Use of dehydrovomifoliol for the preparation of a medicament for improving or promoting wound healing

Abstract

The application discloses application of dehydrodiols in preparation of drugs for improving or promoting wound healing, and belongs to the field of biological medicines. In the application, the improvement or promotion of wound healing includes increase of regenerated epidermis length, increase of collagen area, promotion of wound vascularization, increase of capillary density and / or increase of wound healing rate. Researches find that the dehydrodiols can effectively improve or promote wound healing, and have the advantages of simplicity, safety, reliability and high efficiency, and have wide application prospect in the field of wound healing.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to application of dehydrovomifoliol in preparation of a medicine for improving or promoting wound healing. BACKGROUND

[0002] Wound healing is a complex process involving stages of coagulation, inflammation, removal of damaged matrix components, and subsequent cell proliferation and migration, angiogenesis, matrix synthesis and deposition, re-epithelialization and remodeling. Wounds, such as chronic wounds, severely affect the quality of life and physical and mental health of patients.

[0003] The treatment methods for wounds mainly include traditional wound dressings, hydrogel wound dressings, antibacterial dressings, stem cell therapy, traditional drug transdermal drug delivery methods, etc. These methods still have problems such as poor curative effect, lack of corresponding biological function, difficulty in replacing wound dressings, inability to effectively protect wounds, easy to cause local oxidative stress of wounds, vascular injury and secondary infection, single biochemical function, inability to achieve precise delivery of active substances, long treatment cycle, etc.

[0004] Therefore, there is an urgent need for a simple, safe, reliable and effective medicine for promoting wound healing. SUMMARY

[0005] To solve at least part of the technical problems in the prior art, the present application provides application of dehydrovomifoliol in preparation of a medicine for improving or promoting wound healing. Specifically, the present application includes the following contents.

[0006] In a first aspect, the present application provides application of dehydrovomifoliol in preparation of a medicine for improving or promoting wound healing.

[0007] In some embodiments, the application of dehydrovomifoliol in preparation of a medicine for improving or promoting wound healing according to the first aspect of the present application, wherein the improvement or promotion of wound healing comprises at least one of the following:

[0008] (1) increasing the length of the regenerated epidermis;

[0009] (2) increasing the area of collagen;

[0010] (3) promoting wound vascularization;

[0011] (4) increasing the capillary density;

[0012] (5) increasing the content of alpha-SMA or CD31.

[0013] In some embodiments, the application of dehydrovomifoliol in preparation of a medicine for improving or promoting wound healing according to the first aspect of the present application, wherein the wound comprises a skin wound.

[0014] In some embodiments, the dehydrovomifoliol for use in improving or promoting wound healing according to the first aspect of the present application, wherein the wound comprises acute wound and chronic wound.

[0015] In some embodiments, the dehydrovomifoliol for use in improving or promoting wound healing according to the first aspect of the present application, wherein the acute wound comprises at least one of abrasion, laceration, incised wound, puncture wound and burn, and the chronic wound comprises diabetic wound.

[0016] In a second aspect of the present application, a method for preparing dehydrovomifoliol is provided, comprising the following steps:

[0017] (1) water extraction of Sedum sarmentosum Bunge to obtain a water extract of Sedum sarmentosum Bunge;

[0018] (2) extraction of the water extract of Sedum sarmentosum Bunge using an organic solvent to obtain an extract of Sedum sarmentosum Bunge;

[0019] (3) purification and separation of the extract of Sedum sarmentosum Bunge to obtain the dehydrovomifoliol.

[0020] In some embodiments, the method for preparing dehydrovomifoliol according to the second aspect of the present application, wherein in step (1), the ratio of material to liquid is 1:(5-50).

[0021] In some embodiments, the method for preparing dehydrovomifoliol according to the second aspect of the present application, wherein in step (1), the water extraction time is 0.5-5 h.

[0022] In some embodiments, the method for preparing dehydrovomifoliol according to the second aspect of the present application, wherein the organic solvent comprises at least one of petroleum ether, chloroform, ethyl acetate, n-butanol, ethanol, acetone and cyclohexane.

[0023] In a third aspect of the present application, a method for improving the content of alpha-SMA and / or CD31 of cells in vitro is provided, comprising the step of contacting the cells with dehydrovomifoliol.

[0024] The present application has found that dehydrovomifoliol can effectively improve or promote wound healing, and has the advantages of simplicity, safety, reliability and high efficiency, and has a broad application prospect in the field of wound healing, especially chronic wound healing. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The blood glucose changes of mice in the process of constructing an animal model are shown.

[0026] Figure 2 The effects of different Sedum sarmentosum Bunge extracts on wound healing in animals are shown.

[0027] Figure 3 The effects of different Sedum sarmentosum extracts on wound healing in animals were demonstrated.

[0028] Figure 4 The quantitative analysis results of different Sedum sarmentosum extracts on wound healing in animals are shown.

[0029] Figure 5 The quantitative analysis results of different Sedum sarmentosum extracts in cell experiments are shown. In the figure, A and B are cell viability and cell migration rate of HUVEC cells, respectively; C and D are cell migration rate and cell viability of HaCat cells.

[0030] Figure 6 The effects of different polar components on cell proliferation in cell experiments were demonstrated.

[0031] Figure 7 The results of high performance liquid chromatography analysis of the components in tubes 31-43 are shown.

[0032] Figure 8 The results of high-performance liquid chromatography analysis of the target product are shown.

[0033] Figure 9 The effects of the target product in cell experiments are shown, where A represents the experimental results in HUVEC cells; and B and C represent the experimental results in HaCat cells.

[0034] Figure 10 The target compound is shown. 1 H-NMR analysis results.

[0035] Figure 11 The target compound is shown. 13 C-NMR analysis results.

[0036] Figure 12 The effects of different doses of dehydroepiandrosterol on promoting wound healing in animals were shown, where A represents qualitative analysis results and B represents quantitative analysis results.

[0037] Figure 13 The results of a study on the mechanism of different doses of dehydroemetrol on wound healing in animals are shown. Among them, A is the result of immunohistochemical analysis; B is the effect of different doses of dehydroemetrol on the length of regenerated epidermis; C is the effect of different doses of dehydroemetrol on Col collagen; D is the effect of different doses of dehydroemetrol on the α-SMA vascular area; and E is the effect of different doses of dehydroemetrol on capillary density. Detailed Implementation

[0038] The following detailed description of various exemplary embodiments of the application will not be considered to be limiting of the application, but rather a description of certain aspects, features, and embodiments of the application.

[0039] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, where particular ranges of values are given, understand that each intervening value, to the upper or lower limit of the ranges is also specifically included. The upper and lower limits of these intervening ranges are also specifically included within the scope of the application. These smaller ranges are not insubstantial.

[0040] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict, the content of the present specification will control.

[0041] Applications

[0042] In one aspect of the application, there is provided a use of dehydrovomifoliol in the preparation of a medicament for improving or promoting wound healing. The dehydrovomifoliol can be obtained by chemical synthesis or plant extraction. Examples of the plant include, but are not limited to, Sedum sarmentosum, Premna microphylla, Dendrocalamus latiflorus, Engelhardtia roxburghiana, Matteuccia struthiopteris, Salacia przewalskii, Bidens pilosa, Artemisia vulgaris, and Eupatorium odoratum. The dehydrovomifoliol has a molecular weight of 222, a molecular formula of C 13 H 18 O3, and a chemical structure as shown in Formula I:

[0043] Formula I.

[0044] In a preferred embodiment, the improvement or promotion of wound healing includes, but is not limited to, increasing the length of regenerated epidermis; increasing the area of collagen; promoting wound vascularization; increasing capillary density; increasing the content of a-SMA or CD31; increasing the rate of wound healing, etc. The degree of improvement or promotion is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100%.

[0045] In the present application, the wound includes, but is not limited to, skin wound. The wound includes acute wound and chronic wound. The acute wound includes, but is not limited to, abrasion, laceration, incision, puncture, burn, etc. The chronic wound includes, but is not limited to, diabetic wound. In a preferred embodiment, the chronic wound is diabetic wound.

[0046] The drug of the present application can be prepared into various dosage forms acceptable in clinic, examples of which include, but are not limited to, solution, tincture, ointment, cream, gel, powder, powder, aerosol, spray, patch, transdermal patch, tablet, capsule, granule, suspension, emulsion, syrup, injection, etc. The administration mode of the drug is not particularly limited, and representative administration modes include, but are not limited to, topical administration, oral administration, injection, etc.

[0047] In the present application, the prevention, improvement or treatment is achieved by administering a therapeutically effective amount of the drug to a subject in need. The subject includes, but is not limited to, mammals, examples of which include, but are not limited to, human, mouse, rabbit, cat, dog, cow, sheep, pig, etc.

[0048] The therapeutically effective amount of the present application refers to the effective administration dose recognized in pharmacy, i.e. the amount of the drug of the present application is sufficient to significantly promote wound healing without causing serious side effects. The daily administration dose of the effective component (e.g. dehydrovomifoliol) of the drug of the present application is usually 0.01-500 mg, preferably 1-500 mg, more preferably 10-400 mg, further preferably 30-300 mg, more preferably 60-300 mg, such as 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg, 170 mg, 175 mg, 180 mg, 185 mg, 190 mg, 195 mg, 200 mg, 205 mg, 210 mg, 215 mg, 220 mg, 225 mg, 230 mg, 235 mg, 240 mg, 245 mg, 250 mg, 255 mg, 260 mg, 265 mg, 270 mg, 275 mg, 280 mg, 285 mg, 290 mg, 295 mg, 300 mg, or any dose within the above range. It can be administered as a single dose once a day, can be administered in multiple doses per day, or can be used intermittently.

[0049] Preparation method

[0050] In one aspect of the present application, a preparation method of dehydrovomifoliol is provided, which comprises the following steps:

[0051] (1) Water extraction is performed on the sedum sarmentosum to obtain a sedum sarmentosum water extract;

[0052] (2) Organic solvent is used to extract the sedum sarmentosum water extract to obtain a sedum sarmentosum extract, examples of the organic solvent include but are not limited to petroleum ether, chloroform, ethyl acetate, n-butanol, ethanol, acetone, cyclohexane, etc.;

[0053] (3) Purification and separation are performed on the sedum sarmentosum extract to obtain the dehydrovomifoliol.

[0054] In a preferred embodiment, the preparation method of the present application comprises the following steps:

[0055] (1) Water extraction is performed on the sedum sarmentosum at 50-100℃, preferably 55-100℃, more preferably 60-100℃, further preferably 65-100℃, more preferably 70-100℃, for example 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100℃, for 0.5-5 h, preferably 0.5-4.5 h, more preferably 0.5-4 h, further preferably 0.5-3.5 h, more preferably 1-3 h, for example 1, 1.5, 2, 2.5, 3 h, with a solid-liquid ratio of 1:(5-50), preferably 1:(5-45), more preferably 1:(5-40), further preferably 1:(5-30), for example 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, to obtain a sedum sarmentosum water extract;

[0056] (2) Normal temperature extraction is sequentially performed on the sedum sarmentosum water extract using a first solvent and a second solvent to obtain a sedum sarmentosum extract, wherein the extraction ratio is 1:(1-10), preferably 1:(1-9), more preferably 1:(1-8), further preferably 1:(1-7), more preferably 1:(1-6), for example 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, the first solvent is preferably petroleum ether, and the second solvent is preferably chloroform;

[0057] (3) Column chromatography is used to purify the sedum sarmentosum extract, and high performance liquid chromatography is used for separation to obtain the dehydrovomifoliol.

[0058] It can be understood that the extraction, separation and purification in the preparation method of the present application can be performed once or several times, for example 2, 3, 4, 5 times.

[0059] The present application also provides a method for increasing the content of alpha-SMA or CD31 of cells in vitro, which comprises the step of contacting the cells with dehydroexetol, wherein the cells are not particularly limited, examples of which include but are not limited to RAW264.7 cells, HUVEC cells, HaCAT cells, etc.

[0060] In a preferred embodiment, the method for increasing the content of alpha-SMA or CD31 of cells in vitro comprises culturing the cells using a culture medium, culturing continuously for 2-6 generations, counting (0.5-5)*10 6 cells, plating, adding 0.1-5 mg / ml (preferably 0.1-4.5 mg / ml, further preferably 0.1-4 mg / ml, further preferably 0.1-3.5 mg / ml, more preferably 0.1-3 mg / ml, for example 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.5 mg / ml) dehydroexetol, culturing for 24-100 hours, and detecting the content of alpha-SMA or CD31.

[0061] Examples

[0062] I. Establishment of animal models

[0063] 1. Diabetic mouse model

[0064] 8-week-old C57 mice were fed with high-fat feed for 1 month. Fasting for 12 h before modeling, but not water. Intraperitoneal injection of streptozotocin 100 mg / kg, continuous injection for two days, 50 mg / kg injection for 1 day. Two weeks after the end of administration, monitor the change of blood glucose, use 75% alcohol to disinfect the exposed tail vein of the tail, and use sterile scissors to cut the vein, and use a sterile cotton ball to wipe off the first drop of blood. The second drop of blood was randomly tested for blood glucose, and the fasting blood glucose was ≥11.1 mmol / L for 3 consecutive times, which was considered to be a successful modeling.

[0065] 2. Diabetic wound mouse model

[0066] All mice were anesthetized with 1% isoflurane inhalation, and when the mouse muscle was relaxed, the body was turned to a lateral position and there was no autonomous recovery of the position, it was considered that the mouse was completely anesthetized. Then the skin wound model was prepared. The specific steps are as follows:

[0067] (1) Prepare skin and depilate: fix the mouse on a sterile operating table, depilate the mouse on the dorsal spinal region, and completely expose the skin of the operation area.

[0068] (2) Marking: Mark the area to be excised along the midline of the spine on the back with a drawing ruler and marker, with a diameter of 8 mm.

[0069] (3) Disinfection: Disinfect the skin of the surgical area on the back three times with sterile gauze containing 75% ethanol.

[0070] (4) Wound preparation: Under sterile conditions, ophthalmic scissors and surgical forceps are used to cut away the skin in the marked area to create a circular wound with a diameter of 8 mm.

[0071] (5) Hemostasis and bandaging: Use sterile cotton swabs to stop the bleeding from the wound, clean the wound with saline, seal the wound with Vaseline gauze and medical gauze, and fix the outer layer with adhesive tape to prevent it from falling off. This is the model of a full-thickness skin defect wound.

[0072] (6) Awakening: Replace the bedding and drinking water in the mouse cage, place the mouse on a small electric blanket to warm it, accelerate the metabolism of the anesthetic drug, and promote the mouse's awakening. After awakening, place the mouse in a cage with clean bedding. Once all the mice have awakened, return them to the designated cage rack.

[0073] II. Drug Extraction and Screening

[0074] 1. Drug extraction

[0075] Sedum sarmentosum is a plant belonging to the Crassulaceae family. Sedum sarmentosum The fresh or dried whole herb of *Sedum sarmentosum*. Take *Sedum sarmentosum* and extract with water at a ratio of 1:10, decocting twice for 60 minutes each time to obtain an aqueous extract. Separately, take *Sedum sarmentosum* and extract with 60% ethanol at a ratio of 1:10, decocting twice for 60 minutes each time to obtain a 60% ethanol extract. Then, take another *Sedum sarmentosum* and extract with 100% ethanol at a ratio of 1:10, decocting twice for 60 minutes each time to obtain a 100% ethanol extract.

[0076] 2. Animal experiments

[0077] The solvents of the three types of extracts were evaporated and concentrated to 1 g / ml. 100 μl was administered to each mouse via an 8 mm diameter wound daily for 12 consecutive days. The model group consisted of diabetic mice, and the control group consisted of normal mice. Both groups received 100 μl of physiological saline in the wound daily. Results are as follows: Figure 2 As shown, the water extract yields the best results.

[0078] The water extract with good effect was extracted with petroleum ether, chloroform, ethyl acetate and water saturated n-butanol, respectively, with a ratio of 1:3 each time until the extract was colorless. Five different layer extracts (petroleum ether, chloroform, ethyl acetate, n-butanol and water layer) were obtained. The organic reagents were removed by rotary evaporation and redissolved with water. The extract was prepared into 1 g / ml according to the amount of crude drug, and b-FGF was prepared into 5 μg / ml. Each mouse was given 100 μl of 8 mm diameter wound per day for 8 consecutive days. The positive drug group was configured as 5 μg / ml. The qualitative and quantitative results are shown in Figure 3 and Figure 4 The chloroform layer has the best effect.

[0079] 3. Cell experiment

[0080] RAW264.7 cells, HUVEC cells and HaCAT cells were cultured with high glucose (33 mmol / L) for more than 3 generations. RAW264.7 cells were counted to 1*10 6 cells were added with 5 different layer extracts (according to the same amount of crude drug), and the cell supernatant was collected after 48 hours of culture and centrifuged at 3000 rpm and stored at -80℃.

[0081] The above centrifugate was added to HUVEC cells and HaCAT cells at 10 μl, and CCK-8 was used to detect cell viability, and cell scratch test was used to detect cell migration rate. The results are shown in Figure 5 It is found that the cell experiment results are consistent with the animal experiment results.

[0082] 4. Effective component screening

[0083] 4.1 Silica gel column chromatography

[0084] 200 mesh silica gel was activated in an oven at 110℃ for 2 hours, and then sealed for use. A glass chromatography column (inner diameter 3 cm, length ≥ 30 cm) was used. The silica gel was packed with wet method, and the activated silica gel was mixed with the eluent petroleum ether at a ratio of 1:1 to form a uniform homogenate without air bubbles. The homogenate was slowly poured into the column along the glass rod. The column wall was lightly tapped with a rubber rod or connected to a vacuum pump to extract air, so that the silica gel was tightly packed without discontinuity (the final height was about 15 cm). A 1 cm layer of quartz sand was placed on the surface of the silica gel to prevent disturbance of the silica gel layer during loading.

[0085] The sample was dissolved with ethyl acetate and slowly added to the silica gel column along the wall. The eluent was ethyl acetate and petroleum ether (volume ratio 1:1), and the elution was performed every 5 ml, and 100 tubes were collected.

[0086] Thin layer chromatography was used for identification, and silica gel F 254The plate was activated in an oven at 110°C for 2 hours, and then sealed and stored after cooling. Ethyl acetate: petroleum ether was used as the developing solvent at a ratio of 1:1. Six different components were separated according to polarity, namely tubes 1-30, 31-43, 44-60, 61-70, 71-75, and 76-100.

[0087] 4.2 Cell screening experiment

[0088] Each tube was used for cell experiments according to a unified amount of crude drug. The results are shown in Table 1. Figure 6 The component in tube 31-43 had the best effect.

[0089] 4.3 Purification

[0090] The component in tube 31-43 was further separated using an Agilent A1200 liquid phase. The mobile phase A was water, the mobile phase B was methanol, the chromatographic column was Agilent C18 Plus 4.6*250 mm, the flow rate was 1 ml / min, and B was 5%-100% for 0-50 min.

[0091] The peak with the highest concentration was isolated, and the pure product was obtained by using preparative liquid phase separation.

[0092] 4.4 Effect verification

[0093] The effect of the above pure product was verified by cell experiments, and the results are shown in Table 2. Figure 9

[0094] 4.5 Structure analysis

[0095] The pure product was analyzed by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), and the molecular weight was calculated to be 222, and the molecular formula was C 13 H 18 O3.

[0096] 1 H-NMR (500 MHz, CD3OD) data are shown in Table 3. Figure 10 δ 6.99 (1H, d, J = 10 Hz, H-7), 6.43 (1H, d, J = 10 Hz, H-8), 5.93 (1H, m, H-4), 2.60 (1H, d, J = 15 Hz, H-2a), 2.27 (1H, d, J = 15 Hz, H-2b), 2.30 (3H, s, H-10), 1.89 (3H, s, H-11), 1.06 (3H, s, H-12), 1.01 (3H, s, H-13). 1 ​H-NMR spectra showed four groups of sp3 methyl signals (H-10, H-11, H-12, H-13), one group of sp3 methylene signals (H-2), and three groups of sp2 methine signals (H-4, H-7, H-8).

[0097] 13 C-NMR (125 MHz, CD3OD) data as follows Figure 11 As shown: δ200.69(C-3), 200.39(C-9), 164.68(C-5), 148.38(C-7), 131.76(C-8), 128.08(C-4), 80.01(C-6), 50.57(C-2), 42.69(C-1), 27.67(C-10), 24.78(C-13), 23.56(C-12), 19.20(C-11).

[0098] The above results indicate that the isolated pure product is dehydroepiandrosterol.

[0099] 4.6 Animal Experiments

[0100] Animal experiments were conducted using low (0.35 mg / ml), medium (0.7 mg / ml), and high (1.4 mg / ml) doses of dehydroepiandrosterol. The positive control was achieved using topical recombinant human granulocyte-macrophage stimulating factor gel at a concentration of 20 μg / ml. 2 The result is as follows Figure 12 As shown, the medium dose resulted in the best wound closure. Especially in the first 5 days, the effect was good, indicating that the drug works by targeting the inflammatory phase of post-injury healing. Wounds treated with the medium dose showed the most significant closure effect on day 14. Immunohistochemical results, etc., are as follows... Figure 13 As shown, the medium-dose treatment of well-developed collagen fibers showed the most significant increase in regenerated epidermal length on day 14. In addition, the wounds in the medium-dose treatment group showed enhanced vascularization, such as larger areas of α-smooth muscle actin (α-SMA) vessels, higher density of CD31, and higher capillary density.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions 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. Use of dehydrovomifoliol in the preparation of a medicament for improving or promoting wound healing.

2. Use of dehydroexeterol according to claim 1 for the manufacture of a medicament for improving or promoting wound healing, characterized in that, The wound comprises a skin wound.

3. Use of dehydroexeterol for the preparation of a medicament for improving or promoting wound healing according to claim 1, characterized in that, The wound comprises an acute wound and / or a chronic wound.

4. The use of dehydrodihydrotansyquinol according to claim 3 for the preparation of a medicament for improving or promoting wound healing, characterized in that, The acute wound comprises at least one of a scratch, a laceration, a cut, a stab and a burn, and the chronic wound comprises a diabetic wound.

5. A process for the preparation of dehydrotoxybuproban, characterized in that, The preparation method comprises the following steps: (1) water extracting P. japonica at 50-100 ℃ for 0.5-5 h to obtain a P. japonica water extract; (2) extracting the P. japonica water extract using an organic solvent to obtain a P. japonica extract, the organic solvent being at least one of petroleum ether, chloroform, ethyl acetate and n-butanol, and the extraction ratio being 1:(1-10); (3) purifying and separating the P. japonica extract to obtain the dehydrovomifoliol.

6. The method for preparing dehydroepiandrosterol according to claim 5, characterized in that, In step (1), the solid-liquid ratio is 1:(5-50).

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