia elata gum, Gelatinous GEPs, GGEPs). The two polysaccharides have certain differences in density and volume. They can be separated by floating and then standing. At this time, Gastrodia elata gum is absorbed and separated from the suspension, filtered to remove ethanol, redissolved with water and then recrystallized with ethanol to remove residual small molecular impurities such as gastrodin and balisonglucoside to obtain solid Gastrodia elata gum ( Figure 3 ).

[0063] 1.3 Characterization of Gastrodia elata polysaccharide fragments

[0064] Preliminary testing revealed that the gelatinous polysaccharide fragments in Gastrodia elata exhibit starch-like gelatinization properties. Therefore, we conducted physical and chemical studies on these substances using starch identification and determination methods. Key indicators such as amylose content, starch polymerization degree, molecular weight, viscosity, and gelatinization properties were used to provide accurate and stable data and reliable technical support for research related to the development of starch-based materials derived from Gastrodia elata gelatinous substances.

[0065] 1.3.1 Determination of resistant starch content

[0066] Resistant starch, also known as resistant starch or indigestible starch, cannot be enzymatically hydrolyzed in the small intestine, but can undergo fermentation reactions with volatile fatty acids in the human gastrointestinal colon. To detect the content of resistant starch in a sample, α-amylase and amyloglucosidase are used to remove non-resistant starch. The residue is dissolved with KOH, the acetate buffer is adjusted to neutral, and the starch is hydrolyzed into glucose with amyloglucosidase. The GOPOD reagent buffer reacts with D-glucose to produce quinine dye, and its content is determined by colorimetry. After testing, no resistant starch was detected in the Gastrodia elata polysaccharide fragment.

[0067] Table 1 Detection results of resistant starch content in Gastrodia elata polysaccharide fragments

[0068]

[0069] 1.3.2 Determination of total starch content

[0070] The starch content in the sample was determined by using thermostable α-amylase to hydrolyze the starch into branched and unbranched maltodextrins. Amyloglucosidase then quantitatively hydrolyzed the maltodextrin into D-glucose. GOPOD reagent buffer reacted with D-glucose to produce quinine dye, which was then colorimetrically determined. The total starch content was 49.95%.

[0071] Table 2 Detection results of total starch content in Gastrodia elata polysaccharide fragments

[0072]

[0073] 1.3.3 Determination of amylose content

[0074] The amylose content in the sample is mainly determined by preparing standard solutions of different proportions of amylose and amylopectin, which have different affinities for iodine. The standard curve is then prepared to quantitatively determine the amylose content in the sample ( Figure 4 ). After testing, no amylose was detected in the sample.

[0075] Table 3 Detection results of amylose content in Gastrodia elata polysaccharide fragments

[0076]

[0077] 1.3.4 Molecular weight detection

[0078] The molecular weight test of starch mainly uses gel chromatography, laser light scattering and differential detector to measure the absolute molecular weight of the sample. Gel chromatography can elute the molecules in the solvent in order of weight or size. Larger molecules can only enter the part of the gel pore with larger pore size, while smaller molecules can enter more gel particles. In this way, larger molecules move a shorter distance in the gel bed, while smaller molecules move a longer distance. Large molecules are eluted first, and small molecules are eluted later, thereby achieving the separation effect, thereby achieving the purpose of separation according to molecular size; then, a differential detector is used to detect the concentration information of the sample based on its refractive intensity, and a multi-angle laser light scattering instrument is used to detect the light scattering information of the macromolecules. The molecular weight corresponding to each component is calculated according to the Mark-Houwink equation. The molecular weight of the Gastrodia elata gelatin polysaccharide fragment is Mw = 35581.273kDa.

[0079] The chromatographic data were processed using ASTRA6.1 software. Figures 5-6 Shown are the absolute molecular weight analysis diagram and molecular configuration analysis diagram of the sample, the absolute molecular weight analysis diagram ( Figure 5 ) takes time (time, min) as the horizontal axis and molar mass (MolarMass, g / mol) as the vertical axis; the molecular configuration diagram ( Figure 6 ) takes molar mass (MolarMass, g / mol) as the horizontal coordinate and root mean square radius (RMSRadius, nm) as the vertical coordinate.

[0080] Red line: Multi-angle laser light scattering signal, reflecting the sample molecular size; blue line: differential signal, reflecting the sample concentration; black line: molecular weight fitted from the two signals. The blue line is the differential signal, representing the sample concentration; the red line is the light scattering signal, representing the sample molecular size.

[0081] The slope of the graph, with molecular mass as the horizontal axis and the radius of gyration (RMS Radius) as the vertical axis, can be used as a reference for the molecular configuration. Generally, a slope of 1 indicates a rod-shaped molecule, a slope of 0.5-0.6 indicates an irregular coil, and a slope of 1 / 3 indicates a sphere. (A slope of 0.16 also suggests a dense sphere.)

[0082] Table 4 Gastrodia elata polysaccharide fragment molecular weight detection results

[0083]

[0084] 1.3.5 Gelatinization temperature detection

[0085] Gelatinization temperature: Starch is a series complex of glucose formed by α-1,4 and α-1,6 glycosidic bonds, and has a crystalline and semi-crystalline structure. As the temperature rises, higher-order structures in starch, such as the double helix and crystalline structure, are destroyed. For native starch, this process is called gelatinization. Different starch structures have different phase transition processes, and the temperature and energy required for phase transition also vary. In the study of starch and related polymer derivatives, the phase transition process of starch and its derivatives is measured by differential calorimetry (DSC), which can be used to analyze the structure and physicochemical properties of starch. The gelatinization temperature of Gastrodia elata gum has been tested to have an initial temperature of 52.6°C, a peak temperature of 69.5°C, an end temperature of 94.3°C, and a gelatinization enthalpy of 5.533 J / g.

[0086] Table 5 Gastrodia elata polysaccharide fragment molecular weight detection results

[0087]

[0088]

[0089] 1.3.6 Chain length distribution

[0090] Standards with different chain lengths have different retention times on the chromatogram. The qualitative analysis is based on the retention time of the sample on the chromatographic column, and the concentration is calculated from the standard curve based on the sample peak area. Test results ( Figure 7 ), the average degree of polymerization is 19.7.

[0091] 1.3.7 Summary of Characterization of Gastrodia elata Polysaccharide Fragments

[0092] Characterization revealed that the gelatinous Gastrodia polysaccharides (GGEPs) extracted and separated from Gastrodia elata slices are natural polysaccharides with a certain degree of branching, starch-like properties, and a high molecular weight. The high molecular weight and branched interactions of Gastrodia elata gum provide superior stability when used as a carrier. Furthermore, the gelatinization properties of Gastrodia elata gum indicate that it has limited or slow solubility in water, requiring more time to metabolize in digestive fluids, making it a natural intestinal targeted delivery material.

[0093] 1.4 Sol-gelation of Gastrodia elata

[0094] Polysaccharide hydrogels, three-dimensional network polymer structures constructed from natural or modified polysaccharide molecules through physical or chemical crosslinking, possess the ability to absorb and retain large amounts of water while maintaining a solid state. Their excellent biocompatibility, degradability, and water-retention properties demonstrate significant potential for application in biomedicine, particularly in tissue engineering and drug delivery systems. Furthermore, polysaccharide hydrogels are being actively researched for applications in environmental protection, food preservation, and agricultural production, playing an important role as highly effective functional materials. Their preparation primarily relies on the sol-gel method, which involves chemical reactions and physical structural transformations. Highly active polysaccharide precursors are selected, uniformly mixed, and hydrolyzed and condensed to form a transparent sol. The polysaccharide molecular chains are then dispersed and crosslinked. After aging, the sol particles aggregate to form a three-dimensional network gel, which ultimately solidifies into a hydrogel. During the preparation process, temperature, pH, and reaction time can be manipulated to adjust the hydrogel's properties. Furthermore, the introduction of functional components can achieve multifunctionality. The hydrogels prepared by this method exhibit uniform structure, high porosity, and excellent biocompatibility, making them widely applicable in biomedicine, tissue engineering, and drug delivery.

[0095] Weigh 4g of Gastrodia elata polysaccharide GGEPs and 4ml of water into a 25ml conical flask, ultrasonicate until the Gastrodia elata is evenly dispersed, heat to 50°C, add a magnetic stirrer and stir at 1000r / min. After heating for 4 hours, it was observed that the Gastrodia elata gum and water swelled significantly to form a hydrogel, which was cooled to room temperature to obtain Gastrodia elata hydrogel (GGEPs-HG) ( Figure 8 ).

[0096] 1.5 Freeze-drying of Gastrodia elata hydrogel

[0097] Weigh 0.5g of Gastrodia elata hydrogel, place it in a watch glass of suitable size, equilibrate at room temperature for 0.5h, then freeze the watch glass at -20℃ for 4h. Vacuum dry the frozen Gastrodia elata gel at -80℃ and 0.018bar for 36h, so that the ice crystals are directly sublimated from the solid state to the gaseous state and discharged, avoiding shrinkage and damage during the drying process. In this process, the structure and properties of the sample can be adjusted by controlling the freezing rate, freezing temperature and drying conditions. Finally, a Gastrodia elata aerogel sample with a porous structure and high specific surface area is obtained ( Figure 9 ), refrigerate at 4℃ for later use.

[0098] 1.6 Preparation of Gastrodia elata aerogel-encapsulated bufotoxin complex (impregnation method)

[0099] Weigh 100mg of Gastrodia elata aerogel, grind it repeatedly through a 50-mesh sieve to obtain small particles, set aside, and evenly spread them in a 100ml crystallization dish. Weigh 10mg of toad venom and put it into a centrifuge tube, add 2ml of dichloromethane, and place it in an ultrasonicator for room temperature sonication until toad venom is completely dissolved. The dichloromethane solution in which toad venom is dissolved is slowly and evenly added dropwise to the Gastrodia elata aerogel solid, and the addition is completed in small amounts and multiple times. After the addition is completed, the Gastrodia elata aerogel is aged at 50°C for 3h to allow toad venom to diffuse evenly and tightly combine with the aerogel. After washing the aged aerogel with dichloromethane, remove the excess impregnation liquid on the surface, and dry it to obtain the Gastrodia elata aerogel AB@GGEPs-AG ( Figure 10 ). The quantitative detection by nuclear magnetic resonance internal standard method showed that the drug loading of nervosa bufotoxin was about 3.26w%.

[0100] 1. Characterization of 7AB@GGEPs-AG

[0101] 1.7.1 NMR characterization

[0102] Weigh 0.009 g of AB@GGEPs-AG and dissolve it in 0.6 ml of deuterated DMSO to prepare a test solution with a concentration of 15 mg / mL. Use a 400 Hz nuclear magnetic resonance spectrometer (model: AVANCE400) to detect the 1H NMR spectrum of the test sample. Figure 11 1HNMR characterization showed that bufotoxin could be encapsulated into Gastrodia elata aerogel by the impregnation method, and the AB@GGEPs-AG delivery system was successfully prepared.

[0103] 1.7.2 Infrared test

[0104] At 1600-1650cm -1 A moderate intensity absorption peak appears in the region, corresponding to the stretching vibration of the carbon-carbon double bond in the structure of bufotoxin steroid ( Figure 12 ). At 1700-1750cm -1 There is a strong absorption peak near 1690.69cm, which is mainly attributed to the carbonyl group in the bufotoxin molecule. -1 A new absorption peak appeared, indicating that AB was successfully loaded into GEP.

[0105] 1.7.3 Electron microscopy test

[0106] A certain amount of GEP solution was weighed and freeze-dried to form an aerogel. The drug AB was loaded onto the GEP to form the drug-loaded system AB@GGEPs-AG. A small amount of AB@GGEPs-AG was fixed on the sample stage. The sample was non-conductive and sprayed with gold for 60 seconds at a voltage of 20kV. The microstructure of the sample was observed under a scanning electron microscope (Quattro S) and images were collected. Figure 13 ) It can be seen that the GEP becomes more fluffy and has a rough surface after freeze-drying, forming relatively uniform pores, which is conducive to subsequent drug encapsulation.

[0107] 1.8 Determination of drug content in AB@GGEPs-AG

[0108] 1.8.1 Preparation of standard solution

[0109] Since neribufotoxin has limited solubility in pure water, a phosphate buffer solution containing dimethyl sulfoxide was selected as the solvent. Accurately weigh 1.25 mg of neribufotoxin into a 25 ml volumetric flask and dilute to volume with pH 6.8 phosphate buffer to prepare a 0.05 mg / ml stock solution.

[0110] 1.8.2 Determination of measurement wavelength

[0111] The phosphate buffer solution of bufotoxin has maximum absorption in the ultraviolet region. Accurately measure 5.0 ml of the stock solution in a 10 ml volumetric flask and dilute it to the mark with pH 6.8 phosphate buffer. Using pH 6.8 phosphate buffer as a reference, scan within the wavelength range of 200-700 nm. Figures 14-15 The absorption of solvent is at 210nm. The result shows that there is maximum absorption at 297nm. However, considering that Gastrodia elata has weak absorption interference test between 200-300nm, the detection is carried out at 315nm. At this time, the absorbance is about 80% of the maximum absorption wavelength.

[0112] 1.8.3 Establishment and determination of standard curve

[0113] Accurately pipette 0.625, 1.25, and 2.5 ml of the stock solution into a 10 ml volumetric flask and dilute to the mark with pH 6.8 phosphate buffer. Measure the absorbance at a wavelength of 315 nm using pH 6.8 phosphate buffer as a reference. A linear regression is performed using the concentration C against the absorbance A. The regression equation is A = 13.166*C + 0.016. This indicates that the linear relationship between bufotoxin and fenbufotoxin is observed within the range of 3.125 to 12.5 μg / ml ( Figure 16 ). Standard solutions with different concentration gradients were obtained and UV absorption was measured at a wavelength of 315 nm.

[0114] 1.8.4 Sample testing

[0115] A 3.65 mg sample of AB@GGEPs-AG was added to a 10 ml volumetric flask and diluted to the mark with pH 6.8 phosphate buffer. Ultrasonication was performed for 10 minutes to prepare a 0.365 mg / ml solution. The UV absorbance at 315 nm was 0.078 (control experiments showed that Gastrodia elata gum had no absorption at this wavelength). Based on the standard curve, the concentration of bufotoxin was calculated to be 0.0047 mg / ml, with a drug loading of 1.29%. The drug loading measured by spectrophotometry was lower than the theoretical value. This is because bufotoxin is encapsulated in the high-molecular-weight Gastrodia elata polysaccharide aerogel phase and cannot be completely dissolved into the phosphate buffer in a short time due to diffusion limitations and adsorption. Therefore, the bufotoxin drug loading measured by UV light is lower than the quantitative value determined by nuclear magnetic resonance (NMR). Because DMSO has a strong extraction effect on bufotoxin, the dissolution is faster, making the actual measurement more rapid and accurate.

[0116] 1.9AB@GGEPs-AG in vitro release experiment

[0117] Through continuous release test in simulated medium, the results ( Figure 17 ) It can be seen that the release amount in gastric juice within 2 hours is about 0.5%, indicating that polysaccharides have a protective effect on drug molecules. When the drug enters the small intestinal fluid, the drug release rate is about 3%. When the drug molecules reach the colon, the drug is continuously and rapidly released, and gradually tends to equilibrium in about 32 hours. In contrast, the drug of toad venom is continuously released after entering the small intestine, with a release rate of 7.5% in the small intestine. After entering the colon, the drug is quickly and completely released at the end of the colon. Therefore, using Gastrodia elata gum as a drug carrier can effectively protect the drug from premature release and has a colonic sustained-release effect.

[0118] 1.10AB@GGEPs-AG in vitro cell experiments

[0119] 1.10.1 Cells and main experimental materials

[0120] Caco-2 (human colorectal adenocarcinoma cells) were purchased from Wuhan Punosai Life Science Co., Ltd. LS174T (human colon adenocarcinoma cells) and CT26 (mouse colon carcinoma cells) were kindly provided by Dr. Huang Xiaoqi of Guangzhou University of Chinese Medicine. Cell culture medium, fetal bovine serum, and bispecific antibodies were purchased from Thermo Fisher Scientific. The YO-PRO-1 / PI apoptosis and necrosis detection kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; bufotoxin was provided by Luo Chuan of Anhui China Resources Jinchan Pharmaceutical Co., Ltd.; Gastrodia elata was prepared as a Gastrodia elata aerogel under the "1.5-well Gastrodia elata hydrogel freeze-drying" method, and drug-loaded Gastrodia elata was prepared as AB@GGEPs-AG under the "1.6-well Gastrodia elata aerogel-encapsulated bufotoxin complex" method.

[0121] 1.10.2 Experimental methods

[0122] (1) Cell culture

[0123] Cells were cultured in complete medium (containing 10% fetal bovine serum and 1% double-antibody) at 37°C in a cell culture incubator with 5% CO2. After 80% cell adherence, the supernatant was discarded, the cells were washed twice with PBS, and then trypsinized for 3 minutes. Digestion was terminated by adding culture medium and the cells were resuspended. The cells were collected into a centrifuge tube and centrifuged at 1000×g for 3 minutes before subculturing. Several growing cells were harvested for use in experiments.

[0124] (2) Cell viability detection

[0125] Take cells in the logarithmic growth phase and calculate 2×10 3 Cells were seeded into 96-well plates at 100 μL per well. A blank control group and experimental groups treated with different drugs were set up, with five replicates per group. After the cells adhered on the second day, the original culture medium was removed and the cells were washed twice with PBS. Complete culture medium (100 μL per well) was added to the control group, while complete culture medium containing bufotoxin (final concentrations of 100, 50, and 25 mg / L), gastrodia gum (final concentrations of 100, 50, and 25 mg / L), and drug-loaded gastrodia gum (mixture concentrations of 100, 50, and 25 mg / L, with bufotoxin final concentrations of 3.26, 1.63, and 0.815 mg / L) was added to the experimental groups. After 24 hours of culture, 10 μL of CCK-8 detection reagent was added to each well and incubated at 37°C for 1 hour. The optical density of each well was measured at a wavelength of 450 nm using a microplate reader, and the cell viability was calculated.

[0126] LS174T cells (human colon adenocarcinoma cells) in the logarithmic growth phase were taken and plated at 2×10 3 Cells were inoculated into 96-well culture plates at 100 μL per well. A blank control group and experimental groups treated with different drugs were set up, with 5 replicates per group. After the cells adhered on the second day, the original culture medium was removed and the cells were washed twice with PBS. Complete culture medium was added to the control group at 100 μL per well, while complete culture medium containing bufotoxin (final concentration of 10 mg / L), Gastrodia elata (final concentration of 10 mg / L), and different concentrations of drug-loaded Gastrodia elata (bufotoxin final concentration of 10, 1, and 0.1 mg / L) was added to the experimental groups at 100 μL per well. After 24 hours of culture, 10 μL of CCK-8 detection reagent was added to each well and incubated at 37°C for 1 hour. The optical density of each well was measured at a wavelength of 450 nm using a microplate reader and the cell viability was calculated.

[0127] (3) Detection of cell apoptosis and necrosis

[0128] LS174T (human colon adenocarcinoma cell) cells in the logarithmic growth phase were cultured at a rate of 2×10 3Cells were seeded into 96-well plates at 100 μL per well. A blank control group and experimental groups treated with different drugs were set up, with five replicates per group. After the cells adhered on the second day, the original culture medium was removed and the cells were washed twice with PBS. Complete culture medium (100 μL per well) was added to the control group, while complete culture medium containing 100 mg / mL bufotoxin, Gastrodia elata, or drug-loaded Gastrodia elata was added to the experimental groups, respectively. After 4 hours of culture, apoptosis and necrosis were detected using the YO-PRO-1 / PI Cell Apoptosis and Necrosis Detection Kit.

[0129] YO-PRO-1, also known as oxazole yellow (YP1), is a DNA green fluorescent dye that is impermeable to normal animal cell membranes but permeable to the membranes of apoptotic cells. It is commonly used to detect apoptosis. YO-PRO-1 is a non-cell membrane-permeable carbocyanine monomeric green fluorescent dye with a high affinity for DNA. It exhibits virtually no fluorescence when unbound to DNA, but emits bright green fluorescence upon binding. During apoptosis, cell membrane permeability changes, allowing YO-PRO-1 to enter the cell and bind to DNA, emitting bright green fluorescence. Therefore, this fluorescent dye is often used to analyze and identify apoptotic cells.

[0130] PI, or propidium iodide (PI), is a red fluorescent dye for nucleic acids that only stains necrotic cells that have lost their cell membrane integrity and binds to nucleic acids to emit bright red fluorescence. Therefore, when used in combination with propidium iodide (PI), YO-PRO-1 can simultaneously detect apoptotic and necrotic cells. Apoptotic cells exhibit green fluorescence, while necrotic cells exhibit both red and green fluorescence. Live cells exhibit little or no fluorescence.

[0131] 1.10.3 Experimental Results

[0132] (1) Cell survival rate

[0133] The inhibitory effects of bufotoxin on the proliferation of Caco-2, LS174T and CT26 cells were different. The inhibitory effects on the proliferation of Caco-2 and LS174T cells were more obvious, but there was no significant inhibitory effect on the proliferation of CT26 cells (P<0.05) ( Figures 18-21 ). Gastrodia elata glue can enhance the effect of bufotoxin and reduce the dosage of bufotoxin.

[0134] (2) Cell apoptosis and necrosis

[0135] Observation under an inverted microscope revealed that ( Figure 22). Cells in the blank control and Gastrodia elata gum groups maintained good growth, with tight junctions between cells. Cell proliferation was inhibited in the nervosain and drug-loaded Gastrodia elata gum-treated groups, with a decrease in cell number, increased intercellular spaces, and irregular morphology in some cells. Compared with the blank control group, the nervosain and drug-loaded Gastrodia elata gum-treated groups showed an increase in the number of green-positive cells, indicating increased apoptosis.

[0136] 4 Conclusion

[0137] A method for preparing AB@GGEPs-AG complexes involves extracting and isolating colloidal polysaccharide fragments from crude polysaccharides of the traditional Chinese medicinal herb Gastrodia elata (Tianjin Gastrodia elata). These fragments are then loaded with toad toxin-like small-molecule drugs. The following steps are taken: Crude Gastrodia elata polysaccharides are extracted from Gastrodia elata blocks using a hot water-alcohol precipitation method. The colloidal fraction is then separated by flotation and prepared into a Gastrodia elata gum hydrogel, which is then freeze-dried to form a Gastrodia elata gum aerogel. The Gastrodia elata gum complex AB@GGEPs-AG, loaded with bufotoxin, is then prepared by an impregnation method. In vitro release experiments confirm the sustained-release effect of AB@GGEPs-AG. Cell-based experiments also demonstrate the inhibitory effect of AB@GGEPs-AG on tumor cells and its ability to promote bufotoxin-induced apoptosis. Notably, bufotoxin exhibits a certain selectivity for cancer cells, which has positive implications for its clinical application. The Gastrodia elata gum aerogel carrier system can enhance bufotoxin cellular uptake, reduce bufotoxin dosage, while maintaining efficacy and improving safety.

[0138] In summary, the drug-loaded Gastrodia elata gum complex AB@GGEPs-AG of the present invention can achieve sustained release at the distal end of the colon, demonstrating colon-targeted and sustained-release effects. Furthermore, it can achieve a better anti-colorectal cancer effect while using less bufotoxin. At the same bufotoxin dose, the anti-tumor effect is even greater, creating a synergistic effect. This makes it a safe and effective bufotoxin preparation with practical application value.

Claims

1. A bufotoxin-Gastrodia elata polysaccharide aerogel, characterized by: It is made of a composite of Gastrodia elata aerogel and Sandbufalcon venom; The mass ratio of the gastrodia elata aerogel to bufotoxin is 50 to 150:10; The Gastrodia elata gum aerogel is a solid substance obtained by dispersing the colloidal white solid obtained by water extraction and alcohol precipitation of Gastrodia elata in water, heating it, and then freeze-drying it.

2. The bufotoxin-Gastrodia elata polysaccharide aerogel according to claim 1, characterized in that: The mass ratio of the Gastrodia elata aerogel to the toad venom is 100:

10.

3. A method for preparing the bufotoxin-Gastrodia elata colloid polysaccharide aerogel according to claim 1 or 2, characterized in that: The steps include: 1) taking Gastrodia elata, soaking and extracting it in water, concentrating the extract and adding ethanol, collecting the colloidal white solid to obtain Gastrodia elata colloid polysaccharide; 2) dispersing the Gastrodia elata polysaccharide obtained in step 1) with water, heating and stirring to obtain Gastrodia elata hydrogel, freezing the Gastrodia elata hydrogel at room temperature, and drying to obtain Gastrodia elata aerogel; 3) Gastrodia elata aerogel obtained in step 2) is ground into particles, and then a dichloromethane solution of bufotoxin is evenly added dropwise. After the addition is completed, the mixture is aged, and finally washed with dichloromethane, the dichloromethane is removed, and the mixture is dried to obtain the obtained product.

4. The preparation method according to claim 3, wherein: In step 1), the amount of water added is 15 times the amount of Gastrodia elata; the Gastrodia elata is a Gastrodia elata block with a diameter of less than 1 cm; the soaking time is 4 to 5 hours, the extraction is water bath extraction, the number of water bath extractions is 3, each temperature is 70 to 90° C., and the time is 2 to 4 hours; the concentration is reduced pressure concentration at a temperature of 40 to 60° C., and the concentration is concentrated to a relative density of 1.05; the ethanol is added to the solution until the ethanol concentration reaches 70%.

5. The preparation method according to claim 3, wherein: Step 2) The mass volume ratio of the Gastrodia elata polysaccharide to water is 0.5-1.5 g:1 ml; the heating temperature is 40-60° C., the stirring speed is 500-1500 r / min, and the time is 2-6 h.

6. The preparation method according to claim 3, wherein: Step 2) The Gastrodia elata hydrogel is frozen at a temperature of -20°C for 4 hours; the drying is vacuum drying at a temperature of -70 to -90°C, a pressure of 0.01 to 0.02 bar, and a time of 30 to 40 hours.

7. The preparation method according to claim 3, wherein: Step 3) The mass volume ratio of the Gastrodia elata aerogel to the nervosain dichloromethane solution is 50-150 mg:2 ml; the concentration of the nervosain dichloromethane solution is 3-7 mg / ml.

8. The preparation method according to claim 3, wherein: Step 3) The aging is performed by standing at 40-60° C. for 2-5 hours.

9. Use of the bufotoxin-Gastrodia elata colloid polysaccharide aerogel according to claim 1 or 2 in the preparation of a medicament for treating colorectal cancer.

10. The use according to claim 9, characterized in that: The colorectal cancer includes colorectal adenocarcinoma, colon cancer, and colon adenocarcinoma.

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