Dried toad skin charcoal hemostatic traditional Chinese medicine as well as preparation method and application thereof

Abstract

The invention relates to a dried toad skin charcoal hemostatic traditional Chinese medicine as well as a preparation method and application thereof. The preparation method comprises the following steps: grinding dried toad skin, and calcining the ground dried toad skin in a muffle furnace to obtain toad skin charcoal powder; adding distilled water of which the weight is 8-10 times that of the toad skin charcoal powder into the toad skin charcoal powder, decocting for 15-25 minutes, pouring out filtrate, adding distilled water of which the weight is the same as that of the toad skin charcoal powder again, decocting for 15-25 minutes, and concentrating the filtrate obtained by two times of decoction until the weight is 3-5 times that of the toad skin charcoal powder; s2, the concentrated solution obtained in the step S2 is filtered through a Buchner funnel and a cellulose acetate membrane in sequence and then dialyzed through a 1000 Dalton dialysis membrane for 70-75 h, the dialyzed solution is subjected to centrifugal separation to obtain a toad skin carbon dot (CB-CDs) solution, and the CB-CDs solution is freeze-dried to obtain the dried toad skin carbon hemostasis traditional Chinese medicine. The obtained dried toad skin charcoal hemostatic traditional Chinese medicine has good performance.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and particularly to a traditional Chinese medicine for hemostasis made from charred dried toad skin, its preparation method and application. Background Art

[0002] Traditional Chinese medicines in the form of char (referred to as charred traditional Chinese medicines) generally refer to traditional Chinese medicines that are stir-fried, burned or calcined into a charred state and then used. Charred traditional Chinese medicines are a type of traditional Chinese medicine with a wide range of pharmacological effects and are closely related to the therapeutic effects. However, there is no unified and complete standard for the preparation of charred traditional Chinese medicines in existing research, and it only stays at the control of subjective standards such as color and smell, which brings a lot of difficulties to the scientific research process of modern charred traditional Chinese medicines.

[0003] Dried toad skin is the skin of Bufo gargarizans Cantor or Bufo melanostictus Schneider of the family Bufonidae. It is a traditional Chinese medicine with a long history and remarkable efficacy, and is widely used in clinical practice with low cost.

[0004] Therefore, using dried toad skin as a raw material to prepare charred traditional Chinese medicine (i.e., charred dried toad skin) to provide an efficient and low-cost traditional Chinese medicine for hemostasis has broad application prospects. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a traditional Chinese medicine for hemostasis made from charred dried toad skin, its preparation method and application.

[0006] To solve the above problems, the technical solutions adopted by the present invention are as follows: On the one hand, a preparation method of a traditional Chinese medicine for hemostasis made from charred dried toad skin is provided, and the method includes: Step S1: Grind dried toad skin and then put it into a muffle furnace for calcination to obtain charred toad skin powder; Step S2: Add distilled water equivalent to 8 - 10 times its weight to the charred toad skin powder and decoct for 15 - 25 min, pour out the filtrate, then add the same weight of distilled water again and decoct for 15 - 25 min, and concentrate the filtrates obtained from the two decoctions to 3 - 5 times the weight of the charred toad skin powder; Step S3: Filter the concentrated solution obtained in Step S2 successively through a Buchner funnel and a cellulose acetate membrane, then dialyze through a 1000 - Dalton dialysis membrane for 70 - 75 h, and the dialyzed solution is centrifuged to obtain a solution of carbon dots from charred toad skin ( Cutis Bufonis carbon dots, CB - CDs) solution, and the CB - CDs solution is freeze - dried to obtain the traditional Chinese medicine for hemostasis made from charred dried toad skin.

[0007] As an implementation manner of the present invention, in Step S1, the dried toad skin is put into a muffle furnace and calcined at 300 - 400 °C for 2 - 4 h.

[0008] As an embodiment of the present invention, in step S2, distilled water equivalent to 9.5 times the weight of the powdered toad skin charcoal is added and decocted for 20 minutes. After pouring out the filtrate, the same mass of distilled water is added again and decocted for 20 minutes. The filtrates obtained from the two decoctions are concentrated to a weight equivalent to 4 times that of the powdered toad skin charcoal.

[0009] As an embodiment of the present invention, in step S2, the filtrates obtained from the two decoctions are fed into a rotary evaporator for rotary evaporation and concentration.

[0010] As an embodiment of the present invention, in step S3, the pore size of the cellulose acetate membrane is 0.22 μm.

[0011] Second, a dry toad skin charcoal hemostatic traditional Chinese medicine obtained by the preparation method of the dry toad skin charcoal hemostatic traditional Chinese medicine as described in the first aspect is provided.

[0012] Third, a dry toad skin charcoal hemostatic traditional Chinese medicine suspension is provided, which is obtained by dissolving the dry toad skin charcoal hemostatic traditional Chinese medicine as described in the second aspect in pure water.

[0013] As an embodiment of the present invention, the mass ratio of the dry toad skin charcoal hemostatic traditional Chinese medicine to pure water is 1:(3 - 10).

[0014] Fourth, an application of the dry toad skin charcoal hemostatic traditional Chinese medicine suspension as described in the third aspect in the field of hemostasis and blood coagulation is provided.

[0015] The beneficial effects produced by adopting the above technical solutions are as follows: The dry toad skin charcoal hemostatic traditional Chinese medicine provided by the present invention has only dry toad skin as the raw material, and its raw material source is simple; during preparation, only the dry toad skin needs to be calcined, concentrated, dialysis-separated and freeze-dried to obtain the product dry toad skin charcoal hemostatic traditional Chinese medicine. Its preparation method is simple, the operation is easy, and the reagents used are all non-toxic and harmless reagents, which are environmentally friendly and efficient, can be used for large-scale production, and have good industrialization prospects.

[0016] Therefore, the present invention provides a highly efficient and low-cost hemostatic traditional Chinese medicine with broad application prospects. Description of the Drawings

[0017] Figure 1 TEM images of CB-CDs obtained in Example 2 at different magnifications.

[0018] Figure 2 are the ultraviolet-visible absorption spectrum, X-ray diffraction and infrared spectrum of CB-CDs obtained in Example 2.

[0019] Figure 3 is the X-ray photoelectron spectrum of CB-CDs obtained in Example 2.

[0020] Figure 4 It is the X-ray photoelectron spectrum of the CB-CDs obtained in Example 2. Among them, (A) is the C 1s spectrum, (B) is the O 1s spectrum, (C) is the N 1s spectrum, and (D) is the S 1s spectrum.

[0021] Figure 5 It is the fluorescence spectrum of the CB-CDs obtained in Example 2.

[0022] Figure 6 It is a schematic diagram of the experimental results of the capillary glass tube coagulation experiment on mice provided by the present invention.

[0023] Figure 7 It is a schematic diagram of the experimental results of the recalcification experiment of rat plasma in vitro provided by the present invention.

[0024] Figure 8 It is a schematic diagram of the experimental results of the four coagulation tests on mice provided by the present invention.

[0025] Figure 9 It is a schematic diagram of the experimental results of the rat platelet data and platelet aggregation rate provided by the present invention.

[0026] Figure 10 It is a schematic diagram of the experimental results of the rat platelet function provided by the present invention.

[0027] Figure 11 It is a schematic diagram of the experimental results of the rat fibrinolytic system provided by the present invention. Detailed implementation manners

[0028] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be clearly and completely described below in conjunction with specific embodiments.

[0029] Example 1 A traditional Chinese medicine for hemostasis with dried toad skin carbon, and its preparation method includes: Step S1: Grind the dried toad skin and put it into a muffle furnace for calcination at 300 °C for 4 h to obtain toad skin carbon powder; Step S2: Add 1320 ml of distilled water to 165 g of toad skin carbon powder, decoct for 15 min and then pour out the filtrate. Add 1320 ml of distilled water again and decoct for 15 min. Send the filtrate obtained from the two decoctions into a rotary evaporator for rotary evaporation and concentration until it is concentrated to 495 g; Step S3: Filter the concentrated solution obtained in Step S2 successively through a Buchner funnel and a cellulose acetate membrane, and then dialyze it through a 1000 Dalton dialysis membrane for 70 h. The dialyzed solution is centrifuged to obtain a CB-CDs solution; the CB-CDs solution is freeze-dried to obtain the traditional Chinese medicine for hemostasis with dried toad skin carbon.

[0030] Example 2 A Chinese medicine for hemostasis made from dried toad skin carbon, and its preparation method includes: Step S1: Grind the dried toad skin and put it into a muffle furnace to calcine at 350°C for 3 hours to obtain toad skin carbon powder; Step S2: Add 1500 mL of distilled water to 165 g of toad skin carbon powder, decoct for 20 minutes and then pour out the filtrate. Add another 1500 mL of distilled water and decoct for 20 minutes. Send the 2180 mL of filtrate obtained from the two decoctions into a rotary evaporator for rotary evaporation and concentration until it is concentrated to 660 g; Step S3: Filter the concentrated solution obtained in Step S2 successively through a Buchner funnel and a cellulose acetate membrane, then dialyze it through a 1000 Dalton dialysis membrane for 72 hours. The dialyzed solution is centrifuged to obtain a CB-CDs solution; the CB-CDs solution is freeze-dried to obtain the Chinese medicine for hemostasis made from dried toad skin carbon.

[0031] Example 3 A Chinese medicine for hemostasis made from dried toad skin carbon, and its preparation method includes: Step S1: Grind the dried toad skin and put it into a muffle furnace to calcine at 400°C for 2 hours to obtain toad skin carbon powder; Step S2: Add 1650 mL of distilled water to 165 g of toad skin carbon powder, decoct for 25 minutes and then pour out the filtrate. Add another 1650 mL of distilled water and decoct for 25 minutes. Send the filtrate obtained from the two decoctions into a rotary evaporator for rotary evaporation and concentration until it is concentrated to 825 g; Step S3: Filter the concentrated solution obtained in Step S2 successively through a Buchner funnel and a cellulose acetate membrane, then dialyze it through a 1000 Dalton dialysis membrane for 75 hours. The dialyzed solution is centrifuged to obtain a CB-CDs solution; the CB-CDs solution is freeze-dried to obtain the Chinese medicine for hemostasis made from dried toad skin carbon.

[0032] Comparative Example 1 A Chinese medicine for hemostasis made from dried toad skin carbon, and its preparation method includes: Step S1: Grind the dried toad skin and put it into a muffle furnace to calcine at 350°C for 3 hours to obtain toad skin carbon powder; Step S2: Disperse 165 g of toad skin carbon powder in 1500 ml of distilled water. The obtained solution is successively filtered through a Buchner funnel and a cellulose acetate membrane, then dialyzed through a 1000 Dalton dialysis membrane for 72 hours. The dialyzed solution is centrifuged to obtain a CB-CDs solution; the CB-CDs solution is freeze-dried to obtain the Chinese medicine for hemostasis made from dried toad skin carbon.

[0033] Comparative Example 2 A Chinese medicine for hemostasis made from dried toad skin carbon, and its preparation method includes: Step S1: Grind the dried toad skin and put it into a muffle furnace to calcine at 350°C for 3 hours to obtain toad skin carbon powder; Step S2: Add 1200 mL of distilled water to 165 g of powdered charred toad skin, boil for 20 min, then pour out the filtrate. Add another 1200 mL of distilled water and boil for 20 min. Transfer the 2180 mL of filtrate obtained from the two boilings into a rotary evaporator for rotary evaporation and concentration until it reaches 660 g; Step S3: Filter the concentrated solution obtained in Step S2 successively through a Buchner funnel and a cellulose acetate membrane, then dialyze it through a 1000 Dalton dialysis membrane for 72 h. The dialyzed solution is centrifuged to obtain the CB-CDs solution; The CB-CDs solution is freeze-dried to obtain the hemostatic traditional Chinese medicine of charred toad skin.

[0034] Comparative Example 3 A hemostatic traditional Chinese medicine of charred toad skin, its preparation method includes: Step S1: Grind dried toad skin and put it into a muffle furnace to calcine at 350 °C for 3 h to obtain powdered charred toad skin; Step S2: Add 1500 mL of distilled water to 165 g of powdered charred toad skin, boil for 10 min, then pour out the filtrate. Add the same weight of distilled water again and boil for 30 min. Transfer the filtrate obtained from the two boilings into a rotary evaporator for rotary evaporation and concentration until it reaches 660 g; Step S3: Filter the concentrated solution obtained in Step S2 successively through a Buchner funnel and a cellulose acetate membrane, then dialyze it through a 1000 Dalton dialysis membrane for 72 h. The dialyzed solution is centrifuged to obtain the CB-CDs solution; The CB-CDs solution is freeze-dried to obtain the hemostatic traditional Chinese medicine of charred toad skin.

[0035] Comparative Example 4 A hemostatic traditional Chinese medicine of charred toad skin, its preparation method includes: Step S1: Grind dried toad skin and put it into a muffle furnace to calcine at 350 °C for 3 h to obtain powdered charred toad skin; Step S2: Add 1500 mL of distilled water to 165 g of powdered charred toad skin, boil for 20 min, then pour out the filtrate. Add another 1�00 mL of distilled water and boil for 20 min. Transfer the 2180 mL of filtrate obtained from the two boilings into a rotary evaporator for rotary evaporation and concentration until it reaches 660 g; Step S3: Filter the concentrated solution obtained in Step S2 successively through a Buchner funnel and a cellulose acetate membrane, then dialyze it through a 1000 Dalton dialysis membrane for 72 h. The dialyzed solution is centrifuged to obtain the CB-CDs solution; Step S4: Spin-dry the CB-CDs solution to obtain the hemostatic traditional Chinese medicine of charred toad skin.

[0036] Test Example: Performance Characterization The equipment used in this test example is as follows: Iecai G220 electron transmission microscope (FEI Company, USA); JEN-1230 high-resolution electron transmission microscope (JEOL, Japan); F-4500 fluorescence spectrophotometer (Hitachi, Japan); ultraviolet-visible spectrophotometer (CECIL, UK); Fourier transform infrared spectrometer (Thermo, USA); X-ray photoelectron spectrometer (Thermg, USA); X-ray diffractometer (Bruker, Germany).

[0037] 1. Electron microscopy characterization The CB-CDs solution obtained in Example 2 was diluted and filtered through a microporous membrane, and a copper mesh was used as the observation carrier. An aqueous sample solution was taken with a micro syringe and dropped onto the copper mesh several times and air-dried. Finally, the dispersion and morphology of the sample were observed under a transmission electron microscope (TEM), and the figures at different magnifications are as Figure 1 shown.

[0038] It can be Figure 1 seen that CB-CDs are spherical-like, have good water solubility and good dispersibility, have a crystal structure, with sizes between 2 and 5 nm, and an average particle size of 4.2 ± 0.6 nm.

[0039] The obtained CB-CDs solutions all have obvious bright and dark stripes, and the distance between the bright and dark stripes is the interplanar spacing, and the lattice spacing is about 0.22 nm, corresponding to the (100) crystal plane of carbon.

[0040] Counting more than 300 individual particles provided size distribution data, showing that the CDs are mainly around 6 nm, with an average size of 6.3 nm.

[0041] 2. Characterization by ultraviolet-visible spectrophotometer (UV-Vis), X-ray diffractometer (XRD) and infrared spectrometer (FTIR) The CB-CDs solution obtained in Example 2 was dried, ground into fine powder, dissolved in deionized water, and the ultraviolet-visible absorption spectrum of the sample was measured using an ultraviolet-visible spectrophotometer (UV-Vis), as Figure 2 (A) shown.

[0042] The CB-CDs solution obtained in Example 2 was dried, ground into fine powder, spread evenly on a clean silicon wafer, and analyzed in an X-ray diffractometer (XRD) to perform a structural analysis of the spatial distribution of atoms inside the sample, as Figure 2 (B) shown.

[0043] The CB-CDs solution obtained in Example 2 was dried and ground into a fine powder. The sample powder was mixed evenly with potassium bromide powder stored in a drying oven under an infrared lamp, pressed into a tablet, and scanned with a Fourier transform infrared spectrometer (FTIR) to analyze the surface group distribution of the sample. Figure 2 (C) shown.

[0044] Depend on Figure 2 (A) As can be seen, this shows a clear absorption peak at 322 nm related to the NH bond. The UV spectrum of benzo(α)pyrene has a unique absorption peak at 294 nm, which is in sharp contrast to the HFC CD absorption spectrum, proving that benzo(α)pyrene is not in the prepared HFC CD solution; Figure 2 (B) It can be seen that the X-ray diffraction pattern shows a high-intensity diffraction peak at about 26°, which is consistent with the lattice spacing observed by electron microscopy. Figure 2 (C) It can be seen that OH and NH groups appear at 3450 cm -1 and the CH group appears at 2962 cm -1 and 2826 cm -1 At 1680 cm -1 The peak at 1600 cm was identified as the C=N group, while the peak of the C=O bond appeared at 1600 cm -1 1130 cm -1 and 1050 cm -1 The peak at 1300 cm is attributed to the CO bond, while the peak at 1300 cm -1 and 400 cm -1 The peaks at 1200 cm indicate that these CDs contain some sp2 and sp3 hybridized carbon. -1 The peak at was identified as a CN group.

[0045] Therefore, CB-CDs are mainly composed of sp2 hybridized graphitic carbon with many hydroxyl and carbonyl groups and sp3 carbon on its surface.

[0046] 2. X-ray photoelectron spectrometer (XPS) spectrum characterization The surface elemental composition and functional groups of the CB-CDs obtained in Example 2 were characterized by X-ray photoelectron spectroscopy (XPS). Figure 3 As shown. Figure 4As shown in A - D, significant peaks appear in the high - resolution C1s spectrum at 284.6 eV, 285.6 eV, and 286.3 eV, corresponding to the presence of C = C / C–C, C–N, and C = O / O = C - O groups respectively. Two peaks appear in the O1s spectrum at approximately 532 eV and 530.6 eV, which are related to C–O and C = O groups respectively. In addition, a peak appears in the N1s spectrum at 399.3 eV, corresponding to the C–N bond. The splitting of the S2p peak is deconvoluted to obtain a doublet structure, where the energy difference between the S2p3 / 2 and S2p1 / 2 components is fixed at 1.2 eV and the area ratio is 2:1. The XPS spectrum results indicate that functional groups such as hydroxyl, amino, and carbonyl groups exist on the surface of CB - CDs, which is consistent with the surface groups confirmed by FT - IR spectroscopy.

[0047] 3. Fluorescence Spectrum Analysis Use fluorescence spectrophotometry (FL) to detect whether CB - CDs have fluorescence and analyze their fluorescence properties, namely emission / excitation wavelengths and maximum emission / excitation wavelengths. The specific operation is as follows: After diluting the CB - CDs obtained in Example 2 with deionized water, the spectral scanning wave range is set between 300 nm - 600 nm, and the test is carried out at intervals of 20 nm. The results obtained are as Figure 5 shown.

[0048] As Figure 5 can be seen, its maximum excitation wavelength is 350 nm and the maximum emission wavelength is 460 nm.

[0049] Experimental Example 1: Study on Bleeding Time and Coagulation Time of Mice Establishing a stable and suitable bleeding model is one of the important steps in the study of hemorrhagic diseases and is also a prerequisite for the evaluation and screening of hemostatic drugs. To prove the hemostatic effect of charcoal - processed toad skin, the test examples of the present invention combine common hemostatic models and use the mouse tail - cutting bleeding model and the mouse liver bleeding model to measure the bleeding time of mice in different groups respectively, and use the mouse capillary glass tube coagulation experiment to study the coagulation time of different groups when charcoal - processed toad skin exerts its hemostatic effect.

[0050] (I). Study on Bleeding Time of Mice 1. Experimental Materials (1) Experimental Animals Healthy Kunming mice, SPF - grade, half male and half female, with a body weight of (25 ± 2) g. Under the conditions of temperature (25.0 ± 1.0)°C and relative humidity of 50% - 60%, all experimental animals are adaptively raised, with free access to drinking water and food, and food intake is prohibited within 12 hours before the start of the experiment, but drinking water is not restricted.

[0051] (2) Experimental Reagents The dry toad skin carbon hemostatic traditional Chinese medicine obtained from Example 1 - Example 3, and Comparative Example 1 - Comparative Example 4, Yunnan Baiyao (Yunnan Baiyao Group Co., Ltd.), chloral hydrate (Shaanxi Panlong Yihai Pharmaceutical Co., Ltd.).

[0052] 2. Experimental grouping and administration method Dosage calculation: For an adult with a body weight of 60 kg, the appropriate dose is 6 g of toad skin carbon powder per day. For mice, it is 10 times the adult dosage. So for mice, the appropriate toad skin carbon dose is 60 g / 60 kg, that is, 1 g / kg. The body weight of a mouse is approximately 25 g, so the administration is approximately 25 mg per day.

[0053] In Example 2, finally 44.4 g of dry toad skin carbon hemostatic traditional Chinese medicine was obtained, and when converted, it is 6.8 mg of dry toad skin carbon hemostatic traditional Chinese medicine per day.

[0054] Therefore, the experimental mice were divided into nine groups, with ten mice in each group. The body weight of the mice is approximately 25 g. For the mice in Example 1 group, Example 3 group, and Comparative Example 1 group - Comparative Example 3 group, 272 mg of dry toad skin carbon hemostatic traditional Chinese medicine was dissolved in 10 ml of water, and 0.5 ml was given by gavage each time. Each mouse in each group was gavaged once a day for 7 consecutive days; Example 2 was divided into a low - dose group, a medium - dose group, and a high - dose group. For the mice in the low - dose group of Example 2, 68 mg of dry toad skin carbon hemostatic traditional Chinese medicine was dissolved in 10 ml of water every day, and 0.5 ml was given by gavage each time; for the mice in the medium - dose group of Example 2, 136 mg of dry toad skin carbon hemostatic traditional Chinese medicine was dissolved in 10 ml of water; for the mice in the high - dose group of Example 2, 272 mg of dry toad skin carbon hemostatic traditional Chinese medicine was dissolved in 10 ml of water, and 0.5 ml was given by gavage each time. Each mouse in each group was gavaged once a day for 7 consecutive days.

[0055] The Yunnan Baiyao group was used as the positive control group: 66 mg of Yunnan Baiyao was taken for mice and dissolved in 5 ml of water every day. The administration volume was 0.5 mL per mouse, and it was gavaged once a day for 7 consecutive days. (The administration was approximately 0.264 mg / g) (Calculated based on Yunnan Baiyao at 0.25 g per time, 4 times a day, for an adult with a body weight of 60 kg). The last group of mice was given an equal volume of distilled water for blank control.

[0056] 3. Experimental method (1) Mouse tail - cutting bleeding model One hour after gavaging on the seventh day for each group of mice, fix them with a fixator. After observing a decrease in the mice's autonomous movements, place the mouse's tail flat on the table, disinfect the mouse tail with 70% (v / v) ethanol. After the alcohol has evaporated, cut off the tip of the tail at 5 mm from the end of the mouse tail with a sterilized surgical blade, and immerse the tip of the tail 1.5 cm deep in the physiological saline solution at 37°C. Record the bleeding time of each group of mice.

[0057] (2) Mouse liver bleeding model After anesthetizing each group of mice by intraperitoneal injection of chloral hydrate, cut them open along the ventral midline of the mice with clean surgical scissors to expose the liver. On the premise of preserving the life of the mice, soak the gauze with normal saline and place it under the mouse liver to pad it up. Prick the mouse liver with a 5 ml injection needle to form a small wound, and dip it with a wet filter paper at the wound every half minute until no blood appears on the filter paper. At this time, it can be regarded as the bleeding has stopped. The time required from pricking the mouse liver to start bleeding to the mouse liver stopping bleeding is called the bleeding time, and record the bleeding time of each group of mice.

[0058] 4. Experimental results (1) Experimental results of mouse tail bleeding The results are shown in Table 1. Under the same experimental conditions, compared with the blank control group, the bleeding time of the mice in the other experimental examples was significantly shortened, and the bleeding time of the high-dose groups of mice in Example 1, Example 2 and Example 3 was significantly shorter than that of Yunnan Baiyao, indicating that the Chinese medicine of dried toad skin carbon has good hemostatic effect.

[0059] Table 1 Experimental results of mouse tail bleeding (2) Experimental results of mouse liver bleeding The results are shown in Table 2. In the mouse liver bleeding experiment, under the same experimental conditions, compared with the blank control group, the bleeding time of the mice in the other experimental examples was significantly shortened, and the bleeding time of the high-dose groups of mice in Example 1, Example 2 and Example 3 was not much different from that of Yunnan Baiyao, indicating that the Chinese medicine of dried toad skin carbon has good hemostatic effect.

[0060] Table 2 Experimental results of mouse liver bleeding (II) Research on mouse clotting time 1. Experimental materials (1) Experimental animals Healthy Kunming mice, SPF grade, half male and half female, with a body weight of (25 ± 2) g. Under the conditions of temperature (25.0 ± 1.0) °C and relative humidity of 50% - 60%, all experimental animals were adaptively raised, with free access to drinking water and food, and fasting was prohibited within 12 hours before the start of the experiment, but drinking water was allowed.

[0061] (2) Experimental reagents The Chinese medicine of dried toad skin carbon obtained in Example 2, Yunnan Baiyao (Yunnan Baiyao Group Co., Ltd.), 2.775 g / L CaCl2 solution (Shandong Shangshan Chemical Co., Ltd.).

[0062] (3)Experimental instruments SYSMEX CA-510 fully automatic blood coagulation analyzer (Beijing Pengkun Boyuan Science and Trade Development Co., Ltd.), TDL80-2B tabletop centrifuge (Shanghai Lingyi Biotechnology Co., Ltd.).

[0063] 2. Experimental grouping and administration method Dosage calculation: For an adult with a body weight of 60 kg, the appropriate dose is 6 g of dried toad skin charcoal powder per day. For mice, it is 10 times the adult dosage. So for mice, the appropriate dose of dried toad skin charcoal is 60 g / 60 kg, that is, 1 g / kg. The body weight of a mouse is about 25 g, so the administration is about 25 mg per day.

[0064] In Example 2, finally 44.4 g of dried toad skin charcoal hemostatic traditional Chinese medicine was obtained, and when converted, it is 6.8 mg of dried toad skin charcoal hemostatic traditional Chinese medicine per day.

[0065] Therefore, the experimental mice were divided into five groups, with ten mice in each group. The body weight of the mice is about 25 g. Example 2 was divided into a low-dose group, a medium-dose group, and a high-dose group. For the mice in the low-dose group of Example 2, 68 mg of dried toad skin charcoal hemostatic traditional Chinese medicine was dissolved in 10 ml of water every day, and 0.5 ml was gavaged each time; for the mice in the medium-dose group of Example 2, 136 mg of dried toad skin charcoal hemostatic traditional Chinese medicine was dissolved in 10 ml of water; for the mice in the high-dose group of Example 2, 272 mg of dried toad skin charcoal hemostatic traditional Chinese medicine was dissolved in 10 ml of water, and 0.5 ml was gavaged each time. Each mouse in each group was gavaged once a day for 7 consecutive days.

[0066] The Yunnan Baiyao group was used as the positive control group: Each day, 66 mg of Yunnan Baiyao was taken and dissolved in 5 ml of water for the mice. The administration volume was 0.5 mL per mouse, and it was gavaged once a day for 7 consecutive days. (The administration was about 0.264 mg / g) (Yunnan Baiyao was calculated based on 0.25 g per time, 4 times a day, for an adult with a body weight of 60 kg). The last group of mice was given an equal volume of distilled water for blank control.

[0067] 3. Experimental method (1)Capillary glass tube coagulation experiment For the coagulation experiment, blood was taken from the posterior orbital venous plexus of mice in each group using a fine glass tube with an inner diameter of 1 mm and a length of 10 cm. When the blood filled the glass tube, timing started. Then, the two ends of the capillary glass tube (about 0.5 cm) were broken every 30 seconds and slowly pulled apart to the left and right to determine whether there were blood coagulation filaments at the broken part. The time elapsed until the blood filaments appeared was the coagulation time. The coagulation times of the mice in each group were recorded.

[0068] (2)Statistical analysis method Data were expressed as mean ± standard deviation (SD). Data analysis was performed using GraphPad Prism software. For comparison between groups, one-way ANOVA followed by Dunnett′s test was used. When P < 0.05, it indicated that there were statistical differences in the result data. When P < 0.01, it indicated that there were significant statistical differences in the result data.

[0069] 4. Experimental results The results are shown in Table 3 and Figure 6 as shown in Table 3 and Figure 6 It can be seen that compared with the blank control group and the positive control group, the capillary glass tube clotting time in the high-dose group and the low-dose group was significantly shortened. And there was a statistical difference between the blank control group and the high-dose group (p < 0.05).

[0070] Table 3 Results of capillary glass tube clotting experiment in mice Experimental Example 2: Study on the recalcification time of rat plasma in vitro In this experimental example, the recalcification time of rat plasma in vitro (PRT) was used to verify the blood coagulation effect of the traditional Chinese medicine of dried toad skin carbon for hemostasis.

[0071] 1. Experimental materials (1) Experimental animals Healthy male SD rats, weighing (200 ± 2) g. Under the conditions of temperature (25.0 ± 1.0) °C and relative humidity of 50% - 60%, all experimental animals were adaptively raised, with free access to drinking water and food, and food intake was prohibited within 12 hours before the start of the experiment, while drinking water was allowed.

[0072] (2) Experimental reagents Traditional Chinese medicine of dried toad skin carbon for hemostasis, Yunnan Baiyao (Yunnan Baiyao Group Co., Ltd.), chloral hydrate (Shaanxi Panlong Yihai Pharmaceutical Co., Ltd.), 2.775 g / L CaCI2 solution (Shandong Shangshan Chemical Co., Ltd.).

[0073] (3) Experimental instruments SYSMEX CA-510 fully automatic coagulation analyzer (Beijing Pengkun Boyuan Science and Trade Development Co., Ltd.), TDL80-2B tabletop centrifuge (Shanghai Lingyi Biotechnology Co., Ltd.).

[0074] 2. Experimental grouping and administration method Dosage calculation: For an adult with a body weight of 60 kg, the appropriate dose is 6 g of toad skin carbon powder per day. For the high-dose group, the dose for rats is 12 times that of adults. Therefore, the appropriate dose of toad skin carbon for rats is 72 g / 60 kg, that is, 1.2 g / kg.

[0075] The experimental rats were divided into three groups, with 10 rats in each group. The three groups were: the high-dose group of Example 2, the positive control group, and the blank control group.

[0076] For the high-dose group of Example 2, cardiac blood of the rats was taken for treatment. 0.1 mL of plasma was added to 0.1 mL of the solution of the traditional Chinese medicine of dried toad skin charcoal for hemostasis, corresponding to 2 g of toad skin charcoal powder per kg of rats. Then, the time for fibrin filament formation was recorded. For the positive control group, cardiac blood of the rats was taken for treatment. 0.1 mL of plasma was added to 0.1 mL of the aqueous solution of Yunnan Baiyao at a concentration of 20 mg / L. Then, the time for fibrin filament formation was recorded. For the blank group of rats, cardiac blood of the rats was taken for treatment. 0.1 mL of plasma was added to 0.1 mL of normal saline. Then, the time for fibrin filament formation was recorded.

[0077] 3. Experimental methods Blood was taken from the hearts of the rats and anticoagulated with 3.8% sodium citrate in a ratio of 1:9 (blood: anticoagulant = 9:1). After mixing, it was centrifuged at 1500 r / min for 10 min using a tabletop centrifuge, and the blood collection tube was taken out to separate the plasma (upper layer). Next, the experiment was carried out at 37 °C. 0.1 mL of plasma was added to a 1.5 mL centrifuge tube, and then 0.1 mL of normal saline, the aqueous solution of Yunnan Baiyao, and the solution of the high-dose dried toad skin charcoal hemostatic traditional Chinese medicine of Example 2 were added respectively. After incubation for 1 min each, 0.1 mL of 2.775 g / L CaCl2 solution was added, and after mixing, timing started. Every 15 s, it was picked once with a toothpick to observe whether fibrin filaments were formed. Until fibrin filaments were formed, the required time was the PRT, and the time of each group was recorded.

[0078] (5) Statistical analysis method The data were expressed as mean ± standard deviation (SD). Data analysis was performed using GraphPad Prism software. For between-group comparison, one-way ANOVA followed by Dunnett's test was used. When P < 0.05, it indicated that the result data statistics were different, and when P <  0.01, it indicated that the result data statistics had significant differences.

[0079] 4. Experimental results The results are shown in Table 4 and Figure 7 As shown, compared with the blank group, the plasma recalcification time in the high-dose group of Example 2 was significantly reduced, and there was a statistical difference between the high-dose group of Example 2 and the blank group (p < 0.05).

[0080] Table 4 Experimental results of plasma recalcification time in rats in vitro Experimental Example 3: Study on the Hemostatic Mechanism This experimental example conducts a study on the mechanism of action of the traditional Chinese medicine of dried toad skin charcoal for hemostasis. It is known that the normal hemostatic function of the animal body is related to the blood vessel wall, platelets, coagulation system, and anticoagulation and fibrinolytic systems. The following determinations are carried out in this example: four coagulation tests, and the detection indicators mainly include fibrinogen (FIB), prothrombin time (PT), thrombin time (TT), and activated partial thromboplastin time (APTT), determination of platelet count, aggregation rate and function, and determination of fibrinolytic system function, so as to study the hemostatic mechanism of the traditional Chinese medicine of dried toad skin charcoal for hemostasis and clarify its coagulation factors.

[0081] 1. Experimental Materials (1)Experimental Animals Healthy Kunming mice, SPF grade, half male and half female, with a body weight of (25 ± 2) g. Under the conditions of temperature (25.0 ± 1.0) °C and relative humidity of 50% - 60%, all experimental animals are adaptively raised, with free access to drinking water and food, and food intake is prohibited within 12 hours before the start of the experiment, but drinking water is not prohibited.

[0082] Healthy male SD rats, with a body weight of (200 ± 2) g. Under the conditions of temperature (25.0 ± 1.0) °C and relative humidity of 50% - 60%, all experimental animals are adaptively raised, with free access to drinking water and food, and food intake is prohibited within 12 hours before the start of the experiment, but drinking water is not prohibited.

[0083] (2)Experimental Reagents Traditional Chinese medicine of dried toad skin charcoal for hemostasis, Yunnan Baiyao (Yunnan Baiyao Group Co., Ltd.), chloral hydrate (Shaanxi Panlong Yihai Pharmaceutical Co., Ltd.), 2.775 g / L CaCl2 solution (Shandong Shangshan Chemical Industry Co., Ltd.).

[0084] (3)Experimental Instruments BM830 fully automatic blood cell analyzer (Jinan Oulabo Technology Co., Ltd.), AG800 platelet aggregometer (Shanghai Jumu Medical Devices Co., Ltd.), GL21M high-speed refrigerated centrifuge (Hunan Kaida Scientific Instruments Co., Ltd.), microplate reader (Shandong Santai Instruments Co., Ltd.).

[0085] 2. Experimental Grouping and Administration Methods (1)The body weight of the experimental mice is about 25 g, and they are divided into five groups, with 10 mice in each group. The five groups are: low-dose group of Example 2, medium-dose group of Example 2, high-dose group of Example 2, positive control group, and blank control group.

[0086] In Example 2, for the low-dose group of mice, 68 mg of the dried toad skin carbon hemostatic traditional Chinese medicine was dissolved in 10 ml of water every day, and 0.5 ml was administered by gavage each time; for the medium-dose group of mice in Example 2, 136 mg of the dried toad skin carbon hemostatic traditional Chinese medicine was dissolved in 10 ml of water; for the high-dose group of mice in Example 2, 272 mg of the dried toad skin carbon hemostatic traditional Chinese medicine was dissolved in 10 ml of water, and 0.5 ml was administered by gavage each time. Each mouse in each group was gavaged once a day for 7 consecutive days.

[0087] The Yunnan Baiyao group was used as the positive control group: 66 mg of Yunnan Baiyao was taken and dissolved in 5 ml of water for mice every day. The administration volume was 0.5 mL per mouse, and it was gavaged once a day for 7 consecutive days. (The administration amount was approximately 0.264 mg / g) (Yunnan Baiyao was calculated according to 0.25 g per time and 4 times a day for an adult with a body weight of 60 kg). The mice in the blank group were given an equal volume of distilled water (2 ml) for blank control.

[0088] (2) The body weight of the experimental rats was approximately 200 g, and they were divided into three groups with 10 rats in each group. The three groups were: the high-dose group of Example 2, the positive control group, and the blank control group.

[0089] For the rats in the high-dose group of Example 2, 600 mg of the dried toad skin carbon hemostatic traditional Chinese medicine was dissolved in 20 ml of distilled water, and 2 ml was administered by gavage to each rat each time for 7 consecutive days.

[0090] For the rats in the positive control group, 200 mg of Yunnan Baiyao was taken and dissolved in 20 ml of distilled water every day. The administration volume was 2 mL per rat, and it was gavaged once a day for 7 consecutive days. (Yunnan Baiyao was calculated according to 0.25 g per time and 4 times a day for an adult with a body weight of 60 kg).

[0091] The rats in the blank group were given an equal volume of distilled water (2 ml) for blank control.

[0092] 3. Experimental methods (1) Determination of four coagulation items This determination was carried out with mice as the research object. First, the mice in each group were fixed, the abdominal aorta was exposed and separated, and 1.8 ml of blood was taken with a disposable syringe and dripped into a centrifuge tube pre-filled with 200 uL of 3.8% sodium citrate, and slowly inverted up and down ten times to ensure sufficient anticoagulation. The collected blood sample was immediately centrifuged at 3000 r / min for 15 min: the plasma components after centrifugation were detected in an automatic blood coagulation analyzer, and then the contents of TT, PT, APTT, and FIB in the plasma of the mice in each group were measured in turn.

[0093] (2) Determination of platelet count and aggregation rate This determination was carried out with rats as the research object.

[0094] Platelet count detection method: Two hours after the last administration, rats in each group were intraperitoneally anesthetized with chloral hydrate, fixed, and the abdominal aorta was exposed and separated. 1-2 drops of blood were taken with a disposable syringe and injected into an anticoagulation tube pre-filled with CaCl2. The tube wall was gently flicked to fully mix the blood with the anticoagulant, which served as the sample for platelet count determination. The collected blood sample was detected on a fully automatic blood cell analyzer. Before measuring each sample, it was judged whether the blood sample showed agglutination. Standard operation was carried out according to the instrument instructions, and the platelet count (PLT) in each sample was detected. This determination was carried out within four hours.

[0095] Platelet aggregation rate detection method: First, blood and anticoagulant were added to an anticoagulation tube containing sodium citrate anticoagulant, and the ratio of blood to anticoagulant was 1:9. 3 ml of abdominal aortic blood was taken, slowly mixed, and then centrifuged at 100 g for 10 minutes at room temperature. Subsequently, the upper light yellow suspension was separated, which was the prepared platelet-rich plasma (PRP). After mixing the remaining sample, it was centrifuged at 1600 g for 5 minutes at room temperature to separate the platelet-poor plasma. According to the Born turbidimetry method, a turbidimetric tube containing 250 μL of PRP and a small magnetic bar was placed in a platelet aggregometer. At a temperature of 37 °C, it was incubated for 5 min. After calibration with PPP, while stirring, the inducer ADP (final concentration of 5.6 μg / mL) was added to induce aggregation. The maximum aggregation rate of platelets induced by ADP within 5 min was recorded. Maximum aggregation rate (%) = (OD of PRP after aggregation - OD of PRP before aggregation) / (OD of PPP - OD of PRP before aggregation) × 100%.

[0096] (3)Platelet function determination This determination used rats as the research object. Blood drops from the rat's eyeball were taken with a disposable syringe and injected into a pre-filled centrifuge tube containing 3.2% sodium citrate. During the blood collection process, it was continuously shaken to prevent blood agglutination. After standing at room temperature for ten minutes, the collected blood sample was centrifuged at 3000 r / min for 10 min, and the upper plasma was taken. The contents of platelet activating factors (TXB2 and 6-keto PGF1α) in the rat plasma were measured using a kit.

[0097] (4)Fibrinolytic system function determination This determination used rats as the research object. Blood drops from the rat's eyeball were taken with a disposable syringe and injected into a pre-filled centrifuge tube containing 3.2% sodium citrate. During the blood collection process, it was continuously shaken to prevent blood agglutination. After standing at room temperature for ten minutes, the collected blood sample was centrifuged at 3000 r / min for 10 min, and the upper plasma was taken. The contents of fibrinolytic factors (tPA and PAI) in the rat plasma were measured using a kit.

[0098] (5)Statistical analysis method Data are expressed as mean ± standard deviation (SD). Data analysis was performed using GraphPad Prism software. For comparison between groups, one-way ANOVA was used. When P < 0.05, it indicates that there are statistical differences in the result data. When P < 0.01, it indicates that there are significant statistical differences in the result data.

[0099] 4. Experimental Results (1)Effect of Dry Toad Skin Carbonized Hemostatic Chinese Medicine on Four Coagulation Parameters To further study the hemostatic mechanism of dry toad skin carbonized hemostatic Chinese medicine, we measured the four coagulation parameters of mice in each group. As shown in Table 5 and Figure 8 shown, there were basically no differences in PT among the groups, and there was only a statistical difference between the blank group and the low-dose group, indicating that dry toad skin carbonized hemostatic Chinese medicine does not play a hemostatic role through the exogenous hemostatic pathway. As can be seen from Figure 8 (B), the ATPP value of the positive control group was higher than that of the other groups, but there was no significant difference between the different dose groups and the blank control, indicating that the hemostatic mechanism of dry toad skin carbonized hemostatic Chinese medicine is not significantly related to the endogenous coagulation pathway. From Figure 8 (C), it can be found that there were no statistical differences in the data of each group and the changes were not obvious. TT reflects the time for fibrinogen to be converted into fibrin, representing the common coagulation pathway. The experimental results show that the hemostatic mechanism of dry toad skin carbonized hemostatic Chinese medicine is not closely related to the common coagulation pathway. As can be obtained from Figure 8 (D), compared with the normal group, the plasma FIB values in the high, medium, and low dose groups all increased significantly, and there was a statistical difference between the blank group and the high-dose group (p < 0.05). Thus, it can be seen that the hemostatic mechanism of dry toad skin carbonized hemostatic Chinese medicine is very likely related to the increase in the content of fibrinogen in plasma.

[0100] Table 5 Effect of Dry Toad Skin Carbonized Hemostatic Chinese Medicine on Four Coagulation Parameters (2)Effect of Dry Toad Skin Carbonized Hemostatic Chinese Medicine on Platelet Count and Platelet Aggregation Rate As shown in Table 6 and Figure 9 shown, there were no obvious differences in platelet count between the blank group, Yunnan Baiyao group, and the experimental group, and there was no statistical difference. As can be clearly seen from Figure 9 (B), compared with normal rats, the platelet aggregation rate in the Yunnan Baiyao group and the high-dose group of Example 2 increased significantly, and there was a significant statistical difference between the blank group and the high-dose group of Example 2 (p < 0.01), while there was no statistical difference between the high-dose group of Example 2 and the Yunnan Baiyao group. Therefore, we can speculate that one of the mechanisms by which dry toad skin carbonized hemostatic Chinese medicine plays a hemostatic role is the increase in platelet aggregation rate.

[0101] Table 6 Effect of Dry Toad Skin Carbonized Hemostatic Chinese Medicine on Platelet Count and Platelet Aggregation Rate (3) Effects of dried toad skin carbon hemostatic traditional Chinese medicine on platelet function As shown in Table 7 and Figure 10 it can be seen that platelet aggregation can be achieved by an increase in the content of activated factor TXB2 and a decrease in the content of 6-ketoPGF1α. As Figure 10 (A) shows, compared with the blank group, the content of TXB2 in the high-dose group of Example 2 is slightly decreased, indicating that the dried toad skin carbon hemostatic traditional Chinese medicine does not achieve platelet aggregation through the increase of TXB2. However, from Figure 10 (B), we can see that the contents of 6-ketoPGF1α in the high-dose group of Example 2 and the Yunnan Baiyao group are both significantly lower than those of normal rats, and there is a significant difference between the high-dose group of Example 2 and the normal group (p<0.01), and there is no statistical difference from the positive group. It can be seen that the dried toad skin carbon hemostatic traditional Chinese medicine promotes platelet aggregation and thus plays a coagulation role by reducing the content of 6-ketoPGF1α.

[0102] Table 7 Effects of dried toad skin carbon hemostatic traditional Chinese medicine on platelet function (4) Effects of dried toad skin carbon hemostatic traditional Chinese medicine on fibrinolytic system In plasma, tPA and PAI1 are an important pair of fibrinolytic factors, and the two maintain a relative balance to maintain the stability of the fibrinolytic system. As shown in Table 8 and Figure 11 it can be seen that the tPA value of the high-dose group of Example 2 and the Yunnan Baiyao group is slightly decreased compared with the blank control group, but there is no statistical difference. And from Figure 11 (B), it can be found that the contents of PAI1 in the plasma of rats in the experimental group and the positive control group are both significantly increased compared with the blank group, and there is a statistical difference between the high-dose group of Example 2 and the blank group (p<0.05), while there is no statistical difference from the positive drug group. Therefore, it can be judged that the dried toad skin carbon hemostatic traditional Chinese medicine is similar to Yunnan Baiyao, and affects the fibrinolytic system to play a hemostatic role by increasing the content of tissue-type plasminogen activator inhibitor in plasma to reduce the tPA / PAI1 ratio.

[0103] Table 8 Effects of dried toad skin carbon hemostatic traditional Chinese medicine on fibrinolytic system Therefore, through the experimental determination of the four coagulation factors, platelets, fibrinolytic factors, etc. in the plasma of animals in each group, the following conclusions are obtained: The dried toad skin carbon hemostatic traditional Chinese medicine may specifically play a hemostatic role by increasing the content of fibrinogen, reducing the content of 6-ketoPGF1α to promote platelet aggregation, increasing the content of tissue-type plasminogen activator inhibitor in plasma to reduce the tPA / PAI1 ratio, and affecting the fibrinolytic system and other ways.

[0104] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A preparation method of a traditional Chinese medicine for hemostasis with dried toad skin carbon, characterized in that, The method includes: Step S1: Grind dried toad skin and calcine it in a muffle furnace to obtain toad skin charcoal powder; Step S2: Add distilled water equivalent to 8 - 10 times its weight to the toad skin charcoal powder and decoct for 15 - 25 min. After pouring out the filtrate, add the same weight of distilled water again and decoct for 15 - 25 min. Concentrate the filtrates obtained from the two decoctions to 3 - 5 times the weight of the toad skin charcoal powder; Step S3: Filter the concentrated solution obtained in Step S2 successively through a Buchner funnel and a cellulose acetate membrane, then dialyze it through a 1000 - Dalton dialysis membrane for 70 - 75 h. The dialyzed solution is separated by centrifugation to obtain a toad skin carbon dots CB - CDs solution, and the CB - CDs solution is freeze - dried to obtain the dried toad skin charcoal hemostatic traditional Chinese medicine.

2. The preparation method of a traditional Chinese medicine for hemostasis with dried toad skin carbon as claimed in claim 1, wherein In Step S1, put the dried toad skin into the muffle furnace and calcine it at 300 - 400 °C for 2 - 4 h.

3. The preparation method of a traditional Chinese medicine for hemostasis with dried toad skin carbon as claimed in claim 1, wherein, In Step S2, add distilled water equivalent to 9.5 times its weight to the toad skin charcoal powder and decoct for 20 min. After pouring out the filtrate, add the same mass of distilled water again and decoct for 20 min. Concentrate the filtrates obtained from the two decoctions to be equivalent to 4 times the weight of the toad skin charcoal powder.

4. The preparation method of a traditional Chinese medicine for hemostasis with dried toad skin carbon as claimed in claim 1 or 3, characterized in that, In Step S2, the filtrates obtained from the two decoctions are fed into a rotary evaporator for rotary evaporation and concentration.

5. The preparation method of a traditional Chinese medicine for hemostasis with dried toad skin carbon as claimed in claim 1, wherein, In Step S3, the pore size of the cellulose acetate membrane is 0.22 μm.

6. The dried toad skin charcoal hemostatic traditional Chinese medicine obtained by the preparation method of the dried toad skin charcoal hemostatic traditional Chinese medicine according to any one of claims 1 - 5.

7. A suspension of traditional Chinese medicine for hemostasis with dried toad skin carbon, characterized in that, It is obtained by dissolving the dried toad skin charcoal hemostatic traditional Chinese medicine according to claim 6 in pure water.

8. A dry toad skin carbon hemostatic traditional Chinese medicine suspension according to claim 7, characterized in that, The mass ratio of the dried toad skin charcoal hemostatic traditional Chinese medicine to pure water is 1:(3 - 10).

9. The application of the dried toad skin charcoal hemostatic traditional Chinese medicine suspension according to claim 8 in the field of hemostasis and blood coagulation.

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