Lipopolysaccharide oral absorption enhancer and preparation method thereof

Abstract

The invention belongs to the technical field of biological medicine, and discloses a lipopolysaccharide oral absorption enhancer and a preparation method thereof, the lipopolysaccharide oral absorption enhancer comprises a rutabaga aqueous extract and a second accelerator, and the second accelerator is selected from at least one of chitosan, ethanol or sodium caprylate. The oral lipopolysaccharide absorption enhancer can significantly improve the oral absorption enhancing effect of lipopolysaccharide, and lays a foundation for the protection effect of LPS on tumor-associated macrophages after oral administration.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a lipopolysaccharide oral absorption enhancer and its preparation method. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Traditional cancer treatment primarily involves local and systemic therapies, with drug therapy being one of the main approaches. However, while anti-tumor drugs kill tumor cells, they indiscriminately damage immune cells within the tumor microenvironment (TME), leading to immunosuppression. Furthermore, they attack normal off-target tissues, affecting other rapidly regenerating normal tissues throughout the body, thus causing systemic side effects. Therefore, finding adjuvant drugs to alleviate the side effects of anti-tumor drugs is urgently needed.

[0004] Lipopolysaccharide (LPS) is a key component of the outer membrane of Gram-negative bacteria and is often referred to as endotoxin. Its classic structure consists of three parts: lipid A, core oligosaccharide, and O antigen, and it is an important microbial trigger for stimulating innate immunity. LPS has a protective effect against doxorubicin-damaged RAW 264.7 macrophages in the tumor microenvironment without affecting the killing effect of doxorubicin on tumor cells. Lipid A is the main structure by which LPS exerts its protective effect against chemotherapeutic drug-damaged macrophages. After entering macrophages via endocytosis and phagocytosis, LPS is transported to lysosomes via tubulin, activating lysosome-associated proteins to protect against chemotherapeutic drug-damaged RAW 264.7 macrophages. Simultaneously, LPS significantly upregulates system Xc The expression of the constituent genes SLC3A2 and SLC7A11 promotes the system Xc The expression of GST increases the activity of GST enzyme, thereby promoting GSH synthesis. This allows doxorubicin to bind conjugated with GSH and be excreted into the extracellular space via ABCC1, thus reducing the doxorubicin content in RAW 264.7 and playing a protective role for macrophages.

[0005] However, because LPS is a glycolipid complex with a relatively unique chemical structure and a large molecular weight, its absorption rate in the intestine after oral administration is low, failing to fully exert its protective effect against doxorubicin-damaged RAW264.7 macrophages in the tumor microenvironment. Although existing technologies contain drug absorption enhancers, their absorption-enhancing effects on LPS are poor, still failing to meet the requirements. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide an oral absorption enhancer for lipopolysaccharides and its preparation method.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides a lipopolysaccharide oral absorption enhancer, comprising a water extract of *Rhizopus chinensis* and a second enhancer, wherein the second enhancer is selected from at least one of chitosan, ethanol, or sodium caprylate.

[0008] Secondly, the present invention provides a method for preparing the lipopolysaccharide oral absorption enhancer, comprising the following steps: Clean the thorny bumps thoroughly and crush them for later use; Mix the pulverized raw material with water and perform hydrothermal extraction at 80-100℃ for 2-5 hours to obtain the extract. After repeated extraction, the extracts were combined and dried to obtain the extract. The extract was mixed with the second promoter in a certain proportion to obtain the lipopolysaccharide oral absorption promoter.

[0009] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows: This invention demonstrates the feasibility of oral absorption of lipopolysaccharide (LPS), providing a theoretical basis for future oral administration of LPS to exert its protective effect on doxorubicin-damaged RAW264.7 macrophages in the tumor microenvironment.

[0010] This invention, through screening various absorption enhancers, identified an absorption enhancer that can significantly improve the oral absorption of LPS. A drug composition consisting of two absorption enhancers can induce a significant decrease in transmembrane resistance (TEER) in a Caco-2 cell monolayer model, indicating that tight junctions (TJs) between intestinal epithelial cells are opened, increasing the permeation channels of LPS. Moreover, this effect can be gradually restored after drug removal, demonstrating its safety and reversibility, further laying the foundation for LPS to exert its protective effect on tumor-associated macrophages after oral administration. Attached Figure Description

[0011] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0012] Figure 1 This is a comparative graph showing the effect of adding the drug composition of CS and prickly pear on TEER in Example 1 of the present invention; Figure 2 This is a comparative graph showing the effect of adding the drug composition of CS and prickly pear on TEER in Example 1 of the present invention; Figure 3 This is a comparative graph showing the effect of adding the drug composition of ethanol and prickly pear on TEER in Example 1 of the present invention. Figure 4 This is a comparative graph showing the effect of adding sodium caprylate and the prickly pear drug composition on TEER in Example 1 of the present invention. Figure 5 This is a comparative diagram showing the effect of adding the chitosan and prickly ... Figure 6 This is a comparative graph showing the effect of adding the drug composition of ethanol and prickly pear on LPS transport in Example 3 of the present invention. Figure 7 This is a comparative graph showing the effect of adding the drug composition of sodium caprylate and bursa-pasteurum on LPS transport in Example 3 of the present invention. Detailed Implementation

[0013] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0014] In a first aspect, the present invention provides a lipopolysaccharide oral absorption enhancer, comprising a water extract of *Rhizopus chinensis* and a second enhancer, wherein the second enhancer is selected from at least one of chitosan, ethanol, or sodium caprylate.

[0015] In some embodiments, the mass ratio of the prickly pear water extract and the second promoter is 1:0.5~15.

[0016] In some embodiments, the second promoter is chitosan.

[0017] Preferably, the mass ratio of the water extract of *Rhizopus chinensis* to chitosan is 1:1 to 10.

[0018] Secondly, the present invention provides a method for preparing the lipopolysaccharide oral absorption enhancer, comprising the following steps: Clean the thorny bumps thoroughly and crush them for later use; The pulverized raw material is mixed with water and subjected to hydrothermal extraction at 80-100 ℃ for 1.5-3 hours to obtain the extract. After repeated extraction, the extracts were combined and dried to obtain the extract. The extract was mixed with the second promoter in a certain proportion to obtain the lipopolysaccharide oral absorption promoter.

[0019] In some embodiments, the pulverized raw material is passed through an 80-300 mesh sieve, and the powder particle size is not greater than 180 μm.

[0020] In some embodiments, during hydrothermal extraction, the material-to-liquid ratio is 1:10-30, g:mL.

[0021] In some embodiments, the temperature of hydrothermal extraction is 90-100°C, preferably 95-100°C.

[0022] In some embodiments, the extraction is repeated 1-3 times.

[0023] In some embodiments, the extract is concentrated to 1 / 5 to 1 / 10 of its original volume by rotary evaporation under reduced pressure, and then freeze-dried to obtain the extract.

[0024] In some embodiments, the thorns are collected in September or October.

[0025] The present invention will be further described below with reference to embodiments and experimental examples.

[0026] Example 1 The preparation method of water extract of *Rhizopus chinensis* includes the following steps: Clean the prickly thorns thoroughly, remove impurities, and crush them for later use. The prickly thorns were collected from Yishui County, Linyi City, Shandong Province, during September and October.

[0027] Add the pulverized plant material to a beaker, add 2L of ultrapure water per 100g of plant material, and heat in a 100℃ water bath for 2 hours, stirring constantly to improve extraction efficiency. Repeat the extraction twice under the same conditions and combine the extracts.

[0028] The extract and herbal mixture in the beaker are filtered to remove plant impurities, yielding a hydrothermal extract.

[0029] The obtained hydrothermal extract was concentrated by rotary evaporation under reduced pressure at 50°C until the volume was reduced to 1 / 5 of the original volume. The concentrated solution was then freeze-dried to remove water, yielding the water extract of *Rhizopus spp.*

[0030] Preparation of oral absorption enhancers of lipopolysaccharides: Chitosan CS, sodium octanoate or ethanol and water extract of *Rhizopus chinensis* were mixed with water in a certain proportion to obtain oral absorption enhancers of lipopolysaccharides of different concentrations.

[0031] The experimental method for the effect of oral lipopolysaccharide absorption enhancer on transmembrane resistance in a Caco-2 cell monolayer model includes the following steps: Human colorectal adenocarcinoma cells (Caco-2) were seeded in Transwell chambers at a cell density of 10,000 cells / well and cultured continuously for 7 days, during which their TEER value was continuously monitored. When the TEER was greater than 500 Ω•cm... 2 At that time, the Caco-2 monolayer cell model was successfully constructed and can be used for subsequent detection.

[0032] The upper and lower chamber culture media were aspirated, and each chamber was washed three times with HBSS solution. 400 μL of lipopolysaccharide oral absorption enhancer was added to the top side, and the transepithelium electrical resistance (TEER) was monitored after 180 min. The lipopolysaccharide oral absorption enhancer was then removed, and the chambers were washed three times with HBSS buffer. 400 μL of HBSS buffer was added to the top side, and the TEER value was monitored again in real time for 24 h.

[0033] Figure 1 In this context, "Control" refers to the blank control group, which is the change in the TEER value of the Caco-2 monolayer model without the addition of an absorption promoter over time after the successful construction of the Caco-2 monolayer model. 0.05%CS+800μg / ml refers to the fact that in the oral absorption enhancer of lipopolysaccharide, the mass fraction of chitosan is 0.05% and the concentration of water extract of *Stellaria media* is 800μg / ml. 0.1%CS+800μg / ml refers to the fact that in the oral absorption enhancer of lipopolysaccharide, the mass fraction of chitosan is 0.1% and the concentration of water extract of *Stellaria media* is 800μg / ml. 0.25%CS+800μg / ml refers to the fact that the mass fraction of chitosan in the oral absorption enhancer of lipopolysaccharide is 0.25%, and the concentration of the aqueous extract of *Stellaria media* is 800μg / ml.

[0034] Figure 2 In the above, 0.5%CS+800μg / ml refers to the fact that in the oral absorption promoter of lipopolysaccharide, the mass fraction of chitosan is 0.5% and the concentration of water extract of *Stellaria media* is 800μg / ml. 1%CS+800μg / ml refers to the fact that the mass fraction of chitosan in the oral absorption enhancer of lipopolysaccharide is 1%, and the concentration of water extract of *Stellaria media* is 800μg / ml. Figure 3 In this context, "Control" refers to the blank control group, which is the change in the TEER value of the Caco-2 monolayer model without the addition of an absorption promoter over time after the successful construction of the Caco-2 monolayer model. 10mM ethanol + 800μg / ml thorn refers to the fact that in the oral absorption enhancer of lipopolysaccharide, the molar concentration of ethanol is 10mM and the concentration of water extract of thorn is 800μg / ml. 20mM ethanol + 800μg / ml thorn refers to the fact that in the oral absorption enhancer of lipopolysaccharide, the molar concentration of ethanol is 20mM and the concentration of water extract of thorn is 800μg / ml. 40mM ethanol + 800μg / ml thorn refers to the fact that in the oral absorption enhancer of lipopolysaccharide, the molar concentration of ethanol is 40mM and the concentration of thorn extract in water is 800μg / ml.

[0035] Figure 4 In this context, "Control" refers to the blank control group, which is the change in the TEER value of the Caco-2 monolayer model without the addition of an absorption promoter over time after the successful construction of the Caco-2 monolayer model. 2.5mM sodium caprylate + 800μg / ml thorn refers to the fact that in the oral absorption enhancer of lipopolysaccharide, the molar concentration of sodium caprylate is 2.5mM and the concentration of water extract of thorny thorn is 800μg / ml. 5mM sodium caprylate + 800μg / ml thorn refers to the fact that in the oral absorption enhancer of lipopolysaccharide, the molar concentration of sodium caprylate is 5mM and the concentration of water extract of thorn is 800μg / ml. 10mM sodium caprylate + 800μg / ml thorn refers to the fact that in the oral absorption enhancer of lipopolysaccharide, the molar concentration of sodium caprylate is 10mM and the concentration of thorn extract in water is 800μg / ml.

[0036] The results are as follows Figure 1-4 As shown, treatment of Caco-2 cells with the oral lipopolysaccharide (LPS) absorption enhancer for 3 hours resulted in a significant decrease in the TEER of the cell monolayer. Upon removal of the LPS absorption enhancer, the TEER gradually recovered, returning to its original level after 15 hours. These results indicate that the LPS absorption enhancer can induce the opening of tight junctions (TJs) between Caco-2 cells, and this effect is reversible after the drug composition is removed.

[0037] Example 2 An experimental method for oral absorption of lipopolysaccharide, comprising the following steps: Human colorectal adenocarcinoma cells (Caco-2) were seeded in Transwell chambers at a density of 10,000 cells / well and cultured continuously for 7 days, during which their TEER value was continuously monitored. When the TEER was greater than 500 Ω•cm... 2 At this point, the Caco-2 monolayer cell model was successfully constructed and could be used for subsequent detection. The culture medium in both chambers was aspirated, and both chambers were washed three times with HBSS solution. 400 μL of LPS at different concentrations was added to the upper chamber, and 1000 μL of HBSS solution was added to the lower chamber. The mixture was incubated at 37°C for 120 min. The solution from each lower chamber was then transferred to a black 96-well plate, and the permeability of the test drug was measured.

[0038] In the Caco-2 cell monolayer model, the Papp value of the transported drug showed a good correlation with its absorption in vivo. When Papp > 1 × 10⁻⁶, the absorption rate was significantly higher. -6 When the Papp value is less than 1.0 × 10 cm / s, the drug can be absorbed; conversely, when Papp < ​​1.0 × 10 cm / s, the drug cannot be absorbed. -6 When the flow rate is cm / s, the drug absorption rate is less than 1%. Table 1 shows the oral absorption results of different concentrations of FITC-LPS. The Papp values ​​of LPS at concentrations ranging from 50 ng / mL to 1000 ng / mL are all greater than 1 × 10⁻⁶. -6 The cm / s indicates that LPS can be absorbed, but its permeability is at a low to medium level, and its permeability across Caco-2 is not high.

[0039] Table 1. Transit rate of FITC-LPS across the gut at different concentrations

[0040] Example 3 The application of various lipopolysaccharide oral absorption enhancers in promoting the oral absorption of lipopolysaccharides includes the following steps: Cells were seeded in Transwell chambers at a density of 10,000 cells / well and cultured continuously for 7 days, during which the TEER value was continuously monitored. When the TEER was greater than 500 Ω•cm, the cell line was considered closed. 2 Then, it can be used for subsequent detection. The culture medium in both chambers was aspirated, and both chambers were washed three times with HBSS. 400 μL of a drug combination of different absorption enhancers and plant extracts was added to the upper chamber, and 1000 μL of HBSS solution was added to the lower chamber. The mixture was incubated at 37°C for 3 h. The drug combination was then discarded, and both chambers were washed three times with HBSS. Then, 800 ng / mL FITC-LPS was added, and the mixture was incubated at 37°C for 120 min. After incubation, 200 μL of each solution was transferred to a black 96-well plate, and the fluorescence intensity was measured.

[0041] The intestinal absorption of LPS was promoted by adding different concentrations of oral lipopolysaccharide absorption enhancers. Results were as follows: Figure 5 , Figure 6 and Figure 7 As shown, different concentrations of oral lipopolysaccharide absorption enhancers significantly promoted LPS absorption, and the effect was concentration-dependent.

[0042] The addition of the chitosan (CS) and prickly pear extract drug combination significantly promoted LPS transport. After administration of the chitosan (CS) and prickly pear extract drug combination, the Papp values ​​of FITC-LPS were 1.512 times, 1.606 times, 2.40 times, 2.978 times, and 8.354 times that of the control group, respectively. This indicates that after using the chitosan and prickly pear extract drug combination, the ethanol and prickly pear extract drug combination, and the sodium caprylate and prickly pear extract drug combination, the chitosan and prickly pear extract drug combination was the most significant in promoting LPS absorption.

[0043] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A lipopolysaccharide oral absorption enhancer, characterized in that: It includes a water extract of *Rhizopus chinensis* and a second promoter, wherein the second promoter is selected from at least one of chitosan, ethanol, or sodium octanoate.

2. The lipopolysaccharide oral absorption enhancer according to claim 1, characterized in that: The mass ratio of the water extract of *Rhizopus spp.* to the second accelerator is 1:0.5~15.

3. The lipopolysaccharide oral absorption enhancer according to claim 1, characterized in that: The second promoter is chitosan; the mass ratio of the aqueous extract of *Rhizopus chinensis* to chitosan is 1:1~10.

4. The method for preparing the lipopolysaccharide oral absorption enhancer according to any one of claims 1-3, characterized in that: Includes the following steps: Clean the thorny bumps thoroughly and crush them for later use; The pulverized raw material is mixed with water and subjected to hydrothermal extraction at 80-100℃ for 1.5-3 hours to obtain the extract. After repeated extraction, the extracts were combined and dried to obtain the extract. The extract was mixed with the second promoter in a certain proportion to obtain the lipopolysaccharide oral absorption promoter.

5. The method for preparing the lipopolysaccharide oral absorption enhancer according to claim 4, characterized in that: The pulverized raw material is passed through an 80-300 mesh sieve, and the powder particle size is no greater than 180 μm.

6. The method for preparing the lipopolysaccharide oral absorption enhancer according to claim 4, characterized in that: During hydrothermal extraction, the material-to-liquid ratio is 1:10-30, g:mL.

7. The method for preparing the lipopolysaccharide oral absorption enhancer according to claim 4, characterized in that: The temperature for hydrothermal extraction is 90-100℃, preferably 95-100℃.

8. The method for preparing the lipopolysaccharide oral absorption enhancer according to claim 4, characterized in that: The extraction is repeated 1-3 times.

9. The method for preparing the lipopolysaccharide oral absorption enhancer according to claim 4, characterized in that: The extract was concentrated to 1 / 5 to 1 / 10 of its original volume by rotary evaporation under reduced pressure, and then freeze-dried to obtain the extract.

10. The method for preparing the lipopolysaccharide oral absorption enhancer according to claim 4, characterized in that: The thorny bumps were collected in September and October.

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