Acute gastrorrhagia hemostatic agent based on gardenia carbon nano composite material and preparation method of acute gastrorrhagia hemostatic agent

Abstract

The invention discloses an acute gastrorrhagia hemostatic agent based on a gardenia carbon nano composite material and a preparation method of the acute gastrorrhagia hemostatic agent, and belongs to the technical field of biological medicines. According to the preparation method disclosed by the invention, the gardenia carbon nano composite material is prepared through nanocrystallization transformation, and the average particle size is 2.35 nm + / -0.56 nm. Mouse tail cutting hemostasis and capillary experiments prove that the gardenia charcoal nano composite material has the effects of promoting hemostasis and blood coagulation, and the hemostasis time and the blood coagulation time can be remarkably shortened; through construction of a rat blood-heat gastrorrhagia model, it is verified that the gardenia charcoal nano composite material has a remarkable hemostatic effect; and zebra fish verifies that the gardenia carbon nano composite material can protect gastrointestinal mucosa from being damaged, specifically, the area of a small intestinal cavity is reduced, and the number of neutrophils in the intestinal tract is reduced. The gardenia carbon nano composite material provided by the invention can be used for preparing a medicine for promoting blood coagulation and / or hemostasis, a hemostatic for acute gastrorrhagia and a medicine for relieving gastrointestinal mucosal injury.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an acute gastric bleeding hemostatic agent based on a gardenia charcoal nanocomposite material and a preparation method thereof. Background Art

[0002] Gastrointestinal bleeding refers to bleeding in the digestive tract from the esophagus to the anus, while upper gastrointestinal bleeding refers to bleeding in the digestive tract above the suspensory ligament of the duodenum (mainly including the esophagus, stomach, duodenum, upper jejunum, and bile duct). Symptoms include hematemesis, melena, hematochezia, dizziness, fatigue, and fever.

[0003] Gastric bleeding is a type of gastrointestinal bleeding. When it occurs, the situation is relatively urgent. It refers to bleeding in a section of the gastrointestinal tract starting from the cardia (the junction of the stomach and esophagus) and ending at the pyloric part. The pyloric part is located at the bottom of the stomach and is the passage connecting the stomach and the duodenum. The essence of gastric bleeding is edema and rupture of blood vessels at the fundus of the stomach, or it may be rupture and bleeding of esophageal veins at the fundus of the stomach. Gastric bleeding is a relatively common disease, but there are no exact statistical data in clinical practice. The annual incidence of acute upper gastrointestinal bleeding in adults is 100 / 100,000-180 / 100,000.

[0004] In case of gastric bleeding, the first thing to do is to identify the cause of the bleeding, find the bleeding point, and stop the bleeding promptly and quickly. When there is massive bleeding, the condition is acute and changes rapidly. Blood volume should be replenished quickly and anti-shock should be carried out. Commonly used treatment methods include drug therapy, endoscopic therapy, and surgical treatment. However, the clinical treatment of acute gastric bleeding has problems such as low hemostatic efficiency, poor drug targeting, and the possibility of causing secondary damage. Although traditional carbon materials (such as activated carbon) have adsorption properties, they lack biological activity. Based on this, there is an urgent need to propose a new hemostatic or coagulation material to prepare a hemostatic agent for acute gastric bleeding. Summary of the Invention

[0005] The purpose of the present invention is to provide an acute gastric bleeding hemostatic agent based on gardenia charcoal nanocomposite material and a preparation method thereof, so as to solve the problems existing in the above-mentioned prior art. Experimental verification shows that the gardenia charcoal nanocomposite material prepared by the present invention has the effect of promoting hemostasis and coagulation, and also has the effect of protecting gastrointestinal mucosal damage.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The invention provides an application of a gardenia charcoal nanocomposite material in preparing a hemostatic agent for acute gastric bleeding. The gardenia charcoal nanocomposite material is prepared by pulverizing the gardenia charcoal, extracting with water, dialyzing, concentrating and drying.

[0008] The present invention also provides a method for preparing the gardenia charcoal nanocomposite material, comprising the following steps:

[0009] The gardenia charcoal is pulverized to obtain charcoal powder, the charcoal powder is soaked in water, and heated for extraction to obtain a primary water decoction, and the primary water decoction is filtered to obtain a gardenia charcoal water extract;

[0010] The gardenia charcoal water extract is dialyzed to obtain a dialysate, and the dialysate is heated, concentrated, and freeze-dried to obtain a gardenia charcoal carbon dot solid powder, which is the gardenia charcoal nanocomposite material.

[0011] Optionally, the carbon powder is sieved through a No. 4 sieve;

[0012] The mass ratio of the carbon powder to the water is 1:30.

[0013] Optionally, the heating extraction is performed at a temperature of 100° C., for 1 hour, and for 3 times.

[0014] Optionally, the dialysis is performed on a 1000Da dialysis membrane, and the dialysis time is 72 hours.

[0015] The present invention provides application of the gardenia charcoal nanocomposite material in preparing drugs for promoting blood coagulation and / or hemostasis.

[0016] The present invention provides application of the gardenia charcoal nanocomposite material in preparing a medicine for alleviating gastrointestinal mucosal damage.

[0017] The invention provides a hemostatic agent for acute gastric bleeding, comprising the gardenia charcoal nanocomposite material.

[0018] Optionally, it further comprises pharmaceutically acceptable excipients.

[0019] The present invention discloses the following technical effects:

[0020] The present invention has prepared a gardenia charcoal nanocomposite material through nano-transformation, with an average particle size of 2.35nm±0.56nm. It has been verified by mouse tail-cutting hemostasis and capillary experiments that the gardenia charcoal nanocomposite material has the effect of promoting hemostasis and coagulation, and can significantly shorten the hemostasis time and coagulation time; it has been verified by constructing a rat blood-heat gastric bleeding model that the gardenia charcoal nanocomposite material has a significant hemostatic effect; and it has been verified by zebrafish that the gardenia charcoal nanocomposite material can protect gastrointestinal mucosal damage, which is specifically manifested in reducing the intestinal cavity area and reducing the number of intestinal neutrophils. The gardenia charcoal nanocomposite material provided by the present invention can be used to prepare drugs that promote coagulation and / or hemostasis, prepare hemostatic agents for acute gastric bleeding, and prepare drugs that alleviate gastrointestinal mucosal damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 The flow chart for the preparation of Gardenia charcoal nanocomposites;

[0023] Figure 2 This is the Tyndall effect diagram of Gardenia jasminoides carbon dots;

[0024] Figure 3 Transmission electron microscopy images of gardenia charcoal carbon dots; the scale bar in (a) is 10 nm, and the scale bar in (b) is 5 nm. The inset in the upper left corner shows that the gardenia charcoal carbon dots have a clear graphite lattice structure, and the red lines indicate the lattice fringe spacing;

[0025] Figure 4 This is the particle size distribution diagram of Gardenia charcoal carbon dots;

[0026] Figure 5 Atomic force microscopy observations of Gardenia charcoal carbon dots; (a): Schematic plan view; (b): 3D image of carbon dots; (c): Distribution of radial height of carbon dots along a certain direction; (d): Statistical graph of radial height of the surface;

[0027] Figure 6 This is the X-ray diffraction pattern of Gardenia jasminoides carbon dots;

[0028] Figure 7 This is the UV-visible absorption spectrum of Gardenia jasminoides carbon dots;

[0029] Figure 8 is the fluorescence emission spectrum of gardenia charcoal carbon dots at different excitation wavelengths;

[0030] Figure 9 These are the excitation and emission spectra of Gardenia jasminoides carbon dots;

[0031] Figure 10 This is the FTIR spectrum of Gardenia jasminoides carbon dots;

[0032] Figure 11 X-ray photoelectron spectroscopy analysis of Gardenia jasminoides carbon dots; (a): Full XPS spectrum of Gardenia jasminoides carbon dots; (b): High-resolution XPS spectrum of C1s; (c): High-resolution XPS spectrum of O1s; (d): High-resolution XPS spectrum of N1s; (e): High-resolution XPS spectrum of S2p;

[0033] Figure 12 Figure 2 is the macroscopic observation of the gastric tissues of rats in each group;

[0034] Figure 13 The following are typical images of the zebrafish intestinal lumen area after sample treatment in each group; the red dotted line indicates the analysis site;

[0035] Figure 14 Statistical graph of zebrafish intestinal area after sample treatment in each group;

[0036] Figure 15 The following is a typical graph of the number of neutrophils in the zebrafish intestine after treatment of each group of samples; the yellow dotted line indicates the analysis site, and the green particles are neutrophils;

[0037] Figure 16 The statistical chart shows the number of neutrophils in the zebrafish intestine after treatment of each group of samples. DETAILED DESCRIPTION

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0040] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0041] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0042] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0043] Example 1 Preparation of Gardenia Carbon Nanocomposite Material

[0044] The preparation flow chart of Gardenia charcoal nanocomposites is as follows: Figure 1 .

[0045] 1. Preparation of Gardenia Charcoal

[0046] The sample preparation refers to the 1988 edition of the "National Standards for the Preparation of Traditional Chinese Medicine". The temperature of the stir-frying machine is set to 300℃. After the temperature stabilizes, 500g of gardenia is added and the speed of the stir-frying machine is set to medium speed. Stir-fry for 12 minutes, take out the sample, and wait for the sample temperature to drop naturally. The charcoal is charred black and the inside and outside are consistent. It can be regarded as gardenia charcoal.

[0047] 2. Preparation of Gardenia Carbon Nanocomposites

[0048] Gardenia charcoal was pulverized and passed through a No. 4 sieve. 30.0 g of the charcoal powder was weighed and placed in a 1-liter beaker. 900 mL of deionized water (30 times the weight of the charcoal powder) was added and soaked for 15 minutes. The mixture was then heated to 100°C in a water bath and boiled for 1 hour to dissolve the active ingredients. The charcoal powder was removed by coarse filtration to obtain the initial decoction. The same extraction method was repeated three times. The extract was then filtered through a 0.22 μm microporous membrane to obtain the gardenia charcoal aqueous extract. The gardenia charcoal aqueous extract was concentrated using a rotary evaporator. The concentration of the solution was determined to be 1 g / mL based on the amount of charcoal powder. This yielded 30 mL of gardenia charcoal aqueous extract with a concentration of 1 g / mL.

[0049] 30 mL of the Gardenia charcoal aqueous extract was dialyzed against a 1000 Da dialysis membrane using deionized water in a 1 L beaker for 72 hours. During this process, the dialysis solution needed to be frequently changed to ensure that impurities within the dialysis bag were dialyzed away. Due to the permeability of the dialysis bag, the solution in the dialysis bag increased after dialysis. The resulting solution within the dialysis bag was heated and concentrated, then further quantified to a concentration of 1 g / mL based on the weight of the Gardenia charcoal drug. The Gardenia charcoal carbon dot solution was collected and some was stored at 4°C for future use. Others were freeze-dried in a vacuum freeze dryer to obtain a solid powder of Gardenia charcoal carbon dots (i.e., Gardenia charcoal nanocomposite materials).

[0050] The Tyndall effect is a commonly used physical method to distinguish and identify colloids and solutions. When light passes through a colloidal solution, since the diameter of the dispersed particles in the irradiated solution is generally between 1-100nm, which is smaller than the wavelength of the incident light, scattering occurs and a clear light beam can be seen, thus resulting in the Tyndall effect. Figure 2As shown, the synthesized Gardenia charcoal carbon dots solution is brown and exhibits a significant Tyndall effect. Irradiation of the colloidal solution by an infrared lamp produces a distinct red beam, indicating that the colloidal solution is nanoscale and has a particle size range of 1-100 nm. This demonstrates that the prepared Gardenia charcoal carbon dots are nanoscale.

[0051] 3. Characterization of Gardenia Carbon Nanomaterials

[0052] 3.1 Transmission electron microscopy (TEM)

[0053] The prepared Gardenia charcoal carbon dot solution was diluted to an appropriate concentration and ultrasonically dispersed for 30 minutes. The sample to be tested was filtered with a 0.22 μm microporous filter membrane. Finally, 10 μL was aspirated with a syringe and dropped onto a copper mesh several times, with the liquid falling as much as possible in the middle of the copper mesh to ensure that the solution was completely dry before testing. The sample was repeatedly dried naturally and its morphology, distribution and crystal structure were observed under high and low resolution transmission electron microscopy.

[0054] like Figure 3 As shown in Figure 2, the prepared Gardenia charcoal carbon dots are spherical in shape and well dispersed in aqueous solution. The particle size range is 1.44nm-3.92nm, the average particle size is 2.35nm±0.56nm, and the overall particle size distribution is uniform ( Figure 4 ). Figure 3 The inset in the upper left corner of (b) shows that the gardenia carbon dots have a clear graphite lattice structure and good crystallinity. The lattice fringe spacing is 0.21nm, corresponding to sp 2 (102) diffraction plane of graphitic carbon.

[0055] 3.2 Atomic force microscopy (AFM)

[0056] A uniformly dispersed gardenia charcoal fluorescent carbon dot solution was dropped onto a mica sheet, dried, and placed under an atomic force microscope. The tapping mode was used to scan and image, and the particle size and morphology of the carbon dots were recorded and analyzed.

[0057] Atomic force microscopy is an effective means of analyzing the surface conditions of materials. It can provide three-dimensional images of the surface and can also be used as an auxiliary means of particle size measurement. In order to more clearly understand the surface morphology and height distribution of carbon dots, atomic force microscopy (AFM) was used to characterize them. The characterization results are as follows: Figure 5 As shown. AFM plane image ( Figure 5 (a)) and three-dimensional stereo images ( Figure 5 (b) clearly shows the good dispersion of carbon dots, which is consistent with the morphology seen in transmission electron microscopy. Figure 5 (c) is the distribution of radial height of carbon dots along a certain direction. The radial height is mainly distributed below 4nm. The radial height distribution is statistically analyzed. Figure 5 Middle (d) shows that the particle size of carbon dots is mainly distributed in the range of 2.27±0.13nm, which is basically consistent with the transmission electron microscopy results.

[0058] 3.3 X-ray diffraction (XRD)

[0059] 15 μL of Gardenia charcoal carbon dot solution was drawn up with a microsyringe and dropped onto a clean silicon wafer. The measurement was performed using an Ultima IV and repeated several times. The scanning angle was set to 10-60° and the scanning speed was 2° / min.

[0060] Generally, XRD analysis technology can be used to determine the distribution of atoms or molecules in crystal atoms. Its principle is to use X-rays as probes to study the crystal structure and physical properties of materials. Each substance has a scattering effect on X-rays. During the X-ray diffraction process, the electrons inside the diffracting crystal are attracted and repelled by other atoms, which will produce mutually perpendicular anisotropic forces. The magnitude of this force depends on the distance between each crystal plane, thus affecting the diffraction intensity. Figure 6 As shown in the X-ray diffraction (XRD) pattern of Gardenia charcoal carbon dots, there is a broad diffraction peak at 2θ = 20.5°, which is usually amorphous carbon and organic materials. In addition, the peak at 2θ = 20.5° is consistent with the (002) crystal plane of carbon. This disordered structure is usually composed of sp 2 and sp 3 The hybridized carbon composition indicates that the carbon atoms are not completely arranged into ordered graphite crystals during the carbonization process.

[0061] 3.4 Ultraviolet spectroscopy (UV-vis)

[0062] The Gardenia charcoal fluorescent carbon dot solution was diluted with ultrapure water to an appropriate concentration, and then slowly injected into a clean quartz cuvette to ensure that no bubbles were generated in the cuvette during the injection process. A UV spectrophotometer was used to scan and test the position and intensity of the UV absorption peak of the Gardenia charcoal carbon dots in the wavelength range of 200-600 nm and the scanning speed of 400 nm / min.

[0063] UV spectrum Figure 7 It shows a broad absorption pattern that decreases from 200nm, without showing a clear peak, and a small absorption peak at 240-250nm. The weak absorption peak here is believed to be related to the absorption of the carbon core itself in the nano-type component, so it is speculated that it is mainly derived from the sp 2 The electron transitions of the hybridized carbon atoms also show a long absorption tail, up to 600 nm, which is due to the low-energy transitions within the surface states generated by its surface functional groups.

[0064] 3.5 Fluorescence spectroscopy (FL)

[0065] The excitation and emission spectra of CDs were collected using a fluorescence spectrophotometer. A 0.1 mg / mL solution of Gardenia charcoal carbon dots was prepared. The excitation wavelength was fixed (300 nm to 400 nm), and the fluorescence intensity at different emission wavelengths was measured to obtain the fluorescence emission spectrum. The emission wavelength was then fixed and the fluorescence excitation spectrum was obtained by scanning. The excitation and emission slit widths were 5 nm and 10 nm, respectively, and the scanning speed was 240 nm / min.

[0066] like Figure 8 As shown in Figure 2, within the excitation wavelength range of 300nm-400nm, the gardenia charcoal carbon dots exhibited excitation-dependent fluorescence, with strong fluorescence emission at 489nm. Subsequently, the emission wavelength was fixed at 489nm, and the fluorescence excitation spectrum of CDs was scanned. The optimal excitation wavelength of CDs was found to be 370nm ( Figure 9 ).

[0067] 3.6 Fourier Transform Infrared Spectroscopy (FTIR)

[0068] The infrared spectrum was determined using the potassium bromide tableting method. An appropriate amount of Gardenia charcoal carbon dot powder was added to potassium bromide at a ratio of 1:100 and ground evenly in a mortar. An appropriate amount of the ground powder was placed in a stainless steel mold and tableted using a hydraulic press. The infrared spectrometer was used for determination. The wavelength range was 400-4000 cm -1 , with a resolution of 4cm -1 , scan times 32 times, collect spectral data and analyze.

[0069] The composition of the surface functional groups of Gardenia jasminoides carbon dots was analyzed by FTIR spectroscopy. Figure 10 As shown, 3394.3cm -1 The broad peak centered at 2923.3 cm is attributed to O / NH stretching vibration; -1 The absorption peak at 2343.9 cm is attributed to the CH stretching vibration, which may be caused by the association of methyl or methylene with the aliphatic hydrocarbons present in the carbon dots; -1 The absorption peak at 1622.4 cm can be attributed to C≡C stretching vibration; -1 The absorption peak at 1398.1 cm is the characteristic absorption of C=O; -1 The absorption peak at 1252.3 cm is related to C≡N; -1 、1065.4cm -1 The absorption peak at 796.2 cm indicates that it is related to the CO bond. -1 The absorption peak at 628 cm can be attributed to CS stretching vibration; -1 The absorption peak at may be caused by NH deformation vibration.

[0070] 3.7 X-ray Photoelectron Spectroscopy (XPS)

[0071] X-ray diffraction photoelectron spectroscopy (XPS) was used to determine the elemental composition of the samples using both full and fine spectra. The excitation source was Al Kα (1486.6 eV), the operating voltage was 12 kV, and the binding energy was corrected using surface contamination C1s as the standard.

[0072] XPS was used to analyze the elemental composition and atomic percentage of the Gardenia jasminoides carbon dots. Figure 11 As shown in (a), the XPS full spectrum shows four typical peaks C1s, O1s, N1s and S2p, corresponding to the contents (atomic percentage) of the four elements carbon (C), oxygen (O), nitrogen (N) and sulfur (S) are 71.68%, 25.40%, 2.70% and 0.22% respectively. Based on the high-resolution spectrum, the convolution analysis of the four elements was carried out ( Figure 11 In (b)-(e), the high-resolution spectrum of C1s shows absorption peaks at 284.8eV (CC), 286.0eV (CO), and 285.5eV (C=O), and O1s has absorption peaks at 532.53eV and 530.9eV, corresponding to CO and C=O, respectively. The deconvolution results of N1s show 399.8eV (pyrrolic nitrogen) and 396.0eV (CN). S2p has two absorption peaks at 168.1eV and 164.0eV, corresponding to RSO3 - And C=S.

[0073] Example 2 Hemostatic effect of gardenia charcoal nanocomposite material

[0074] 1. Effects on hemostasis time and coagulation time in mice

[0075] Thirty ICR mice were randomly divided into a blank group, a Yunnan Baiyao group, and a Gardenia charcoal nanocomposite group, with 10 mice in each group, half male and half female. After three days of adaptive feeding, the blank group was gavaged with normal saline, while each treatment group was gavaged with the corresponding drug (dosage: 0.015g / g) once daily for seven consecutive days.

[0076] 1.1 Determination of hemostasis time in mice

[0077] One hour after the last dose, the hemostasis time of each group of mice was measured using the tail snip method. After the mice were anesthetized, the tails were snipped 5 mm away from the mice. The tails were observed for bleeding. The timer was started when the blood overflowed spontaneously. The filter paper was used to absorb the blood every 15 seconds until no more blood flowed out (no blood was found when the filter paper was absorbed). The timer was stopped, and the hemostasis time was calculated.

[0078] 1.2 Determination of mouse coagulation time

[0079] One hour after the last administration, the coagulation time of mice was measured by the capillary method. After the mice were anesthetized, blood was collected from the retroocular venous plexus of the mice using a capillary tube (10 cm long, 1 mm inner diameter). Timing began when the blood entered the capillary tube. The capillary tube was broken about 0.5 cm every 15 seconds and slowly pulled apart to observe whether there was a blood clot at the break. When a blood clot appeared, it was immediately broken at the other end for verification. Timing was stopped when blood clots appeared at both ends. This was the coagulation time.

[0080] Table 1 Hemostasis time and coagulation time of each group

[0081]

[0082] Note: Compared with the blank group, **p<0.01; compared with the Yunnan Baiyao group, #p<0.05, ##p<0.01.

[0083] The in vitro hemostatic and coagulation effects of the gardenia charcoal nanomaterial were observed using tail-snip hemostasis and capillary tube tests in mice. As shown in Table 1, compared with the blank group, the hemostatic and coagulation times of the gardenia charcoal nanocomposite group were reduced, preliminarily indicating that the gardenia charcoal nanocomposite has a certain effect in promoting hemostasis and coagulation. There were extremely significant differences between the Yunnan Baiyao and gardenia charcoal nanocomposite groups (p < 0.01). Compared with the Yunnan Baiyao group, the hemostatic and coagulation times of the gardenia charcoal nanocomposite group were also shortened, with an extremely significant difference in hemostasis time and a significant difference in coagulation time.

[0084] 2. Effect on blood-heat and gastric bleeding in rats

[0085] 2.1 Construction of blood-heat gastric bleeding model and drug treatment

[0086] Male Sprague-Dawley rats were acclimated for 5 days in an animal housing environment maintained at a temperature of 18-25°C and a relative humidity of 45%-75%. Rats with abnormal body temperatures (less than 36.5°C or greater than 38.5°C) or temperature fluctuations greater than 0.5°C between two consecutive measurements were excluded before grouping. The rats were randomly divided into six groups: a blank group, a model group, a Yunnan Baiyao group (positive drug group), a raw gardenia group, a gardenia charcoal group, and a gardenia charcoal nanocomposite (GFCCDs) group, with 10 rats in each group. All rats were fasted for 12 hours before modeling. After measuring basal body temperature, rats in all groups except the blank group received a subcutaneous injection of a 15% dry yeast suspension, while rats in the normal group received an equal volume of saline (1 mL / 100 g). Four hours after the fever model was established, temperature was measured and the first dose of drug was administered. Rats in the normal and model groups received an equal volume of saline. The raw gardenia group, gardenia charcoal group, and GFCCDs group received a dose of 4.6 g / kg (10 mL / kg). Rats in the positive drug group received a dose of 0.5 g / kg in a 10 mL / kg volume. When the rats' body temperature remained stable at a high temperature (i.e., 6 hours after induction of the fever model), all rats in the normal group were gavaged with anhydrous ethanol at a volume of 0.5 mL / 100 g to establish an acute gastric bleeding model. One hour after gavage with anhydrous ethanol, each group received a second dose of the drug. One hour after the second dose, the rats in each group were anesthetized with 10% chloral hydrate intraperitoneally. Blood was collected from the abdominal aorta and anticoagulated with 3.8% sodium citrate (sodium citrate to blood volume ratio of 1:9). Whole blood was centrifuged at 3000 rpm for 10 minutes to obtain plasma. Plasma exogenous coagulation function parameters (fibrinogen, prothrombin time, activated partial thromboplastin time, and thrombin time) were measured. The rats were sacrificed after blood was drawn, and the stomach tissues were removed by laparotomy, rinsed with physiological saline, and immersed in 10% formalin. The bleeding and damage of the gastric mucosa were observed with the naked eye and photographed.

[0087] The basal body temperature of the rats before modeling and the changes in rectal temperature of the rats 1 to 8 hours after modeling were recorded. The body temperature change value (△T) = body temperature at each time point - basal body temperature.

[0088] The TXB2 and 6-keto-PGF1a levels in serum were determined by enzyme-linked immunosorbent assay according to the kit requirements, and the data were used in the study of gardenia charcoal nanocomposites.

[0089] 2.2 Results

[0090] Table 2 shows the changes in rectal temperature of rats in each group from 1 to 8 hours after the fever model was established. The body temperature of the model group rats showed an upward trend after model establishment, reaching a maximum at 6 hours, 2.3°C higher than the basal body temperature at 0 hours. The rats were given oral administration at 4 hours, before the temperature peaked. One hour after administration (5 hours after model establishment), the temperature of the rats in the different Gardenia jasminoides treatment groups increased less significantly than that of the model group (p < 0.01, p < 0.05). This indicates that when administered before the peak temperature after model establishment, raw Gardenia jasminoides, Gardenia charcoal, and GFCCDs all reduced fever in the rats, with no significant differences among the treatment groups.

[0091] Table 2 Changes in rectal temperature at each time point compared to the initial rectal temperature of rats in each group (n=9)

[0092]

[0093] Note: Compared with the blank group, the model group ## p<0.01, ### p<0.001. *p<0.05, **p<0.01 compared with the model group.

[0094] The four coagulation parameters, TXB2, and 6-keto-PGF1a were used as efficacy indicators to compare the differences in hemostatic efficacy. The results showed (Table 3) that compared with the blank group, the PT of rats in the model group was significantly shortened (p<0.05), TT (p<0.05) and APTT were significantly prolonged (p<0.01), and the FIB level was significantly increased (p<0.01); compared with the model group, Yunnan Baiyao significantly shortened TT and APTT (p<0.05), significantly reduced the FIB level (p<0.01), and prolonged PT (p<0.05); compared with the model group, the Gardenia charcoal nanocomposite material significantly shortened TT and APTT (p<0.01, p<0.05), significantly reduced the amount of FIB (p<0.05), and prolonged PT (p<0.05), showing better effects than raw Gardenia or Gardenia charcoal.

[0095] Compared with the blank group, the model group showed increased serum TXB2 (p < 0.01) and decreased 6-keto-PGF1a (p < 0.05) in rats. Yunnan Baiyao significantly decreased TXB2 (p < 0.01) and increased 6-keto-PGF1a (p < 0.01) compared with the model group. The Gardenia charcoal nanocomposite had the strongest effect, outperforming Yunnan Baiyao, raw Gardenia, or Gardenia charcoal (Table 4). This suggests that the Gardenia charcoal nanocomposite possesses significant hemostatic efficacy.

[0096] Table 3 Effects of each group on the four coagulation parameters

[0097]

[0098] Note: Compared with the blank group, *p<0.05, **p<0.01; compared with the model group, #p<0.05, ##p<0.01.

[0099] Table 4 Effects of each group on TXB2 and 6-keto-PGF1a

[0100]

[0101] Note: Compared with the blank group, *p<0.05, **p<0.01; compared with the model group, #p<0.05, ##p<0.01.

[0102] like Figure 12 As shown, the gastric lining surface of rats in the normal group was normal, with an intact and shiny mucosal surface and no signs of bleeding, ulceration, or other lesions. Compared with the blank group, the gastric tissue surface of rats in the model group was dark red, with dilated capillaries and severe streak-like hemorrhages. Intragastric administration of Yunnan Baiyao produced a hemostatic effect on gastric bleeding lesions, inhibiting gastric mucosal bleeding lesions. In the raw gardenia group, the gastric lining surface was red and swollen, with localized streak-like hemorrhages. Treatment with gardenia charcoal and GFCCDs significantly improved gastric bleeding lesions. These results suggest that gardenia exhibits varying degrees of hemostatic effects on gastric bleeding in rats with blood-heat hemorrhage. Gardenia charcoal and GFCCDs effectively alleviated bleeding, demonstrating a significantly stronger improvement than raw gardenia.

[0103] Example 3 Effect of Gardenia Carbon Nanocomposite Material on Alleviating Gastric Mucosal Damage

[0104] 1. Sample preparation information

[0105] Gardenia charcoal carbon dot solution, the solvent is standard dilution water.

[0106] Positive control: Mesalazine enteric-coated tablets (hereinafter referred to as mesalazine), batch number L23260A, Losan Pharma GmbH, solvent is standard dilution water.

[0107] 2. Experimental Animals

[0108] Transgenic neutrophil green fluorescent zebrafish MPX strain zebrafish were raised in fish farming water at 28°C (water quality: 200 mg of instant sea salt was added to each 1 L of reverse osmosis water, conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / L CaCO3), and were bred and provided by the fish farming center of Zhejiang Huante Biotechnology Co., Ltd. The experimental animal use license number is: SYXK (Zhejiang) 2022-0004. The breeding and management complies with the requirements of the international AAALAC certification (certification number: 001458), and the IACUC ethics review number is: IACUC-2025-11933-01.

[0109] 3. Instruments, consumables and reagents

[0110] A dissecting microscope (SZX7, OLYMPUS, Japan); a CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); a precision electronic balance (CP214, OHAUS, USA); a motorized focus continuous zoom fluorescence microscope (AZ100, Nikon, Japan); a 6-well plate (Zhejiang Bellanbo Biotechnology Co., Ltd., China); an automatic sample rapid grinder (JXFSTPRP-24L, Shanghai Jingxin Laboratory Equipment Technology Department, China); and an ultrasonic cleaner (JP-010T, Shenzhen Jiemeng Cleaning Equipment Co., Ltd., China).

[0111] Methylcellulose (Batch No. C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China); TNBS (Batch No. 0000296416, Sigma, USA).

[0112] 4. Detection

[0113] 4.1 Maximum detectable concentration (MTC) determination

[0114] Transgenic neutrophil green fluorescent zebrafish (MPX) strains at 3 days post-fertilization (dpf) were randomly selected and placed in culture dishes. All groups, except the normal control group, were treated with TNBS in water to establish a zebrafish gastrointestinal mucosal injury model. After 2 days of treatment at 28°C, TNBS was removed and the zebrafish were distributed into 6-well plates, with 30 zebrafish treated per well (experimental group). Gardenia charcoal carbon dots were then treated with water at concentrations of 62.5, 125, 250, 500, and 1000 μg / mL, respectively. A normal control group and a model control group were also set up, with the volume per well being 3 mL. After a further 2 days of treatment at 28°C, the median toxicity (MTC) of the samples in the model zebrafish was measured.

[0115] Results: The MTC for the protective effect of Gardenia charcoal carbon dots on gastrointestinal mucosal injury was 500 μg / mL. The poorer condition compared to the model control group indicated that zebrafish were intolerant to this concentration and that this concentration was not suitable (Table 5).

[0116] Table 5: Concentrations for protecting gastrointestinal mucosal damage (n=30)

[0117]

[0118]

[0119] 4.2 Intestinal lumen area

[0120] 3-day-old transgenic neutrophil green fluorescent zebrafish (MPX) strain zebrafish were randomly selected and plated in culture dishes. All experimental groups, except the normal control group, were treated with TNBS in water to establish a zebrafish gastrointestinal mucosal injury model. After two days of treatment at 28°C, the TNBS was removed and the zebrafish were distributed into six-well plates, with 30 zebrafish treated in each well (experimental group). The Gardenia charcoal carbon dot group was treated with Gardenia charcoal carbon dots at concentrations of 125, 250, and 500 μg / mL, respectively. The positive control group was given mesalamine at a concentration of 500 μg / mL. A normal control group and a model control group were also established. The volume per well was 3 mL. After another two days of treatment at 28°C, 10 zebrafish were randomly selected from each group and photographed under a dissecting microscope. Images were saved and data were acquired using NIS-Elements D 3.20 advanced image processing software. The intestinal lumen area of ​​the zebrafish was analyzed statistically to evaluate the efficacy of the samples in protecting against gastrointestinal mucosal injury in zebrafish. Statistical analysis was performed using SPSS software, with p < 0.05 indicating statistically significant differences.

[0121] Results: Gardenia charcoal carbon dots have the effect of assisting in protecting gastrointestinal mucosal damage, specifically by reducing the intestinal lumen area (Table 6, Figure 13 、 Figure 14 ).

[0122] Table 6 Evaluation of the efficacy of protecting gastrointestinal mucosal damage (intestinal area) (n=10)

[0123]

[0124] Note: Compared with the model control group, *p<0.05, ***p<0.001.

[0125] 4.3 Intestinal neutrophil count

[0126] 3-day-old transgenic neutrophil-producing green fluorescent zebrafish (MPX) strain were randomly selected and plated in culture dishes. All experimental groups, except the normal control group, were treated with TNBS in water to establish a zebrafish gastrointestinal mucosal injury model. After two days of treatment at 28°C, the TNBS was removed and the zebrafish were distributed into six-well plates, with 30 zebrafish treated in each well (experimental group). The Gardenia charcoal carbon dot group was treated with Gardenia charcoal carbon dots at concentrations of 125, 250, and 500 μg / mL, respectively. The positive control group was treated with mesalamine at a concentration of 500 μg / mL. A normal control group and a model control group were also established. The volume per well was 3 mL. After another two days of treatment at 28°C, 10 zebrafish were randomly selected from each group and photographed under a fluorescence microscope. Images were saved and data were acquired using NIS-Elements D 3.20 advanced image processing software. The number of intestinal neutrophils in the zebrafish was analyzed, and statistical analysis of this indicator was used to evaluate the efficacy of the samples in protecting against gastrointestinal mucosal injury in zebrafish. Statistical analysis was performed using SPSS software, with p < 0.05 indicating statistically significant differences.

[0127] Results: Gardenia charcoal carbon dots have the effect of assisting in protecting gastrointestinal mucosal damage, which is specifically manifested by reducing the number of intestinal neutrophils (Table 7, Figure 15 and Figure 16 ).

[0128] Table 7 Evaluation of efficacy in protecting gastrointestinal mucosal damage (number of intestinal neutrophils) (n=10)

[0129]

[0130] Note: Compared with the model control group, *p<0.05, ***p<0.001.

[0131] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. Application of Gardenia charcoal nanocomposite material in the preparation of hemostatic agent for acute gastric bleeding, characterized in that: The gardenia charcoal nanocomposite material is prepared by pulverizing the gardenia charcoal, extracting with water, dialyzing, concentrating and drying.

2. The method for preparing the gardenia charcoal nanocomposite material as claimed in claim 1, characterized in that: The following steps are involved: The gardenia charcoal is pulverized to obtain charcoal powder, the charcoal powder is soaked in water, and heated for extraction to obtain a primary water decoction, and the primary water decoction is filtered to obtain a gardenia charcoal water extract; The gardenia charcoal water extract is dialyzed to obtain a dialysate, and the dialysate is heated, concentrated, and freeze-dried to obtain a gardenia charcoal carbon dot solid powder, which is the gardenia charcoal nanocomposite material.

3. The preparation method according to claim 2, wherein The carbon powder is passed through a No. 4 sieve; The mass ratio of the carbon powder to the water is 1:

30.

4. The preparation method according to claim 2, wherein The heating extraction was performed at a temperature of 100° C., for 1 h, and for 3 times.

5. The preparation method according to claim 2, wherein The dialysis was performed on a 1000Da dialysis membrane for 72 hours.

6. Use of the gardenia charcoal nanocomposite material as claimed in claim 1 in the preparation of drugs for promoting blood coagulation and / or hemostasis.

7. Use of the gardenia charcoal nanocomposite material as claimed in claim 1 in preparing a drug for alleviating gastrointestinal mucosal damage.

8. A hemostatic agent for acute gastric bleeding, characterized in that: The invention comprises the gardenia charcoal nanocomposite material as claimed in claim 1.

9. The hemostatic agent for acute gastric bleeding according to claim 8, characterized in that Also contains pharmaceutically acceptable excipients.

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