Disclosure of Invention
The invention provides a preparation method of aescin sodium-hydroxy carthamin yellow A hydrogel, and the first aim is to provide the aescin sodium-hydroxy carthamin yellow A hydrogel prepared by the preparation method, and the third aim is to provide the application of the hydrogel in preparing medicines for treating sepsis.
The above object of the present invention is achieved by the following technical scheme:
A process for preparing the aqueous gel of aescin sodium-hydroxy safflor yellow A includes such steps as self-assembling the aescin sodium and hydroxy safflor yellow A in alkaline aqueous solution to obtain aqueous gel, or dissolving aescin sodium and hydroxy safflor yellow A in organic solvent, removing organic solvent to obtain vesicles, and self-assembling in alkaline aqueous solution to obtain aqueous gel.
Preferably, the alkaline aqueous solution comprises NaOH solution, KOH solution and PBS buffer solution.
Preferably, the organic solvent includes methanol and ethanol.
More preferably, the method for self-assembling the aescin sodium and the hydroxysafflor yellow A into the hydrogel directly in an alkaline aqueous solution comprises the following steps of weighing the aescin sodium and the hydroxysafflor yellow A, dissolving in the alkaline aqueous solution to obtain an aqueous solution, heating the obtained aqueous solution at a constant temperature, standing and cooling at the constant temperature to obtain the aescin sodium-hydroxysafflor yellow A hydrogel.
More preferably, the method for preparing the hydrogel by self-assembling the aescin sodium and the hydroxysafflor yellow A in an alkaline aqueous solution comprises the steps of weighing aescin sodium and hydroxysafflor yellow A, dissolving the aescin sodium and hydroxysafflor yellow A in the organic solvent to obtain an organic solution, removing the organic solvent from the obtained organic solution to obtain vesicles, dissolving the obtained vesicles in the alkaline aqueous solution, heating at constant temperature, standing and cooling at normal temperature to obtain the aescin sodium-hydroxysafflor yellow A hydrogel.
More preferably, the ratio of the aescin sodium to the hydroxysafflor yellow A in the alkaline aqueous solution or the organic solvent is (0.5-100): 0.5-50 g/L. In a specific embodiment, the ratio of the aescin sodium to the hydroxysafflor yellow A in the alkaline aqueous solution or the organic solvent is (8-60): 1-30 g/L.
More preferably, the pH of the alkaline aqueous solution is 8.0 to 14.0.
More preferably, the constant temperature heating temperature is 30-100 ℃.
The aescin sodium-hydroxy carthamin yellow A hydrogel prepared by the preparation method is provided.
Application of the aescin sodium-hydroxy carthamin yellow A hydrogel in preparing medicines for treating sepsis is provided.
The beneficial effects are that:
1. The invention provides a preparation method of aescin sodium-hydroxy carthamin yellow A hydrogel, which is simple and stable, and can be used for preparing the injectable aescin sodium-hydroxy carthamin yellow A hydrogel with high stability, and the aescin sodium-hydroxy carthamin yellow A hydrogel has remarkable treatment effect on sepsis of mice;
2. The aescin sodium-hydroxy safflor yellow A hydrogel obtained by the invention adopts natural products as raw materials, does not need to add other auxiliary materials or complex reagents, catalysts and the like, has high safety and good biocompatibility, can be biodegraded, and is suitable for the pharmaceutical field;
3. The applicant previously prepares an aescine injectable hydrogel (see patent CN 111249226A), the principle is that aescine is dissolved in alkaline aqueous solution, and the aescine is self-assembled to form nano fibers through hydrogen bonds, pi-pi stacking, electrostatic acting force, van der Waals force, coordination bonds and other non-covalent acting forces, and the nano fibers are further self-assembled to form the hydrogel with a three-dimensional network structure. Before the technical scheme of the invention is put forward, the applicant tries a plurality of other natural products and aescin sodium hydrogels, but the expected effect is not achieved, mainly because the natural products and aescin sodium coexist to influence the non-covalent acting forces such as hydrogen bonds, pi-pi stacking, electrostatic acting forces, van der Waals forces, coordination bonds and the like among aescin molecules, and interfere the self-assembly of the natural products and the aescin sodium to form nanofibers so as to form the hydrogels. The applicant has found that the hydroxy safflower yellow A and the aescin sodium can form hydrogel, can be injected, has high stability, realizes slow release effect, and can simultaneously exert the drug effects of the two natural products of the hydroxy safflower yellow A and the aescin sodium;
4. The aescin sodium-hydroxy carthamin yellow A hydrogel can be injected intravenously and does not block blood vessels. The injectable hydrogel in the prior art is mainly prepared by locally administering high polymers such as biological polysaccharide in situ, and few supermolecular hydrogels composed of nano particles, liposome, polypeptide and the like are reported at present compared with the hydrogel which can not block blood vessels by intravenous injection, wherein the research on tail vein injection is almost not available.
Detailed Description
The following describes the essential aspects of the present invention in detail with reference to examples, but is not intended to limit the scope of the present invention.
EXAMPLE 1 preparation example
The aescin sodium 8 mg and the hydroxy carthamin yellow A1 mg are weighed and dissolved in NaOH solution with the 1mL pH of 8 to obtain aqueous solution. Then heating the obtained water solution at a constant temperature of 30 ℃ for 48 h, standing and cooling at normal temperature to obtain aescin sodium-hydroxy carthamin yellow A hydrogel (ES-HSYA@gel-1).
The NaOH solution can also be replaced by other alkaline aqueous solutions, such as KOH solution and PBS buffer solution, and the pH is preferably 8.0-14.0.
EXAMPLE 2 preparation example
Weighing aescin sodium 35 mg, and dissolving hydroxysafflor yellow A20 mg in 15mL ethanol to obtain ethanol solution. And removing the organic solvent from the obtained ethanol solution by using a rotary evaporator (water bath can be used at the temperature of 30-100 ℃) to obtain the vesicle-1. Then the vesicle-1 is dissolved in 1mL NaOH solution with pH of 12, heated for 12h at the constant temperature of 60 ℃, and kept stand and cooled at the normal temperature to obtain aescin sodium-hydroxy carthamin A hydrogel (ES-HSYA@gel-2).
The NaOH solution can also be replaced by other alkaline aqueous solutions, such as KOH solution and PBS buffer solution, and the pH is preferably 8.0-14.0.
EXAMPLE 3 preparation example
Weighing aescin sodium 60 mg, and dissolving hydroxysafflor yellow A30 mg in 30 mL methanol to obtain methanol solution. And (3) keeping the temperature of the obtained methanol solution at a constant temperature of 1h at 70 ℃, and removing the organic solvent by using a rotary evaporator (all water baths can be used at 30-100 ℃), so as to obtain the vesicle-2. Then, the vesicle-2 obtained was dissolved in 1mL of NaOH solution having a pH of 14, heated at a constant temperature of 100℃for 0.5 hour, and cooled at a constant temperature to obtain sodium aescinate-hydroxy carthamin A hydrogel (ES-HSYA@gel-3).
The NaOH solution can also be replaced by other alkaline aqueous solutions, such as KOH solution and PBS buffer solution, and the pH is preferably 8.0-14.0.
Example 4 characterization of morphology and Performance detection
1. Appearance characterization
The appearance of the aescin sodium-hydroxy carthamin yellow A hydrogel (ES-HSYA@gel-2) prepared in example 2 was recorded with a digital camera, as shown in FIG. 1 (a is an aqueous alkali solution before gel formation, b is the gel formation of the hydrogel for 1 day, and c is the gel formation of the hydrogel for 30 days), and no significant change was observed after gel formation, indicating that the hydrogel had good stability. The sodium aescinate-hydroxy carthamin yellow A hydrogel prepared in other examples has similar characteristics.
2. Microcosmic topography characterization
The microstructure of the aescin sodium-hydroxy carthamin yellow A hydrogel (ES-HSYA@gel-2) prepared in example 2 was observed by using a Scanning Electron Microscope (SEM) and a Transmission Electron Microscope (TEM). As shown in fig. 2a, SEM shows that the microscopic morphology of the hydrogel sample presents a three-dimensional network of hollow spherical shells with a size of 0.1-1 μm and micron-sized fibers, which may be related to the self-assembly of aescin sodium-hydroxysafflor yellow a vesicles into fibers. TEM shows that the sample of the aqueous solution of the vesicle alkali (i.e. the aqueous solution of the alkaline water before gel formation) is spherical vesicle with the diameter of about 110 nm (b in fig. 2), which is probably formed by self-assembly of amphipathic molecule aescin sodium and hydroxysafflor yellow A in water, and the vesicle structure is favorable for cell uptake through endocytosis, and has important significance for improving the bioavailability of the medicine and prolonging the acting time of the medicine.
The sodium aescinate-hydroxy carthamin yellow A hydrogel prepared in other examples has similar characteristics.
3. Infrared spectrogram and ultraviolet spectrogram
FIG. 3 is an infrared spectrum and an ultraviolet spectrum of sodium aescinate-hydroxy carthamin yellow A hydrogel (ES-HSYA@gel-2) prepared in example 2. In the infrared spectrogram (fig. 3 a), sodium aescinate (ES), hydroxysafflor yellow a (HSYA), ES-hsya@gel all showed O-H stretching peaks (about 3350 cm-1), C-H stretching peaks of methyl and methylene (about 2920 cm-1、2850 cm-1) and bending peaks thereof (about 1420 cm-1、1380 cm-1), and alkene=c-H out-of-plane bending peak (about 890 cm-1). In addition, C-O stretching vibration peaks (1240 cm-1、1170 cm-1 or so) of ether bond and hydroxyl group in aescin sodium are reserved in ES-HSYA@gel, and benzene ring skeleton vibration peaks (1560 cm-1 or so) in hydroxyl carthamin A are reserved in ES-HSYA@gel. In the UV-Vis spectrum (fig. 3 b), the characteristic absorption peaks of sodium aescinate are located at 200 nm, 220 and nm, and are related to the n→pi transition of c= C, C =o in its molecular structure. The characteristic absorption peaks of the hydroxysafflor yellow A are 195 nm, 223 nm, 260 nm, 330 nm and 400 nm, and are because the molecular structure contains benzene rings and C= C, C =O and is conjugated, and the absorption in ultraviolet and visible light regions is strong. The positions of the ES-HSYA@gel at the positions of 207 nm ultraviolet absorption peaks are red shifted, the intensities of 260 nm and 330 nm ultraviolet absorption peaks are relatively increased, and the intensity of 400 nm ultraviolet absorption peaks is relatively reduced, probably due to the molecular structure changes of aescin sodium and hydroxy carthamin A in the gelling process.
The sodium aescinate-hydroxy carthamin yellow A hydrogel prepared in other examples has similar characteristics.
4. Injectability of
Fig. 4 shows that the aescin sodium-hydroxy carthamin yellow A hydrogel (ES-HSYA@gel-2) prepared in example 2 has smooth injection process, no obvious blocking phenomenon, can quickly recover to form stable gel at normal temperature, maintains a good shape, has the potential of local administration, and provides a good platform for drug delivery and tissue engineering. The sodium aescinate-hydroxy carthamin yellow A hydrogel prepared in other examples has similar characteristics.
5. Rheological behavior
The sodium aescinate-hydroxysafflor yellow A hydrogel (ES-HSYA@gel-2) prepared in example 2 was subjected to strain scanning, and as shown in FIG. 5 a, the storage modulus (G ') and loss modulus (G ' ') exhibited typical gel behavior as a function of strain. Constant in a low strain range, and G ' is higher than G ' ', exhibits an elastic-based solid behavior, and has a stable network structure. When the strain exceeds 10%, G' decreases and g″ increases, the hydrogel transitions from elastic behavior to viscous behavior until the intersection point hydrogel structure is broken and flow occurs. The hydrogel was subjected to viscosity scanning, as shown in fig. 5 b, and at low shear rates, the hydrogel exhibited higher viscosity, with progressive decrease in apparent viscosity as the shear rate increased, dissociation of the gel occurred, and flowability was enhanced. The sodium aescinate-hydroxy carthamin yellow A hydrogel prepared in other examples has similar characteristics.
6. XRD pattern
The sodium aescinate-hydroxysafflor yellow A hydrogel (ES-HSYA@gel-2) prepared in example 2 was subjected to X-ray diffraction, as shown in FIG. 6. Sodium aescinate (8 °, 12 °, 40 °), hydroxysafflor yellow a (8 °, 23 °), and hydrogel (8 °, 12 °, 23 °) show diffraction peaks corresponding to those of single component, but the peak intensity is significantly reduced, probably due to the formation of a large number of amorphous regions in the hydrogel, resulting in reduced crystallinity. The sodium aescinate-hydroxy carthamin yellow A hydrogel prepared in other examples has similar characteristics.
7.1 H NMR spectra
FIG. 7 is a1 H NMR spectrum of sodium aescinate-hydroxysafflor yellow A hydrogel (ES-HSYA@gel-2) prepared in example 2. The1 H NMR spectrum of sodium aescinate shows a number of characteristic peaks corresponding to complex proton environments in its steroidal structure, H in the higher field region (δ0.5-2.0 ppm) being-CH3、-CH2 -on the alicyclic ring and chain, H in the lower field region (δ3.0-5.0 ppm) being-O-CH2 -, -O-CH-, -ch=ch-, and H being chemically displaced by adjacent groups (e.g. c=o). The sodium aescinate-hydroxy carthamin yellow A hydrogel prepared in other examples has similar characteristics.
Example 5 animal experiment
1. Experimental animal
75C 57BL/6 mice of 6-8 weeks old, male, body weight 22+ -1 g, from Henan Seebeck laboratory animal Co., ltd, animal eligibility number SCXK (Yu) 2020-0005. The animal house of the Changsha medical college is fed with the feed, the environmental temperature is controlled to be 20+/-1 ℃, the relative humidity is controlled to be 50+/-10%, the illumination period is 12 hours of illumination/12 hours of darkness, and standard feed and sterilized drinking water are provided for free feeding and drinking water.
2. Experimental reagent
Dexamethasone sodium phosphate injection (Hubei jin pharmaceutical industry Co., ltd., national medicine standard H42020019), hydroxy safflower yellow A (Nanjing spring and autumn bioengineering Co., ltd., purity 98%), aescin sodium (Shanghai source leaf biotechnology Co., ltd., purity 98%), ES-HSYA@gel hydrogel prepared in example 2, and the preparation method of ES-HSYA@gel hydrogel is the same as that of ES-HSYA@gel except that HSYA is not added. An air-hemp machine. All reagents are prepared and used according to specifications or experimental requirements, and the preservation conditions meet the requirements of the reagents.
3. Experimental method
After the mice were adaptively bred for one week, they were randomly divided into 5 groups, including 15 groups of model group (CLP group), hydroxysafflor yellow a aqueous solution group (HSYA group), aescine sodium hydrogel group (es@gel group), aescine sodium-hydroxysafflor yellow a hydrogel group (ES-hsya@gel group), dexamethasone group (DEX group), respectively. Modeling method sepsis models were created by anesthetizing mice, exposing the midline incision in the abdomen, ligating the cecum (about 60% length), puncturing the cecum with a sterile 21G needle, squeezing out a few stool, and then retracting the cecum and closing the abdomen. Postoperative subcutaneous injection of physiological saline 1ml promotes recovery. After molding, 4 h, each group was given 1 dose by tail vein injection, and then no more doses were given, mice were continuously observed for 7 day survival rate, and mice body temperature and body weight were recorded. Statistical analysis was performed using GRAPHPAD PRISM 9.5.0 statistical software, with P <0.05 statistically significant for differences.
CLP group model mice were injected with the same volume of PBS solution by tail vein, and after molding for 4 hours, were injected by tail vein for 1 time, and observed continuously for 7 days.
HSYA group, in which the model mice were given an aqueous solution of hydroxysafflor yellow A by intravenous injection at a dose of 4.5mg/kg based on the weight of the mice, and were given by tail intravenous injection for 1 time after molding for 4 hours, followed by continuous observation for 7 days;
ES@gel group, namely, model mice are injected with sodium aescinate hydrogel (ES@gel) intravenously, the composition is administrated according to the weight of the mice, the dose is 6.8mg/kg based on sodium aescinate, the composition is administrated by tail vein injection for 1 time after molding for 4 hours, and the composition is continuously observed for 7 days;
The ES-HSYA@gel group is characterized in that the model mouse tail is injected with ES-HSYA@gel intravenously, the dose is 11.3 mg/kg according to the weight of the mouse, wherein the content of aescin sodium is 6.8mg/kg, the content of hydroxysafflor yellow A is 4.5mg/kg, the tail is injected with the model mouse tail for 1 time after molding for 4 hours, and the model mouse tail is observed continuously for 7 days;
Dexamethasone group, model mice were given a solution of dexamethasone Mi Songshui by intravenous injection at a dose of 2mg/kg according to body weight of the mice, and after molding for 4 hours, were given by intravenous injection at the tail for 1 time, and continuous observation was carried out for 7 days.
① Body temperature analysis body temperature changes were recorded for each group of mice 7 days after dosing.
② Body weight analysis body weight changes were recorded for each group of mice 7 days after dosing.
4. Experimental results
Fig. 8 shows the survival rate of 7d (n=15) for different groups of mice. Compared with the CLP group, the hydroxyl carthamin yellow A aqueous solution (HYSA) group and the Dexamethasone (DEX) group have no obvious improvement on the survival rate of 7d of mice (P > 0.05), the esculin sodium hydrogel (ES@gel) group and the esculin-hydroxyl carthamin yellow A hydrogel (HSYA-ES@gel) group can improve the survival rate of 7d of mice (P < 0.05), and the effect of the HSYA-ES@gel group is the best (P < 0.0001), so that the hydrogel has obvious treatment effect on sepsis of mice.
Fig. 9 shows the body temperature (n=15) of different groups of mice. Except for the more constant body temperature of the HSYA-ES@gel group, the remaining groups exhibited a relaxation Zhang Re (difference in early and late body temperatures >2 ℃), indicating that the HSYA-ES@gel group can maintain sepsis mouse body temperature.
Figure 10 shows the body weights of the different groups of mice (n=15). The weight change of each group of mice is almost the same, and the mice are in good condition.
It can be seen that, in summary:
1. The invention provides a preparation method of aescin sodium-hydroxy carthamin yellow A hydrogel, which is simple and stable, and can be used for preparing the injectable aescin sodium-hydroxy carthamin yellow A hydrogel with high stability, and the aescin sodium-hydroxy carthamin yellow A hydrogel has remarkable treatment effect on sepsis of mice;
2. The aescin sodium-hydroxy safflor yellow A hydrogel obtained by the invention adopts natural products as raw materials, does not need to add other auxiliary materials or complex reagents, catalysts and the like, has high safety and good biocompatibility, can be biodegraded, and is suitable for the pharmaceutical field;
3. The applicant previously prepares an aescine injectable hydrogel (see patent CN 111249226A), the principle is that aescine is dissolved in alkaline aqueous solution, and the aescine is self-assembled to form nano fibers through hydrogen bonds, pi-pi stacking, electrostatic acting force, van der Waals force, coordination bonds and other non-covalent acting forces, and the nano fibers are further self-assembled to form the hydrogel with a three-dimensional network structure. Before the technical scheme of the invention is put forward, the applicant tries a plurality of other natural products and aescin sodium hydrogels, but the expected effect is not achieved, mainly because the natural products and aescin sodium coexist to influence the non-covalent acting forces such as hydrogen bonds, pi-pi stacking, electrostatic acting forces, van der Waals forces, coordination bonds and the like among aescin molecules, and interfere the self-assembly of the natural products and the aescin sodium to form nanofibers so as to form the hydrogels. The applicant has found that the hydroxy safflower yellow A and the aescin sodium can form hydrogel, can be injected, has high stability, realizes slow release effect, and can simultaneously exert the drug effects of the two natural products of the hydroxy safflower yellow A and the aescin sodium;
4. The aescin sodium-hydroxy carthamin yellow A hydrogel can be injected intravenously and does not block blood vessels. The injectable hydrogel in the prior art is mainly prepared by locally administering high polymers such as biological polysaccharide in situ, and few supermolecular hydrogels composed of nano particles, liposome, polypeptide and the like are reported at present compared with the hydrogel which can not block blood vessels by intravenous injection, wherein the research on tail vein injection is almost not available.
The above-described embodiments serve to describe the substance of the present invention in detail, but those skilled in the art should understand that the scope of the present invention should not be limited to this specific embodiment.