Cassia twig and cassia bark decoction powder as well as preparation method and application thereof

Abstract

The invention provides cassia twig and cassia bark decoction powder as well as a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicine compositions. The cassia twig and cassia bark decoction powder is prepared from 8-12 parts of cassia twig, 4-8 parts of Chinese herbaceous peony, 4-8 parts of fresh ginger and 3-5 parts of liquorice through soaking, decocting, filtering, vacuum drying and freeze drying. The cassia twig and cassia twig decoction powder can improve the protection rate of thyroid follicular cells, relieve migration of damaged thyroid follicular cells, accelerate inflammation regression and improve damage of TgAb to thyroid follicular cells.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine compositions, and in particular to a Guizhi Jiagui decoction powder, a preparation method and application thereof. Background Art

[0002] Hashimoto's thyroiditis (HT), also known as chronic lymphocytic thyroiditis and lymphocytic goiter, is a common autoimmune disease of the thyroid gland. Its pathological characteristics are diffuse infiltration of lymphocytes and plasma cells in the thyroid gland, destruction of thyroid follicles, and new capillaries in the glandular tissue, which eventually lead to decreased thyroid function. The clinical characteristics are normal thyroid function, hyperthyroidism (hyperthyroidism) or hypothyroidism (hypothyroidism). At present, the incidence of Hashimoto's thyroiditis is increasing year by year. It is more common in women aged 30 to 50 years old. The onset is insidious, the development is slow, and the course is long. The main manifestation is thyroid enlargement, most of which are diffuse, and a few can be localized. Some of them start with swelling of the face and limbs. The disease has become an important disease that affects health.

[0003] At present, Western medicine mainly believes that the onset of this disease is related to factors such as cells, viruses, genetics, and the environment. In terms of treatment, thyroid hormone replacement therapy is mainly adopted according to the patient's thyroid function, but there are often large side effects of hormones in clinical practice, and it is easy to relapse after stopping the drug. Surgery and radiotherapy often cause hypoglycemia and other surgical sequelae. The current new therapy is gene therapy. Studies have confirmed that it has a decisive role in immune regulation and pathological changes, but the required dose is large.

[0004] In recent years, traditional Chinese medicine has achieved good results in the treatment of Hashimoto's thyroiditis. Traditional Chinese medicine's syndrome differentiation and treatment has certain flexibility, few side effects, and high safety. In the treatment of Hashimoto's thyroiditis, traditional Chinese medicine starts from the patient's constitution, combines the patient's emotions, external environment and other factors, judges the symptom characteristics according to the changes in the condition, and uses targeted individualized medication. While treating the primary disease, it improves the patient's immunity. However, since the pathogenic mechanism of HT is still unclear, there is currently no treatment prescription for the cause. Clinical treatment plans often use symptomatic treatment of antithyroid drugs and glucocorticoids. In the diagnosis and treatment of clinical diseases, traditional Chinese medicine's syndrome differentiation and treatment are similar to targeted treatment, and the prescriptions and medicines have definite efficacy and few toxic side effects. Therefore, it is an urgent problem to clarify the effective prescriptions and medicines of traditional Chinese medicine in the treatment of Hashimoto's thyroiditis and explore the significance of its principles, methods and prescriptions. Summary of the invention

[0005] The purpose of the present invention is to provide a Guizhi Jiagui Decoction powder and a preparation method and application thereof. The present invention reveals the pathogenic mechanism of Hashimoto's thyroiditis. Guizhi Jiagui Decoction is a therapeutic prescription targeting the pathogenic mechanism and has the advantages of reliable efficacy, reasonable dosage form and small side effects.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing Guizhi Jiagui Decoction powder, comprising the following steps:

[0008] (1) Soak the Chinese medicine raw materials in water for 25 to 35 minutes;

[0009] (2) heating the water soaked with the Chinese medicinal materials in step (1) to boiling, decocting over low heat for 50 to 70 minutes, and collecting the decoction 1;

[0010] (3) adding water to the residue, continuing to boil over low heat, and collecting the boiling liquid 2;

[0011] (4) mixing the infusion solution 1 and the infusion solution 2, and concentrating the mixture to 1 / 12 to 1 / 8 of the original volume to obtain a concentrated Chinese medicine solution;

[0012] (5) filtering the concentrated Chinese medicine solution and centrifuging it to obtain a supernatant;

[0013] (6) vacuum drying the supernatant, pre-freezing it, and then freeze-drying it to obtain Guizhi Jiagui Decoction powder;

[0014] The Chinese medicinal raw materials include the following components in parts by weight: 8-12 parts of cassia twig, 4-8 parts of peony root, 4-8 parts of ginger and 3-5 parts of liquorice.

[0015] Preferably, the mass volume ratio of the Chinese medicinal material to water in step (1) is 19-31 g:600 mL; the mass volume ratio of the Chinese medicinal material to the second added water is 19-31 g:200 mL.

[0016] Preferably, the simmering time in step (3) is 30 to 35 minutes.

[0017] Preferably, the centrifugal speed is 2700-3200 rpm, and the centrifugal time is 23-27 min.

[0018] Preferably, the vacuum degree of the vacuum drying is -0.09 to -0.095 MPa, the temperature of the vacuum drying is 56 to 60° C., and the time of the positive vacuum drying is 23 to 25 hours.

[0019] Preferably, the pre-freezing temperature is -22 to -18°C, and the pre-freezing time is 10 to 15 hours; the freeze-drying temperature is -60 to -65°C, and the freeze-drying time is 36 to 48 hours.

[0020] The invention also provides Guizhi Jiagui Decoction powder prepared by the preparation method.

[0021] The present invention also provides the use of the Guizhi Jiagui Decoction powder in preparing a medicine for treating Hashimoto's thyroiditis.

[0022] By adopting the above technical scheme, the present invention has the following beneficial effects: the Guizhi Jiagui Decoction powder of the present invention can improve the protection rate of Nthy-ori3-1 cells, alleviate the migration of damaged thyroid follicular cells, accelerate the disappearance of inflammation, promote the development of tissue regeneration and repair, improve the damage of TgAb to thyroid follicular cells, and can be used to prepare drugs for the treatment of Hashimoto's thyroiditis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a dose-effect diagram of the damage rate of Nthy-ori3-1 cells with different concentrations of TgAb.

[0024] Figure 2 This is the time-effect relationship diagram of TgAb on the damage rate of Nthy-ori3-1 cell model (****: P<0.0001).

[0025] Figure 3 This is a graph showing the effects of the blank control group and TgAb model group on cell migration rate in Experimental Example 1 (**: P<0.05).

[0026] Figure 4 The figure shows the migration ability of Nthy-ori3-1 cells (microscope image 100×).

[0027] Figure 5 The morphology of thyroid follicular cells in the blank control group and the model group (image under microscope 400×).

[0028] Figure 6 This is the result of flow cytometry double staining to detect the damage rate of TgAb in the blank control group and model group cells at 8 ng / mL.

[0029] Figure 7 This is the result of detecting the antibody-antigen binding site by immunofluorescence labeling.

[0030] Figure 8 This is the result of ELISA detection of the secretion level of inflammatory cytokine T4 and the expression of IL-6.

[0031] Fig. 9 This is a dose-effect diagram of the protective effect of Guizhi Jia Gui Tang on normal cells and cell models; left: normal cells, right: model cells.

[0032] Fig.10 The graphs show the cell migration ability of the TgAb damaged cell model in the model group at 0h and 24h.

[0033] Fig.11The graphs of cell migration ability of the model drug-administered group at 0h and 24h.

[0034] Fig.12 This is a graph showing the effect of Guizhi Jia Gui Tang on the migration ability of the cell injury model.

[0035] Fig.13 This is the cell morphology diagram of the blank control group and model group in Experimental Example 4.

[0036] Fig.14 This is the cell morphology diagram of the blank-administered group and the model-administered group of Guizhi Jia Gui Tang in Experimental Example 4.

[0037] Fig.15 This is a graph showing the damage rate results of the blank control group and model group cells in Experimental Example 5.

[0038] Fig.16 This is the result graph of the cell damage rate in the Guizhi Jia Gui Tang group in Experimental Example 5.

[0039] Fig.17 This is a graph showing the effect of Guizhi Jia Gui Tang on T4 secretion.

[0040] Fig.18 This is a graph showing the effect of Guizhi Jia Gui Tang on the expression of inflammatory cytokine IL-6. DETAILED DESCRIPTION

[0041] The present invention provides a method for preparing Guizhi Jiagui Decoction powder, comprising the following steps:

[0042] (1) Soak the Chinese medicine raw materials in water for 25 to 35 minutes;

[0043] (2) heating the water soaked with the Chinese medicinal materials in step (1) to boiling, decocting over low heat for 50 to 70 minutes, and collecting the decoction 1;

[0044] (3) adding water to the residue, continuing to boil over low heat, and collecting the boiling liquid 2;

[0045] (4) mixing the infusion solution 1 and the infusion solution 2, and concentrating the mixture to 1 / 12 to 1 / 8 of the original volume to obtain a concentrated Chinese medicine solution;

[0046] (5) filtering the concentrated Chinese medicine solution and centrifuging it to obtain a supernatant;

[0047] (6) vacuum drying the supernatant, pre-freezing it, and then freeze-drying it to obtain Guizhi Jiagui Decoction powder;

[0048] The Chinese medicinal raw materials include the following components in parts by weight: 8-12 parts of cassia twig, 4-8 parts of peony root, 4-8 parts of ginger and 3-5 parts of liquorice.

[0049] In the Chinese medicinal material of the present invention, the weight portion of the cassia twig is 8 to 12 parts, more preferably 9 to 11 parts, and more preferably 10 parts;

[0050] The weight fraction of the peony is 4 to 8 parts, more preferably 5 to 7 parts, and more preferably 6 parts;

[0051] The weight proportion of the ginger is 4 to 8 parts, more preferably 5 to 7 parts, and more preferably 6 parts;

[0052] The weight proportion of the licorice is 3 to 5 parts, more preferably 3.5 to 4.5 parts, and even more preferably 4 parts.

[0053] The present invention mixes Chinese medicinal raw materials with water and soaks them, wherein the mass volume ratio of the Chinese medicinal raw materials to water is 19-31 g:600 mL, more preferably 22-28 g:600 mL, and more preferably 25 g:600 mL; the soaking time is 25-35 min, more preferably 27-32 min, and more preferably 30 min; the soaking is preferably carried out in a beaker, and then the water soaked with the Chinese medicinal raw materials is heated to boiling, and the heating is preferably carried out on an electric stove covered with an asbestos net, and then the water is adjusted to a slow fire for decocting, and the decocting time is 50-70 min, more preferably 55-65 min, and more preferably 60 min. The decocting is accompanied by stirring, and the stirring is preferably carried out by stirring the bottom of the container with a glass rod, and the decocting liquid 1 is collected after decocting. Then add water to the medicinal residue and continue to decoct over low heat. The mass volume ratio of the Chinese medicinal raw material to the second water addition is 19-31g:200mL, more preferably 23-27g:200mL, more preferably 25g:200mL, and the second decoction time is 30-35min, more preferably 31-34min, and more preferably 33min; then collect the boiling liquid 2, the purpose of the second decoction is to precipitate the remaining effective ingredients of the Chinese medicinal pieces as much as possible. The boiling liquid 1 and the boiling liquid 2 are mixed and concentrated to obtain a Chinese medicine concentrated liquid; concentrated to 1 / 12-1 / 8 of the original volume, more preferably 1 / 11-1 / 9, and more preferably 1 / 10. The present invention filters the Chinese medicine concentrated liquid and centrifuges it to obtain a supernatant; the filtration preferably uses a stacked four-layer gauze to cover the funnel, pours the medicinal liquid on the upper mouth of the funnel, and removes impurities by negative pressure suction filtration. The collected filtrate is preferably collected in a centrifuge tube and then centrifuged. The centrifugal rotation speed is 2700-3200rpm, more preferably 2900-3100rpm, and more preferably 3000rpm; the centrifugal time is 23-27min, more preferably 24-26min, and more preferably 25min; the centrifugation is preferably carried out in a low-temperature high-speed centrifuge, and the centrifugation is preferably carried out by adjusting the low-temperature high-speed centrifuge to room temperature.The supernatant after centrifugation is vacuum dried, and the vacuum degree of the vacuum drying is -0.09 to -0.095 MPa, more preferably -0.092 to -0.094 MPa, and more preferably -0.093 MPa; the temperature of the vacuum drying is 56 to 60°C, more preferably 57 to 59°C, and more preferably 58°C; the time of the vacuum drying is 23 to 25 hours, more preferably 23.5 to 24.5 hours, and more preferably 24 hours; the supernatant after vacuum drying is pre-frozen, and the pre-freezing temperature is -22 to -18°C, more preferably -21 to -19°C, and more preferably -20°C; the pre-freezing time is 10 to 15 hours, more preferably 12 to 14 hours, and more preferably 13 hours; the temperature of the freeze drying is -60 to -65°C, more preferably -61 to -64°C, and more preferably -62°C; the time of the freeze drying is 36-48 hours, more preferably 40 to 45 hours, and more preferably 43 hours.

[0054] The invention also provides Guizhi Jiagui Decoction powder prepared by the preparation method.

[0055] The present invention also provides the use of the Guizhi Jiagui Decoction powder in preparing a medicine for treating Hashimoto's thyroiditis.

[0056] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0057] The Nthy-ori3-1 cells of the present invention were purchased from Guangzhou Geneo Biotechnology Co., Ltd.

[0058] Example 1

[0059] Take 8g of cinnamon twig, 4g of peony root, 4g of ginger, and 3g of licorice, add 600mL of ultrapure water and soak for half an hour, place the beaker soaked with medicine on an electric stove covered with asbestos mesh and heat until the water boils, then adjust to a low heat and continue to decoct for 50min (during this period, stir the bottom of the beaker with a glass rod) to collect the liquid, add 200mL of ultrapure water and repeat the decoction for 30min, and concentrate the decoction liquid twice to a volume of 60mL. Cover the funnel with four layers of gauze, pour the liquid into the upper mouth of the funnel, filter out impurities with negative pressure suction, and collect the remaining liquid in a 50mL centrifuge tube. Adjust the low-temperature high-speed centrifuge to room temperature, centrifuge at 2900rpm for 23min, pour the obtained supernatant into an evaporating dish and vacuum dry for 23h. The vacuum degree of vacuum drying is -0.09MPa, and the temperature of vacuum drying is 56℃. Cover with plastic wrap, put the evaporating dish into a refrigerator at -22℃ and freeze it for 10h, then transfer it to a low-temperature freeze dryer and freeze-dry it at -60℃ until it becomes a crystalline powder. After crushing in the evaporating dish, scrape the drug into a 5mL EP tube and place it in the refrigerator for later use.

[0060] Example 2

[0061] Take 10g of cinnamon twig, 6g of peony root, 6g of ginger, and 4g of licorice, add 600mL of ultrapure water and soak for half an hour, place the beaker soaked with medicine on an electric stove covered with asbestos mesh and heat until the water boils, then adjust to low heat and continue to decoct for 1h (during this period, stir the bottom of the beaker with a glass rod) to collect the liquid, add 200mL of ultrapure water and repeat the decoction for 32min, and concentrate the decoction liquid twice to a volume of 60mL. Cover the funnel with four layers of gauze, pour the liquid into the top of the funnel, filter out impurities with negative pressure, and collect the remaining liquid in a 50mL centrifuge tube. Adjust the low-temperature high-speed centrifuge to room temperature, centrifuge at 3000rpm for 25min, pour the obtained supernatant into an evaporating dish and vacuum dry for 24h. The vacuum degree of vacuum drying is -0.093MPa, and the temperature of vacuum drying is 58℃. Cover with plastic wrap, put the evaporating dish into a refrigerator at -20℃ for 12h, transfer it to a low-temperature freeze dryer and freeze it at -62℃ until it becomes a crystalline powder. After crushing in the evaporating dish, scrape the drug into a 5mL EP tube and place it in the refrigerator for later use.

[0062] Example 3

[0063] Take 12g of cinnamon twig, 8g of peony root, 8g of ginger, and 5g of licorice, add 600mL of ultrapure water and soak for half an hour, place the beaker soaked with medicine on an electric stove covered with asbestos net and heat until the water boils, then adjust to low heat and continue to decoct for 70min (during this period, stir the bottom of the beaker with a glass rod), collect the liquid, add 200mL of ultrapure water and repeat the decoction for 35min, and concentrate the decoction liquid twice to a volume of 60mL. Cover the funnel with four layers of gauze, pour the liquid into the top of the funnel, filter out impurities with negative pressure, and collect the remaining liquid in a 50mL centrifuge tube. Adjust the low-temperature high-speed centrifuge to room temperature, centrifuge at 3200rpm for 27min, pour the obtained supernatant into an evaporating dish and vacuum dry for 25h. The vacuum degree of vacuum drying is -0.095MPa, the temperature of vacuum drying is 60℃, cover with plastic wrap, put the evaporating dish in a refrigerator at -18℃ for 14h, transfer it to a low-temperature freeze dryer and freeze it at -65℃ until it becomes a crystalline powder. After crushing in the evaporating dish, scrape the drug into a 5mL EP tube and place it in the refrigerator for later use.

[0064] Experimental Example 1 Establishment of thyroid follicular cell injury model

[0065] Normally cultured Nthy-ori3-1 cells were operated according to the routine cell passage steps, and 1×10 6 The cells were added to 20 mL of RPMI-1640 medium containing 20% ​​fetal bovine serum. After the cell solution was evenly mixed, it was transferred to the sample loading tank. Each well had a volume of 200 μl and contained 1×10 4The cell solution of cells was cultured in a 96-well plate and placed in a carbon dioxide cell culture incubator for 24 hours. The cell growth was observed. The 96-well plate was washed twice with 210μl / well PBS every other day to avoid interference of the low-level antibodies contained in the complete culture medium on the experiment. 200μl of TgAb solution diluted with basic culture medium at a concentration of 8ng / mL was added to each well to establish a "model group". At the same time, the cells were grown under normal conditions, washed with PBS and added with an equal volume of basic culture medium as a "blank control group" and placed in the incubator for continued culture.

[0066] 1. MTT assay

[0067] The cells modeled by the above modeling method were inoculated in a 96-well plate, and 3 sets of duplicate wells were set for the model group and the blank control group. Each group of the TgAb model group was added with 200 μl of serum-free medium containing TgAb at different concentrations (antibody concentrations were 1, 2, 4, 8, 16, and 32 ng / mL), and placed in a carbon dioxide constant temperature incubator for 24 hours. The next day, after observing the cell situation in the well, the supernatant was removed, and 100 μl of the MTT working solution containing 10% prepared in advance was added to each well with a spray gun. After incubation in a constant temperature incubator for 3.5h-4h, the supernatant was removed, and 150 μl of DMSO solution was added to each well, and the crystal violet was moved to a horizontal shaker and shaken for 20min to completely dissolve the crystal violet. The absorbance OD value at 570nm was measured with an enzyme marker, and the cell damage rate was calculated. The experiment was repeated 3 times, and the plateau concentration was selected to continue the later experiment. Subsequently, the viability of thyroid cells was detected at 6, 12, 24, 36, and 48h using the same experimental method and grouping. The cell damage rate of the thyroid gland is calculated as follows:

[0068] Cell damage rate = (blank control group OD value - model group OD value) / blank control group OD value × 100%

[0069] (1) Dose-effect relationship of TgAb in inhibiting thyroid follicular cell proliferation

[0070] By detecting the viability of Nthy-ori3-1 cells in the blank control group and the model group by MTT, it was found that the thyroid cell damage rate increased with the increase of TgAb concentration, showing a dose-effect dependence. In addition, when the TgAb working solution was 8 ng / mL, the cell damage effect reached a plateau, as shown in Table 1, Figure 1 shown.

[0071] Table 1 The dose-effect relationship of different TgAb concentrations on Nthy-ori3-1 cell damage

[0072]

[0073] (2) Time-effect relationship of TgAb inhibiting thyroid follicular cell proliferation

[0074] The TgAb working solution was selected to have a concentration (8 ng / mL) that caused the thyroid cell damage rate to reach a plateau, and then the thyroid cell damage model was tested for time effects. The results are shown in Table 2. Figure 2 The results showed that TgAb tended to be stable after 24 hours of action on the thyroid cell model, and the difference between the model group and the blank control group was statistically significant, P<0.0001.

[0075] Table 2 Time-effect relationship of TgAb on the damage rate of Nthy-ori3-1 cell model

[0076]

[0077] 2. Cell scratch assay

[0078] Use a marker to mark two parallel straight lines of moderate width on the bottom of each well of the 6-well plate in advance. Normally cultured Nthy-ori3-1 cells were routinely operated according to the cell passaging steps, and 1×10 6 Cells were cultured for 24h to ensure that the cells covered 90% of the bottom of the dish. A 200μl gun tip was used to scratch each well. The gun tip was evenly applied to make it perpendicular to the thin line marked on the bottom of the dish. Each well was scratched twice. After scratching, PBS was washed twice to wash away the cells that fell off after mechanical damage. The control group was cultured in serum-free medium, while the model group was cultured in serum-free medium with an appropriate concentration of TgAb. The cell growth was observed and photographed at 0 and 24h, respectively. The same scratch position was taken at each time point as much as possible. Finally, Image J software was used to analyze and calculate the cell migration rate = (initial area - 24h area) / initial area × 100%.

[0079] The cell scratch test was used to detect the damage of Nthy-ori3-1 cells by TgAb for 24 hours, and the changes in the scratch healing rate were observed. The results showed that the scratch healing rate of the blank control group was (67.37% ± 0.025), and the scratch healing rate of the TgAb model group was (36.49% ± 0.021). Compared with the blank control group of the experiment, the scratch healing rate of the TgAb model group was significantly reduced, and the difference was statistically significant P < 0.01. (See Figure 3 , Figure 4 ). This indicates that TgAb can inhibit cell growth and tissue healing.

[0080] 3. Observe cell morphology using an inverted phase contrast microscope

[0081] Nthy-ori3-1 cells in the logarithmic growth phase were digested with 0.25% EDTA-containing trypsin solution, and after counting the cells, they were diluted to a cell density of 5×10 5After the cell suspension of cells / mL was inoculated in a 6-well plate, it was incubated in a carbon dioxide constant temperature incubator for 24 hours, the supernatant in the well plate was aspirated, and it was washed twice with PBS solution to set up a blank control group and a model group. The blank control group was added with 1mL / well of basic culture medium, and the model group was added with 1mL / well of TgAb working solution (8ng / mL) that reached the plateau concentration after dose-effect screening. The 6-well plate was placed in a constant temperature incubator and continued to be cultured for 24 hours, and then transferred to an inverted phase contrast microscope to observe the morphological changes of each group of cells and take pictures and record them.

[0082] The observation revealed that the thyroid follicular cells in the blank control group were mostly cubic, with smooth and complete cell membranes, clear boundaries, transparent cytoplasm, good adhesion to the wall, and normal nuclear division under high magnification; while the cells in the model group were cubic, oval or irregular, with blurred cell boundaries, less cytoplasm, poor adhesion to the wall, and cell vacuolization (see Figure 5 ).

[0083] 4. Flow Cytometry

[0084] After observing the status of Nthy-ori3-1 cells under a microscope, the cell subculture step was performed, the cells were centrifuged and counted, and 1 mL of the medium containing 1×10 6 cells were added to each well of a 6-well plate, and a small amount of fetal bovine serum was added to each well. After gently shaking the 6-well plate, the plate was placed in a constant temperature incubator for incubation for 24 hours. After observing the cells, they were first washed with PBS for shaking, and then the blank control group was added with RPMI-1640 medium without serum; the model group was added with an equal amount of serum-free medium containing TgAb at the plateau concentration, and the 6-well plate was transferred to an incubator for incubation for 24 hours. According to the operating instructions of the Annexin V-FITC cell apoptosis detection kit, the cells were washed once with PBS, and the cells were digested with an appropriate amount of EDTA-free trypsin digestion solution (EDTA may affect the binding of Annexin V to phosphatidylserine), the digestion was terminated, the cells were gently blown, and the cells were collected by centrifugation at 2000 rpm / min for 5 minutes, the supernatant was discarded, and the cells were resuspended twice in precooled 4 mL PBS, and 1-10×10 5 cells, centrifuge again and discard the supernatant, resuspend each group of cells in 500μl of 1×Binding Buffer into flow tubes, add 5μl of AnnexinV-FITC dye to each tube, store at room temperature in the dark for 5min after resuspending, add 10μl of PI to each tube, gently flick to mix, incubate at room temperature in the dark for 5min, detect cell apoptosis rate by flow cytometer within 1 hour, and then analyze the results using Flow Jo software. The cell apoptosis rate is shown in Table 3, and the results are analyzed as shown in Table 3. Figure 6 shown.

[0085] Table 3 Flow cytometry double staining method to detect the damage rate of TgAb to the cell model at 8 ng / mL

[0086]

[0087] Compared with the blank control group without TgAb treatment, the apoptosis of Nthy-ori3-1 cells in the model group was significantly increased. The results showed that TgAb promoted the apoptosis process of thyroid follicular cells and induced apoptotic damage.

[0088] 5. Indirect Immunofluorescence

[0089] Nthy-ori3-1 cells in the logarithmic growth phase were treated according to the subculture procedure and 2×10 4 The cell suspension of cells was added to a clean 24×24mm cover glass and placed in a 6-well plate in a constant temperature incubator overnight. After 24 hours, the cells grew well. After absorbing the supernatant, the cells were washed twice with PBS solution. 1mL of tissue fixative was added to each experimental well of the six-well plate. After standing for 10 minutes, the cells were aspirated. After washing twice with PBS solution, the cells were aspirated. Then, rabbit serum was fully added and blocked at room temperature for 30 minutes. A blank control group and a model group were set up. An appropriate amount of PBS solution was added to the wells of the blank control group, and an appropriate amount of TgAb working solution was added to the wells of the model group. The cells were cultured overnight at 4°C. The next day, the cell well plate was placed on a decolorizing shaker and the slides were washed 3 times with PBS solution, each time for 5 minutes. After drying slightly, FITC-labeled rabbit anti-goat IgG antibody was added to the slides and covered, and incubated at room temperature for 50 minutes. Wash with PBS 3 times, 5 min / time, and after absorbing the excess liquid, add DAPI staining solution to the cell slide, incubate at room temperature and avoid light for 10 min, place the cell well plate on a decolorizing shaker and wash the slide with PBS solution 3 times, 5 min each time. Take the cell slide out of the 6-well plate, dry it slightly, seal it with PBS sealing agent, and then put it on. Finally, observe it under a fluorescence microscope and collect and take pictures. The results are as follows Figure 7 shown.

[0090] The results showed that the cell nuclei stained with DAPI appeared blue under ultraviolet excitation, and the positively expressed FITC fluorescent markers emitted green light. The image results showed that Tg antigen was expressed in the colloid of thyroid follicular cells. The fluorescence signal labeled with FITC in the blank control group was lower than that in the model group, indicating that TgAb promoted the production and secretion of Tg in thyroid follicular cells, and there was a specific binding of Tg and TgAb during in vitro cell culture.

[0091] 6. ELISA

[0092] After Nthy-ori3-1 cells were subcultured, 1 mL of 1×10 6cells were added to each well of a 6-well plate, and a small amount of fetal bovine serum was added to each well. After slightly shaking the 6-well plate, the plate was placed in a constant temperature incubator for incubation for 24 hours. After observing the cells, they were first washed with PBS by shaking, and then the blank control group was added with RPMI-1640 medium without serum; the model group was added with an equal amount of serum-free medium containing TgAb at the plateau concentration, the 6-well plate was transferred to an incubator and cultured for 24 hours, the cell supernatant was taken and transferred to 2 mL EP tubes respectively, and after centrifugation at 2500 rpm / min for 20 minutes at low temperature of 4°C, the supernatant was aspirated into a new spare EP tube and prepared as a sample for the next experiment, or stored in a -80°C refrigerator.

[0093] Before the experiment officially begins, place the ELISA kit at room temperature for 30 minutes in preparation. Then follow the instructions in the relevant manual to operate in accordance with the specifications. Use the corresponding detection reagents for human thyroxine (T4) and inflammatory factors (IL-6), and do not confuse them. Set up blank wells, standard wells, and sample wells to dilute and add concentrated washing solution and standard samples, then (incubate and wash) add enzyme-labeled reagents except for the blank wells, incubate and wash, add colorimetric reagents and mix, incubate at 37°C in the dark for 15 minutes, terminate the reaction with the stop solution, and finally adjust the blank well to zero. Within 15 minutes, quickly detect the absorbance OD value at a wavelength of 450nm. Then, based on the known standard concentration and the detected OD value, use Excel software to find the linear regression equation and calculate the sample concentration, and use GraphPadPrism 8 software for statistical analysis. The results are shown in Table 4. Figure 8 shown.

[0094] Table 4 T4 content in supernatant of thyroid follicular cells damaged by Tg antibody

[0095]

[0096] The results of ELISA test showed that the T4 level in the model group was lower than that in the blank control group, while the actual expression of IL-6 was significantly higher than that in the blank control group, with a statistically significant difference of P<0.05. This indicated that TgAb treatment promoted the expression of inflammatory factors in thyroid follicular epithelial cells, induced inflammatory damage to thyroid follicular cells, and the cell model showed mild hypothyroidism.

[0097] In summary, TgAb can induce inflammatory damage such as proliferation inhibition, apoptosis, and growth inhibition in thyroid follicular epithelial cells Nthy-ori3-1; the proliferation inhibition of thyroid follicular cells by TgAb varies with time and antibody concentration, and the maximum inhibition rate can reach 80-90%. In the in vitro culture of monolayer cells without the formation of thyroid follicular functional structure, Tg is stored in thyroid follicular epithelial cells and has the ability to specifically bind to TgAb. This shows that the inflammatory injury model of thyroid follicular cells was successfully constructed by TgAb induction.

[0098] Experimental Example 2 MTT experiment

[0099] 1 g of the drug powder prepared in Example 2 was weighed by an electronic balance and placed in a 1.5 mL EP tube. 0.25 mL of RPMI-1640 medium was added and vortexed to mix well. The drug concentration was 4000 μg / mL. The drug solution was then filtered through a 0.22 μm micropore bacterial filter into a new 1.5 mL EP tube and stored in a refrigerator for later use.

[0100] Nthy-ori3-1 cells were used as experimental research objects, and the MTT experiment was used to observe the effect of Guizhi Jiagui Decoction on the proliferation of Nthy-ori3-1 cells under the state of TgAb-induced Nthy-ori3-1 cell damage.

[0101] Observe Nthy-ori3-1 cells under an inverted microscope. When the cell morphology is good and the density reaches about 80%, subculture the cells in the logarithmic growth phase. After counting the cells, use 200 μl of complete medium at 1×10 4 Cells / well were inoculated in a 96-well plate, and after culturing in a cell culture incubator for 24 hours, the supernatant was aspirated, and the 96-well plate was washed twice with 210 μl / well PBS and then the liquid was aspirated to avoid interference of the original low-level antibodies in the experiment. The 96-well plate was divided into 4 groups: blank control group, model group, model administration group, and blank administration group, and 3 rows of duplicate wells were set in each group. Then the following treatments were performed respectively: First, 100 μl of serum-free culture medium was added to each well of the blank control group and the blank administration group, and at the same time, 100 μl of TgAb working solution with a concentration of 8 ng / mL was added to each well of the model group and the model administration group. After the working solution of each group fully acted on the cells for 15 minutes, 100 μl of serum-free culture medium was added to each well of the blank control group and the model group, and at the same time, 100 μl of the traditional Chinese medicine solution with a concentration of 100 μg / mL was added to each well of the model administration group and the blank administration group, and the cells were placed in a cell culture incubator for continued cultivation.

[0102] After 24 hours of incubation, take out the 96-well plate and observe it under a microscope, then transfer it to a clean bench to discard the supernatant, add 100 μl of the pre-prepared MTT working solution to each well, and continue incubating in the incubator for 3.5-4 hours. Discard the MTT solution, add 150 μl of DMSO solution to each well, move it to a horizontal shaker and shake it for 20 minutes to completely dissolve the crystal violet, and measure the absorbance OD value at 570 nm with an enzyme marker. Repeat the experiment 3 times. Calculate the cell protection rate of each group under the action of a drug concentration of 100 μg / mL. The calculation formula is as follows:

[0103] ① Blank medication group: drug protection rate = (blank medication group OD value - blank control group OD value) / blank control group OD value × 100%;

[0104] ② Model medication group: drug protection rate = (OD value of model medication group - OD value of model group) / OD value of model group × 100%;

[0105] ③Net protection rate of model drug administration group = drug protection rate of model group - drug protection rate of blank group

[0106] After calculation, the protection rates of Guizhi Jia Gui Tang for the model medication group and the blank medication group were 29.03%±0.03 and 0.26%±0.09, respectively.

[0107] Then, the blank control group, model group, model administration group and blank administration group were subjected to the dose-effect experiment of Guizhi Jiagui Decoction powder by MTT experiment. The protection rates of cells in the model administration group and blank administration group were calculated when the concentrations of Guizhi Jiagui Decoction were 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL and 400 μg / mL, respectively. The concentration curve of drug dose-effect was drawn using GraphPadPrism software. The results are shown in Table 5. Fig. 9 shown.

[0108] Table 5 The dose-effect relationship of Guizhi Jiagui Decoction on the model administration group, blank administration group and net protection rate of the model administration group

[0109]

[0110] From the above results, it can be seen that when the concentration of Guizhi Jiagui Tang powder is 200 μg / mL, the protection rate of the cells in the model administration group is the highest. Therefore, Guizhi Jiagui Tang powder with a concentration of 200 μg / mL was selected for subsequent experiments.

[0111] Experimental Example 3 Cell scratch test

[0112] Use a marker to mark two parallel straight lines of moderate width on the bottom of each well of the 6-well plate in advance. Normally cultured Nthy-ori3-1 cells were routinely operated according to the cell passaging steps, and 1×10 6Cells were cultured for 24h to ensure that the cells covered about 90% of the bottom of the dish. A 200μl gun tip was used to scratch each well. The gun tip was evenly applied to make it perpendicular to the thin line marked on the bottom of the dish. Two scratches were made in each well. After scratching, PBS was washed twice to wash away the cells that fell off after mechanical damage. Then PBS was aspirated. The blank control group was cultured twice, and 500μl of serum-free medium was added to the model group. At the same time, 500μl of serum-free medium containing TgAb at a concentration of 8ng / mL was added to the model group. After 15min of antibody action, 500μl of serum-free medium was added to the model administration group. Similarly, 500μl of serum-free medium containing TgAb at a concentration of 8ng / mL was first added, and 500μl of medium containing 200μg / mL of Guizhi Jiagui Decoction powder was added 15min later for culture. The cell growth was observed and photographed at 0 and 24 h, and the same scratch was taken at each time point. Image J software was used for analysis and calculation: cell migration rate = (initial area - 24 h area) / initial area × 100%.

[0113] Migration diagram of cells in different groups Fig.10 , Fig.11 As shown in Figure 2, the effect of Guizhi Jiagui Decoction powder on the migration ability of cell injury models is as follows Fig.12 The results showed that the scratch healing rate of the model group was (36.49±2.09), and the scratch healing rate of the Guizhi Jiagui Tang group was significantly reduced, and the difference was statistically significant (P<0.01). This shows that Guizhi Jiagui Tang powder can alleviate the migration of damaged thyroid follicular cells, accelerate the disappearance of inflammation, and facilitate the development of tissue regeneration and repair.

[0114] Experimental Example 4: Observation of cell morphology using an inverted phase contrast microscope

[0115] Nthy-ori3-1 cells in the logarithmic growth phase were digested with 0.25% EDTA-containing trypsin solution, and after counting the cells, they were diluted to a cell density of 5×10 5 After the cell suspension of cells / mL was prepared, it was inoculated in a 6-well plate. After incubation in a carbon dioxide constant temperature incubator for 24 hours, the supernatant in the well plate was aspirated, and the plate was washed twice with PBS solution. Four groups were set up: blank control group, model group, model treatment group and blank treatment group. 500μl of basic culture medium was added to each well of the blank control group and the blank treatment group, while 500μl of TgAb working solution with a concentration of 8ng / mL was added to each well of the model group and the model treatment group. After the action time was 15min, 500μl of basic culture medium was continued to be added to each well of the blank control group and the model group, while 500μl of Guizhi Jiagui Tang powder solution with a concentration of 200μg / mL was added to each well of the blank treatment group and the model treatment group. After the 6-well plate was placed in a constant temperature incubator and cultured for 24 hours, it was transferred to an inverted phase contrast microscope to observe the morphological changes of cells in each group and enlarged and photographed for record. The results are shown as follows Fig.13 , Fig.14 shown.

[0116] The results showed that compared with the blank control group, in the blank treatment group of Guizhi Jia Gui Tang, there was no significant change in the morphology of epithelial-like cells, the cell boundaries were clear, and the cytoplasm was transparent; the cell gaps were wider than those in the blank control group, but the edges were clear and the growth was good.

[0117] Compared with the blank administration group, the cells in the model administration group had unclear boundaries, blurred edges, less cytoplasm, bubble-like cell structure protrusions, and visible cell fragments.

[0118] Experimental Example 5 Flow Cytometry

[0119] The apoptosis rate of cells in the blank control group, model group and model administration group (Guizhi Jiagui Decoction group) was detected by flow cytometry combined with Annexin V-FITC / PI double staining. The apoptosis detection steps were the same as those in Experimental Example 1, and the apoptosis results were analyzed using Flow Jo software. The results are shown in Table 6; the software analysis results are shown in Table 6. Fig.15 , Fig.16 shown.

[0120] Table 6 Cell viability and apoptosis rate in different groups

[0121]

[0122] The results showed that compared with the blank control group, the cell viability rate of the model group was significantly decreased, and the apoptosis rate was significantly increased, indicating that TgAb indeed induced excessive apoptosis of thyroid follicular cells; compared with the model group, the cell apoptosis rate of the model medication group (Guizhi Jia Gui Tang group) did not show significant improvement, but the cell activity rate was significantly increased. It can be seen that after administration, Guizhi Jia Gui Tang powder can increase the number of viable cells, alleviate the occurrence of cell apoptosis, and protect the thyroid follicular cells damaged by TgAb.

[0123] Experimental Example 6 Enzyme-linked immunosorbent assay

[0124] After Nthy-ori3-1 cells were subcultured according to the procedure, 1 mL of 1×10 6cells were added to each well of a 6-well plate, and a small amount of fetal bovine serum was added to each well. After gently shaking the 6-well plate, the plate was placed in a constant temperature incubator for 24 hours and then observed. After that, the cells were first washed with PBS and then the blank control group was added with serum-free RPMI-1640 medium twice; the model group was added with TgAb working solution and serum-free medium successively; the model medication group (Guizhi Jiagui Tang group) was added with serum-free medium containing TgAb working solution and 200μg / mL Guizhi Jiagui Tang powder successively. After the 6-well plate was transferred to the incubator and cultured for 24 hours, the cell supernatant was taken and transferred to 2mL EP tubes respectively. After centrifugation at 2500rpm / min for 20min at low temperature of 4℃, the supernatant was aspirated into a new spare EP tube and prepared as a sample for the immediate experiment, or stored in a -80℃ refrigerator.

[0125] Before the experiment officially begins, place the ELISA kit at room temperature for 30 minutes in preparation. Then follow the instructions in the relevant manual to operate in accordance with the specifications. Use the corresponding detection reagents for human thyroxine (T4) and inflammatory factors (IL-6), and do not confuse them. Set up blank wells, standard wells, and sample wells to dilute and add concentrated washing solution and standard substances, and then (incubate and wash) add enzyme-labeled reagents except for the blank wells, incubate and wash, add the color developer and mix, incubate at 37°C in the dark for 15 minutes, terminate the reaction with the stop solution, and finally adjust the blank well to zero. Within 15 minutes, quickly detect the absorbance OD value at a wavelength of 450nm. Then, based on the known standard concentration and the detected OD value, use Excel software to find the linear regression equation and calculate the sample concentration. Use GraphPadPrism 8 software to statistically analyze the results as shown in Table 7. Fig.17 , Fig.18 shown.

[0126] Table 7 T4 and IL-6 content in cell supernatant of each group

[0127]

[0128]

[0129] The results showed that the supernatant content of T4 in the model administration group was higher than that in the model group and lower than that in the blank control group; while the expression level of inflammatory factor IL-6 in the model administration group was lower than that in the model group. This indicates that Guizhi Jiagui Decoction powder can alleviate the inflammatory damage caused by overexpression of inflammatory factor IL-6 in the thyroid follicular cell injury model and regulate the secretion level of T4.

[0130] From the above examples, it can be seen that the present invention provides a Guizhi Jiagui Decoction powder and its preparation method and application. The Guizhi Jiagui Decoction powder of the present invention can improve the protection rate of Nthy-ori3-1 cells, alleviate the migration of damaged thyroid follicular cells, accelerate the disappearance of inflammation, promote the development of tissue regeneration and repair, and improve the damage of TgAb to thyroid follicular cells.

[0131] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing Guizhi Jiagui Decoction powder, characterized in that: The following steps are involved: (1) Soak the Chinese medicine raw materials in water for 25 to 35 minutes; (2) heating the water soaked with the Chinese medicinal materials in step (1) to boiling, decocting over low heat for 50 to 70 minutes, and collecting the decoction 1; (3) adding water to the residue, continuing to boil over low heat, and collecting the boiling liquid 2; (4) mixing the infusion solution 1 and the infusion solution 2, and concentrating the mixture to 1 / 12 to 1 / 8 of the original volume to obtain a concentrated Chinese medicine solution; (5) filtering the concentrated Chinese medicine solution and centrifuging it to obtain a supernatant; (6) vacuum drying the supernatant, pre-freezing it, and then freeze-drying it to obtain Guizhi Jiagui Decoction powder; The Chinese medicinal raw materials include the following components in parts by weight: 8-12 parts of cassia twig, 4-8 parts of peony root, 4-8 parts of ginger and 3-5 parts of liquorice.

2. The preparation method according to claim 1, characterized in that: The mass volume ratio of the Chinese medicinal material to water in step (1) is 19-31 g:600 mL; the mass volume ratio of the Chinese medicinal material to the second added water is 19-31 g:200 mL.

3. The preparation method according to claim 1 or 2, characterized in that: The time for continuing to simmer over low heat in step (3) is 30 to 35 minutes.

4. The preparation method according to claim 3, characterized in that: The centrifugal speed is 2700-3200 rpm, and the centrifugal time is 23-27 min.

5. The preparation method according to claim 4, characterized in that: The vacuum degree of the vacuum drying is -0.09 to -0.095 MPa, the temperature of the vacuum drying is 56 to 60° C., and the time of the vacuum drying is 23 to 25 hours.

6. The preparation method according to claim 1, characterized in that: The pre-freezing temperature is -22 to -18°C, and the pre-freezing time is 10 to 15 hours; the freeze-drying temperature is -60 to -65°C, and the freeze-drying time is 36 to 48 hours.

7. Guizhi Jiagui Tang powder prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the Guizhi Jiagui Tang powder according to claim 7 in preparing a medicine for treating Hashimoto's thyroiditis.

9. Use of the powder according to claim 7 in preparing a drug for improving the protection rate of thyroid follicular cells or alleviating the migration of damaged thyroid follicular cells.

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