Tenuigenin intravenous injection and preparation method thereof

Abstract

According to the tenuigenin intravenous injection and the preparation method thereof, the sodium bicarbonate solution is used for dissolving the tenuigenin, the solubility and stability of the tenuigenin are improved, and the prepared tenuigenin intravenous injection has high safety and good effectiveness, can be directly used for intravenous injection, and can be used for preparing the tenuigenin intravenous injection. The medicine utilization rate is high and the effect is quick.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and particularly relates to a polygala sapogenin intravenous injection and a preparation method thereof. Background Art

[0002] Senegenin (SEN) is an extract of the root of Polygala tenuifolia, a traditional Chinese medicine. 30 H 45 ClO6 is a white crystalline powder at room temperature, easily soluble in hot water, hot methanol and hot ethanol, and insoluble in ether, chloroform and benzene. It is often used to treat neurological diseases such as insomnia and Alzheimer's disease. SEN can also inhibit the occurrence and development of inflammatory reactions through various pathways, such as inhibiting the production and release of interleukin-1β, reducing the number and activity of alveolar macrophages, and promoting the scavenging of free radicals in the lungs. In existing studies, SEN plays a vital role in neuroprotection, myocardial repair, anti-oxidation and anti-inflammatory, and inhibition of tumor cells.

[0003] Polygala sapogenin is easy to form Polygala saponin in solution, which can combine with cholesterol on red blood cells, destroy red blood cell membranes, and produce hemolysis, which limits the use of total Polygala sapogenin as an intravenous injection. In order to inhibit the formation of total Polygala saponin from Polygala sapogenin, dimethyl sulfoxide is often used for dissolution. However, dimethyl sulfoxide has certain irritation and corrosiveness to tissues and is not suitable as a solvent for intravenous injection.

[0004] Currently, there is no injection formulation of Polygala tenuifolia sapogenin, and no intravenous injection of Polygala tenuifolia sapogenin has been marketed. Summary of the invention

[0005] In order to solve the above technical problems, the first aspect of the present invention provides a method for preparing polygala sapogenin intravenous injection, comprising the following steps:

[0006] Step S1, preparing a sodium bicarbonate solution using physiological saline to make the sodium bicarbonate concentration be 5 mg / mL to 15 mg / mL; the sodium chloride concentration of the physiological water is 8 mg / mL to 9 mg / mL;

[0007] Step S2, adding polygala sapogenin to a sodium bicarbonate solution, stirring and dissolving, the dissolving temperature is 4° C. to 65° C., and the stirring time is 1 hour to 3 hours;

[0008] Step S3, standing for 12 to 16 hours to completely dissolve the polygala sapogenin, making the concentration of polygala sapogenin reach 0.5 mg / mL to 33 mg / mL, and adjusting the pH value to 6.5 to 8.5;

[0009] Step S4, filtering and packaging.

[0010] As a preferred embodiment, the pH value of the sodium bicarbonate solution is 8.3.

[0011] As a preferred embodiment, the physiological saline is of injection grade.

[0012] As a preferred embodiment, the sodium bicarbonate is of pharmaceutical grade.

[0013] As a preferred embodiment, the polygala sapogenin is of pharmaceutical grade.

[0014] As a preferred solution, the pore size of the filter material used for the filtration is 0.45 microns.

[0015] As a preferred embodiment, the filtration is sterilizing filtration.

[0016] As a preferred embodiment, the preparation process is carried out under a sterile environment.

[0017] As a preferred solution, step S4 also includes sterilization.

[0018] Polygala sapogenin has a higher solubility in sodium bicarbonate solution. Because Polygala sapogenin has a lower solubility in physiological saline, the solvent of Polygala sapogenin commonly used at present is dimethyl sulfoxide (DMSO), although DMSO is called "universal solvent", it has certain cytotoxicity. The inventor unexpectedly found in the experiment that sodium bicarbonate solution was used to dissolve Polygala sapogenin, which greatly improved the solubility of Polygala sapogenin. Clinically, the sodium bicarbonate solution of 50g / L is a commonly used drug for intravenous injection, and sodium bicarbonate is used as a solvent with higher safety, and experiments in mice have also verified this viewpoint. Using sodium bicarbonate solution as the solvent of Polygala sapogenin makes Polygala sapogenin suitable for intravenous injection, and the drug utilization rate is high, the onset is fast, and the irritation and corrosivity of DMSO to tissue are eliminated.

[0019] Polygala sapogenin has good compatibility with sodium bicarbonate. Polygala sapogenin is extremely stable in sodium bicarbonate solution, does not produce other impurities, does not change the physical and chemical properties and molecular structure of Polygala sapogenin, and improves the stability of Polygala sapogenin intravenous injection. Its stability has been verified by stability tests, its molecular structure has been verified by liquid chromatography-mass spectrometry, and its low impurity characteristics have been verified by liquid chromatography analysis.

[0020] Polygala tenuifolia sapogenin with sodium bicarbonate solution as solvent has high safety and effectiveness. Its safety and effectiveness have been verified through animal experiments.

[0021] The second aspect of the present invention provides a polygala sapogenin intravenous injection, which is prepared by any of the aforementioned preparation methods.

[0022] As a preferred embodiment, the polygala sapogenin intravenous injection is a colorless, odorless, transparent liquid.

[0023] As a preferred embodiment, the concentration of the polygala sapogenin intravenous injection is 2 mg / ml

[0024] Polygala tenuifolia sapogenin intravenous injection can be directly injected intravenously, with high drug utilization rate and rapid onset of effect.

[0025] Through the above technical solution, the present invention produces the following beneficial effects:

[0026] (1) Sodium bicarbonate solution was used to dissolve Polygala tenuifolia sapogenin, thereby increasing the solubility of Polygala tenuifolia sapogenin.

[0027] (2) Polygala tenuifolia sapogenin is more stable in sodium bicarbonate solution.

[0028] (3) Polygala tenuifolia sapogenin intravenous injection has high safety and good efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The HPLC spectrum of 50ug / ml Polygala tenuifolia sapogenin injection of Example 2;

[0030] Figure 2 The HPLC spectrum of 75ug / ml Polygala tenuifolia sapogenin injection of Example 2;

[0031] Figure 3 The HPLC spectrum of 100ug / ml Polygala sapogenin injection of Example 2;

[0032] Figure 4 The HPLC spectrum of 150ug / ml Polygala tenuifolia sapogenin injection of Example 2;

[0033] Figure 5 The HPLC spectrum of 500ug / ml Polygala tenuifolia sapogenin injection of Example 2;

[0034] Figure 6 The standard curve of the polygala sapogenin content of Example 2;

[0035] Figure 7 The HPLC-MS / MS spectra of the polygala sapogenin of Example 2;

[0036] Figure 8 It is the MS mass spectrum of the polygala sapogenin of Example 2;

[0037] Fig. 9 It is the MS mass spectrum of the polygala sapogenin of Example 2;

[0038] Fig.10The results of the effect of intravenous injection of Polygala tenuifolia sapogenin on the body weight of mice in Example 3 (n=5); wherein the NS group is a normal saline control group, the RG group is a sodium bicarbonate solvent control group, and the SEN (30 mg / kg) group, the SEN (60 mg / kg) group, the SEN (120 mg / kg) group, and the SEN (240 mg / kg) group represent the intravenous injection of Polygala tenuifolia sapogenin 30 mg / kg group, the 60 mg / kg group, the 120 mg / kg group, and the 240 mg / kg group, respectively;

[0039] Fig.11 The effect of intravenous injection of Polygala sapogenin on the organ-brain ratio of important organs in mice in Example 3 (n=5); SEN (30 mg / kg) group, SEN (60 mg / kg), SEN (120 mg / kg) group, SEN (240 mg / kg) group represent intravenous injection of Polygala sapogenin 30 mg / kg, 60 mg / kg, 120 mg / kg, 240 mg / kg, respectively;

[0040] The figures show the mass ratios of the heart, liver, spleen, lung, kidney and brain of mice in each group 7 days after injection;

[0041] Fig.12 The figure shows the effect of intravenous injection of Polygala tenuifolia sapogenin on the ratio of the weight of important organs to body weight of mice in Example 3 (n=5); the SEN (30 mg / kg) group, the SEN (60 mg / kg) group, the SEN (120 mg / kg) group, and the SEN (240 mg / kg) group represent the intravenous injection of Polygala tenuifolia sapogenin at 30 mg / kg, 60 mg / kg, 120 mg / kg, and 240 mg / kg, respectively; the figure shows the ratio of the weight of the brain, heart, liver, spleen, lung, and kidney to body weight of mice in each group 7 days after injection;

[0042] Fig.13 The effect of intravenous injection of Polygala sapogenin on the wet-to-dry ratio of important organs of mice in Example 3 (n=5); SEN (30mg / kg) group, SEN (60mg / kg), SEN (120mg / kg) group, SEN (240mg / kg) group represent intravenous injection of Polygala sapogenin 30mg / kg, 60mg / kg, 120mg / kg, 240mg / kg respectively. The figure shows the wet-to-dry ratio of brain, heart, liver, spleen, lung and kidney of mice in each group 7 days after injection;

[0043] Fig.14The effect of intravenous injection of Polygala sapogenin on the pathological changes of important organs in mice of Example 3 (n=5); SEN-A, SEN-B, SEN-C, and SEN-D represent the groups injected with 30 mg / kg, 60 mg / kg, 120 mg / kg, and 240 mg / kg of intravenous injection of Polygala sapogenin, respectively; A, B, C, D, E, and F represent HE-stained sections of the brain, heart, liver, spleen, lung, and kidney of mice 7 days after injection of intravenous injection of Polygala sapogenin;

[0044] Fig.15 The therapeutic effect of intravenous injection of Polygala tenuifolia sapogenin in Example 4 on CLP mice (n=30); the sepsis score (MSS) of each group of mice was performed within 24 hours after surgery. If the mice died, the highest score of 28 points was calculated; there was no statistically significant difference between the CLP group and the CLP+RG group (P>0.05), and there was a statistically significant difference between the CLP group and the CLP+RG group and the CLP+SEN group (P<0.05);

[0045] Fig.16 The effect of intravenous injection of Polygala tenuifolia sapogenin on the survival rate of CLP mice in Example 4; there was no significant difference between the CLP group and the CLP+RG group (P>0.05), and there was a significant difference between the CLP group and the CLP+RG group and the CLP+SEN group (P<0.05);

[0046] Fig.17 This is the effect of intravenous injection of Polygala tenuifolia sapogenin on the body weight of CLP mice in Example 4; the body weights of the mice that survived for 7 days were counted, including 10 mice in the sham group, 4 mice in the CLP group, 5 mice in the CLP+RG group, and 14 mice in the CLP+SEN group. DETAILED DESCRIPTION

[0047] In order to more clearly and completely understand the purpose, technical scheme and advantages of the present invention, the technical scheme of the present invention and the technical effects achieved by the technical scheme of the present invention and the technical effects achieved by the technical scheme of the present invention and the technical effects achieved by the technical scheme of the present invention will be described in detail below through embodiments. Obviously, the described embodiments are only examples of a part of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present invention.

[0048] Example 1

[0049] Take normal saline, add sodium bicarbonate, stir and dissolve thoroughly to prepare a 10 mg / ml sodium bicarbonate solution. According to the preparation requirements of Table 1, add polygala sapogenin to the 10 mg / ml sodium bicarbonate solution, continue stirring at room temperature for 30 seconds, let it stand for 12 hours, and observe the dissolution of the drug.

[0050] According to the preparation requirements in Table 1, after adding polygala sapogenin to dimethyl sulfoxide, continue stirring, let it stand for 12 hours, and observe the dissolution of the drug.

[0051] The results are shown in Table 1. The maximum solubility of Polygala sapogenin in sodium bicarbonate solution is about 32 mg / ml, while the solubility of Polygala sapogenin in dimethyl sulfoxide (DMSO) is about 10 mg / ml. The above experimental results show that using sodium bicarbonate solution as solvent increases the solubility of Polygala sapogenin.

[0052] In addition, when the inventors used sodium bicarbonate solution to dissolve Polygala sapogenin, they found that the dissolution of Polygala sapogenin was significantly promoted by standing.

[0053] Table 1: Solubility test results

[0054]

[0055]

[0056] After dissolution, the sample was filtered through a 0.45 micron microporous filter membrane and packaged to prepare polygala sapogenin intravenous injection. All operations were performed under a sterile environment.

[0057] The 10mg / ml and 20mg / ml polygala sapogenin intravenous injection solutions were placed at room temperature, 4°C, and 65°C for stability testing. On the first day and the 30th day, the drug solutions at room temperature, 4°C, and 65°C were all clear and transparent, and there was no obvious change. This shows that the quality of the prepared polygala sapogenin intravenous injection solution is stable and uniform.

[0058] Example 2

[0059] 1. Purpose of the experiment

[0060] It was proved that polygala sapogenin was the only effective active ingredient in the polygala sapogenin intravenous injection with sodium bicarbonate solution as solvent.

[0061] 2. Experimental materials and instruments

[0062] 2.1 Experimental Materials

[0063] Polygala tenuifolia sapogenin was from Beijing Solebao Technology Co., Ltd., sodium bicarbonate was from Beijing Solebao Technology Co., Ltd., methanol (chromatographic grade) was from Beijing Zeping Technology Co., Ltd., acetonitrile was from Beijing Zeping Technology Co., Ltd., water and phosphoric acid were from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0064] 2.2 Experimental instruments and equipment

[0065] Shimadzu SHIMADZU-LC20A liquid chromatograph, Agilent 1290 liquid phase-6550qtof mass spectrometer, Thermo Fisher U3000 liquid phase-QE mass spectrometer, Waters iclass ab5500 liquid phase mass spectrometer.

[0066] 2.3 Sample preparation

[0067] First, take 5 ml of normal saline to prepare 5 ml of 10 mg / ml sodium bicarbonate, then add 2.5 mg of polygala sapogenin, stir at room temperature for about 30 seconds, then let it stand for 12 hours. After the polygala sapogenin is completely dissolved, the pH value is measured to be 8.1. Use a pipette to aspirate 1 ml, and dilute it in a gradient manner: 150 ug / ml, 100 ug / ml, 75 ug / ml, 50 ug / ml.

[0068] 3. Experimental methods

[0069] 3.1 Liquid chromatography analysis

[0070] According to the method in the paper "Simultaneous analysis of polygala acid, senegenin and 3,6'-disinapoylsucrose in rat plasma by liquid chromatography-tandem massspectrometry: application to a pharmacokinetic study after oral administration" (2014) by Lin L et al., the prepared polygala senegenin intravenous injection was analyzed by high performance liquid chromatography. The analysis method is as follows:

[0071] The wavelength is 210nm; the chromatographic column is C18, and the column model is 4.6mm×250mm 5um; the mobile phase is a mixture of methanol and 0.05% phosphoric acid solution in a mass ratio, and the volume ratio of methanol and 0.05% phosphoric acid solution in a mass ratio is 7:3; the flow rate is 1ml / min; the column temperature is 30℃; and the injection volume is 10ul.

[0072] 3.2 Liquid-mass analysis

[0073] Mobile phase A is 0.1% formic acid aqueous solution by volume; mobile phase B is acetonitrile solution; flow rate is 0.3 ml / min, injection volume is 5 ul; chromatographic column is waters BEH C18, column model is 2.1×100mm 1.7um; mass spectrometry scanning range is primary 70-1500 m / z, sheath gas temp is 350℃; sheath gas flow is 12 L / min, ESI+ mode voltage is 4000 V.

[0074] 4. Experimental results and analysis

[0075] 4.1 Liquid chromatography results

[0076] The results of liquid chromatography of different concentrations of polygala sapogenin intravenous injection are as follows Figure 1 to Figure 5 As shown in the chromatogram, it can be seen that the polygala sapogenin peaks appear at similar times and have good separation.

[0077] Figure 6 is the standard curve, the standard curve formula is y=1119.8x-16354, R 2 It is 0.9994, where y is the peak area. By substituting the value of the peak area, the actual concentration of polygala sapogenin can be calculated.

[0078] The actual concentration of the prepared Polygala sapogenin intravenous injection solution calculated according to the above method is shown in the table below. Polygala sapogenin is dissolved in sodium bicarbonate solvent, and the actual measured concentration is basically consistent with the theoretical concentration, proving that Polygala sapogenin does not degrade or polymerize in the sodium bicarbonate solvent, and the loss is extremely small.

[0079] Table 2: Actual concentration of polygala sapogenin injection dissolved in sodium bicarbonate

[0080] Peak area Actual concentration (ug / ml) Theoretical concentration (ug / ml) 31457 42.696 50 72797 79.613 75 98128 102.234 100 153002 151.237 150 542676 499.221 500

[0081] 4.2 Analysis results of liquid quality

[0082] The results of HPLC-MS / MS are shown in Figure 7 The MS spectrometry results are shown in Table 3 and Figure 8 , Fig. 9 There are four mass-to-charge ratios (M / Z) in the intravenous injection of Polygala tenuifolia sapogenin using sodium bicarbonate solution as solvent, which are 537.2837, 538.2889, 539.2985 and 540.3025, and their molecular formulas are C 30 H 45 ClO6(M+H)+, no other impurities appeared. It shows that the intravenous injection of Polygala tenuifolia sapogenin with sodium bicarbonate solution as solvent has good stability and does not change the physicochemical properties and molecular structure of Polygala tenuifolia sapogenin.

[0083] Table 3 MS mass spectrum peak list

[0084]

[0085]

[0086] Example 3

[0087] 1. Purpose of the experiment

[0088] To evaluate the safety of polygala tenuifolia sapogenin intravenous injection.

[0089] 2. Experimental Materials

[0090] 2.1 Experimental animals

[0091] Healthy SPF c57BL / 6 male mice, 6-7 weeks old, weighing about 20 g, were purchased from Hangzhou Hangsi Biotechnology Co., Ltd. The feeding and experimental procedures complied with the relevant work operation regulations of the Animal Ethics Committee.

[0092] 2.2 Reagents and consumables

[0093] Reagents and consumables are listed in the table below.

[0094] Table 3: Reagents and consumables information

[0095]

[0096]

[0097] 2.3 Instruments and Equipment

[0098] The instruments and equipment are shown in the table below.

[0099] Table 4: Instrument information

[0100] name factory Dehydrator (ASP300) Shanghai Leica Instruments Co., Ltd. Embedding machine (JB-L5) Wuhan Junjie Electronics Co., Ltd. Pathology Slice Machine (RM2235) Shanghai Leica Instruments Co., Ltd. Freezing Table (JB-L5) Wuhan Junjie Electronics Co., Ltd. Tissue Spreading Machine (JK-5) Wuhan Junjie Electronics Co., Ltd. Oven (101-313) Shanghai Lichen Bangxi Instrument Technology Co., Ltd. Glass slides Servicebio Upright optical microscope (CM2000) WALINOVEA

[0101] 2.4 Experimental samples

[0102] Under a sterile environment, first take 10 ml of normal saline to prepare 10 mg / ml sodium bicarbonate, then add 20 mg of Polygala tenuifolia sapogenin, stir at room temperature for about 30 seconds, then let stand for 12 hours, and after the Polygala tenuifolia sapogenin is completely dissolved, filter with a 0.45 um filter membrane to prepare Polygala tenuifolia sapogenin intravenous injection.

[0103] 3. Experimental methods

[0104] 3.1 Experimental Grouping

[0105] SPF male c57BL / 6 mice weighing about 20 g were selected and randomly divided into normal saline group (NS group), solvent (10 g / L sodium bicarbonate solution) control group (RG group), and polygala sapogenin intravenous injection group. The polygala sapogenin intravenous injection groups were divided into group A (30 mg / kg), group B (60 mg / kg), group C (120 mg / kg), and group D (240 mg / kg).

[0106] 3.2 Experimental process and administration method

[0107] Each group of mice was adaptively fed for one week and divided into 6 groups according to body weight, with 5 mice in each group. Each mouse in the drug group was injected with an intravenous injection of the corresponding concentration of Polygala tenuifolia sapogenin according to body weight, and the saline group and the solvent control group were injected with equal amounts of saline and solvent, respectively, and the injection method was injection from the tail vein. Then, each group of mice was returned to the mouse breeding room and freely obtained drinking water and food. The body weight was continuously monitored for 7 days, and samples (brain, heart, liver, spleen, lung, and kidney) were taken on the seventh day. The mice were weighed before anesthesia. Before taking samples, each mouse was injected with 10g / L sodium pentobarbital according to body weight. After anesthesia, blood was taken from the heart, and then 20ml of 40g / L paraformaldehyde was used for cardiac perfusion. When the mouse muscle contraction, chest arch, tail swinging and other phenomena were observed, and the limbs became stiff, it indicated that the perfusion was successful. After removing the mouse organs, wipe off the surface moisture and immediately weigh them with an animal tissue scale. Then, the organs are divided into two parts. One part is placed in paraformaldehyde fixative to prepare paraffin-embedded sections, and the other part is used for drying to measure the wet-to-dry ratio of each mouse organ.

[0108] 3.3 Body weight changes of mice in each group

[0109] After each group of mice were injected with drugs or saline via the tail vein, the weight of the mice was recorded every day starting from that day and monitored continuously for 7 days to observe whether the vital signs of the mice were affected.

[0110] 3.4 Measurement of organ-brain ratio, organ-body ratio, and wet-to-dry ratio of brain, heart, liver, spleen, lung, and kidney tissues

[0111] The mice in each group were weighed before sampling, and the wet weight of each tissue was weighed after sampling. They were then placed in a 65°C oven for 24 h until the weight no longer changed, and then the dry weight of each tissue was weighed.

[0112] The calculation formula is as follows:

[0113] Organ-brain ratio = mouse organ mass / brain mass

[0114] Organ-to-body ratio = mouse organ mass / mouse body weight

[0115] Wet-to-dry ratio = wet weight of mouse organs / dry weight of mouse organs

[0116] 3.5 HE staining of paraffin-embedded sections of important organs of mice

[0117] 3.5.1 Dewax the paraffin sections to water: sequentially place the sections in xylene I for 10 min, xylene II for 10 min, xylene III for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 85% alcohol by volume for 5 min, 75% alcohol by volume for 5 min, and pure water for 5 min.

[0118] 3.5.2 Hematoxylin staining: Stain the sections with hematoxylin solution for 3-5 minutes, wash with tap water, differentiate with differentiation solution, wash with tap water, reblue with bluing solution, and rinse with running water.

[0119] 3.5.3 Eosin staining: The sections were dehydrated in 85% and 95% alcohol by volume for 4 min each, and then stained in eosin solution for 10 s.

[0120] 3.5.4 Dehydration and sealing: The sections were placed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene I for 5 min, xylene II for 5 min, and then sealed with transparent sealing medium.

[0121] 3.5.5 Microscope examination, image acquisition and analysis.

[0122] 3.6 Routine blood tests of mice in each group

[0123] After anesthesia, blood was collected from the heart of each group of mice, immediately placed in an EDTA anticoagulant tube, and carefully shaken to allow the blood sample and EDTA anticoagulant to be fully mixed to prevent blood coagulation.

[0124] 3.7 Data Analysis

[0125] The experimental data are expressed as mean ± standard deviation, and n represents the number of samples. GraphPad Prism software was used for statistical analysis. The differences between the groups were analyzed using one-way ANOVE. P < 0.05 was considered statistically significant (*).

[0126] 4. Experimental results

[0127] 4.1 Changes in mouse body weight

[0128] The results of the mouse weight change experiment are shown in Fig.10 Compared with the saline group and the solvent control group, there was no statistically significant difference in the weight change of mice in groups A, B, C, and D over 7 days (n=5, P>0.05). This indicates that intravenous injection of polygala sapogenin with sodium bicarbonate as solvent does not affect the physical development of mice.

[0129] 4.2 Results of organ-brain ratio, organ-body ratio, and wet-to-dry ratio of brain, heart, liver, spleen, lung, and kidney tissues

[0130] The results of the brain-to-brain ratio experiment are shown in Fig.11 , the results of the organ-body ratio experiment are shown in Fig.12 , wet-to-dry ratio test results are shown in Fig.13Compared with the saline group and the solvent control group, there were no significant differences in the organ-brain ratio, organ-body ratio, and wet-to-dry ratio of mice in groups A, B, C, and D (n=5, p>0.05). This indicates that intravenous injection of polygala sapogenin with sodium bicarbonate as solvent did not cause congestion, edema, hyperplasia, or atrophy of the mouse organs.

[0131] 4.3 HE staining results of important organs of mice

[0132] HE staining of organs Fig.14 Compared with the saline group, there was no significant difference in the tissue structure of the main organs (brain, heart, liver, spleen, lung, and kidney) of the mice in the drug group and the solvent control group. This shows that intravenous injection of polygala sapogenin solution with sodium bicarbonate solution as the solvent will not cause obvious pathological damage to important organs.

[0133] 4.4 Routine blood tests of mice in each group

[0134] The blood routine test results of each group of mice are shown in the table below. Compared with the normal saline group (NS group) and the solvent control group (RG group), the blood routine test indicators of the Polygala tenuifolia sapogenin intravenous injection group were within the normal reference value range, indicating that the intravenous injection of Polygala tenuifolia sapogenin intravenous injection with sodium bicarbonate solution as the solvent will not affect the blood of the body.

[0135] Table 5: Blood routine test results of mice

[0136]

[0137]

[0138] The above experimental results show that the intravenous injection of Polygala tenuifolia sapogenin using sodium bicarbonate solution as solvent is relatively safe.

[0139] Example 4

[0140] 1. Experimental purpose

[0141] To investigate whether intravenous injection of Polygala tenuifolia sapogenin can improve the survival rate of septic mice.

[0142] 2. Experimental Animals

[0143] One hundred healthy SPF c57BL / 6 male mice, aged 6 to 7 weeks and weighing about 20 g, were purchased from Hangzhou Hangsi Biotechnology Co., Ltd. The feeding and experimental procedures complied with the relevant work operation regulations of the Animal Ethics Committee.

[0144] 3. Experimental methods and process

[0145] After one week of adaptive feeding, the mice were randomly divided into four groups according to their body weight: sham group (10 mice), cecal ligation and puncture group (CLP group) (30 mice), cecal ligation and puncture + solvent control group (CLP+RG group) (30 mice), and cecal ligation and puncture + polygala sapogenin intravenous injection group (CLP+SEN group) (30 mice). The mice of CLP model were intraperitoneally injected with sodium pentobarbital (60 mg / kg), then fixed on the operating table, the midline of the abdomen was shaved and the surgical area was disinfected with iodine, the abdominal cavity was incised about 1 cm along the linea alba, the cecum was carefully separated and exposed, and the 1 / 2 of the length of the cecum was ligated with No. 4 thread. After ligation, a 21G needle was used to puncture about 0.5 cm above the blind end of the cecum, and the needle was pierced twice and a small amount of cecal contents were squeezed out from the needle hole, and then the cecum was carefully put back into the abdominal cavity. The mesentery should be carefully avoided during the whole process, and finally the surgical incision was sutured layer by layer. After surgery, the mice were returned to the warming pad and each mouse was subcutaneously injected with 0.5 ml of 37°C preheated saline for resuscitation. 4 h before surgery, mice in the CLP+SEN group received tail vein injection of polygala sapogenin (12 mg / kg) according to body weight. Mice in the CLP and CLP+RG groups received tail vein injection of equal amounts of saline and solvent (10 mg / ml sodium bicarbonate solution), respectively. In the sham operation group, the cecum was exposed for 1 minute after laparotomy and returned to the abdominal cavity without ligation and puncture. 4 h after surgery, mice in the CLP, CLP+RG, and CLP+SEN groups received tail vein injection of equal amounts of saline, solvent, and SEN (12 mg / kg), respectively. Thereafter, mice in the CLP, CLP+RG, and CLP+SEN groups received tail vein injection of equal amounts of saline, solvent, and SEN (12 mg / kg) once every 24 h until the fourth day after surgery. The vital signs of mice in each group were observed every day, and the weight changes of mice were recorded.

[0146] 24 hours after CLP surgery, the mouse sepsis score (MSS) of each group of mice was evaluated, and the weight of the mice and the mortality of the mice in each group were recorded every day.

[0147] 4. Experimental results and analysis

[0148] 4.1 Measurement of mouse sepsis score (MSS)

[0149] According to the method in the paper "Aloe-Emodin Ameliorates Cecal Ligation and Puncture-Induced Sepsis" (2023) by Su J et al., sepsis scores were performed on each group of mice 24 hours after surgery.

[0150] The measurement results of mouse sepsis score (MSS) are shown in the table below and Fig.15The Sham group was the sham operation group, the CLP group was the sepsis model group, the CLP+RG group was the sepsis+solvent control group, and the CLP+SEN group was the sepsis+polygala sapogenin intravenous injection group. Compared with the Sham group, the CLP+SEN group a P<0.05; CLP+SEN group was compared with CLP group. b P<0.05; CLP+SEN group was compared with CLP+RG group. c P<0.05. GraphPad Prism software was used for statistical analysis. One-way ANOVE was used to analyze the differences between the groups. P<0.05 was considered statistically significant.

[0151] Compared with the CLP group and CLP+RG group, the sepsis score of mice in the CLP+SEN group was significantly reduced (p<0.05), indicating that the intravenous injection of Polygala tenuifolia sapogenin with sodium bicarbonate solution as solvent has a good therapeutic effect and its drug effectiveness is high.

[0152] Table 6: Measurement results of mouse sepsis score (MSS)

[0153]

[0154] 4.2 Changes in 7-day mortality and body weight of mice in each group

[0155] The changes in the 7-day mortality rate of each group of mice are shown in the table below, and the changes in the survival rate are shown in Fig.16 ; The changes in body weight of mice in each group for 7 days are shown in Fig.17 .

[0156] After CLP surgery, mice in each group had a high mortality rate within 24-48 hours. Compared with the CLP group and CLP+RG group, the survival rate of mice in the CLP+SEN group was significantly increased (p<0.05). Except for the sham operation group, the weight of mice in each group dropped sharply after surgery. There was no statistical difference in the weight changes of mice in the CLP group, CLP+RG group, and CLP+SEN group. However, it can be seen that from the fourth day after surgery, the weight of mice in the CLP+SEN group began to increase slowly, while the weight of mice in the CLP group and CLP+RG group remained unchanged or was still decreasing.

[0157] Table 7: Changes in the 7-day mortality rate of mice in each group

[0158]

[0159]

[0160] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A method for preparing a polygala sapogenin intravenous injection, characterized in that: The following steps are involved: Step S1, preparing a sodium bicarbonate solution using physiological saline to make the sodium bicarbonate concentration be 5 mg / mL to 15 mg / mL; The sodium chloride concentration of the physiological water is 8 mg / mL to 9 mg / mL; Step S2, adding polygala sapogenin to a sodium bicarbonate solution, stirring and dissolving, the dissolving temperature is 4° C. to 65° C., and the stirring time is 1 hour to 3 hours; Step S3, standing for 12 to 16 hours to completely dissolve the polygala sapogenin, making the concentration of polygala sapogenin reach 0.5 mg / mL to 33 mg / mL, and adjusting the pH value to 6.5 to 8.5; Step S4, filtering and packaging.

2. The preparation method according to claim 1, characterized in that: The pH value of the sodium bicarbonate solution is 8.

3.

3. The preparation method according to claim 2, characterized in that: The physiological saline is of injection grade; the sodium bicarbonate is of pharmaceutical grade; and the polygala sapogenin is of pharmaceutical grade.

4. The preparation method according to claim 3, characterized in that: The pore size of the filter material used in the filtration is 0.45 microns.

5. The preparation method according to claim 2, characterized in that: The filtration is sterilizing filtration.

6. The preparation method according to claim 5, characterized in that: The preparation process is carried out under a sterile environment.

7. The preparation method according to claim 6, characterized in that: The step S4 also includes sterilization.

8. A polygala sapogenin intravenous injection, characterized in that: The polygala sapogenin intravenous injection is prepared by any one of the preparation methods described in claims 1 to 8.

9. The polygala sapogenin intravenous injection according to claim 8, characterized in that The polygala sapogenin intravenous injection is a colorless, odorless, transparent liquid.

10. The polygala sapogenin intravenous injection according to claim 8, characterized in that: The concentration of the polygala sapogenin intravenous injection is 2 mg / ml.

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