Ussuriwaw extractive, its preparation method and application

Abstract

The application discloses a ussurian wavy extract, which is prepared by the following method steps: taking dried whole plant of ussurian wavy, weighing; adding 85% ethanol with a ratio of 1g:10mL, and soaking for 24h; heating and ultrasonic extracting at constant temperature of 70-90 DEG C, extracting for 3h for 3 times, filtering while hot, discarding the filter residue, and combining the filtrate; reducing pressure and concentrating the filtrate to extract, and drying in an oven; taking the dried extract, and obtaining the ussurian wavy extract. The application further discloses a preparation method and application of the ussurian wavy extract. The application has the advantages of simple process, fast extraction speed, high extraction rate, strong activity and the like, and has good extraction effect. The ussurian wavy extract has the effect of resisting septicemia, and can be used for preparing anti-septicemia drugs and the like.

Description

[0001] This invention belongs to the field of plant extraction technology, specifically relating to an extract of Ussurivavir, its preparation method, and its application. Background Technology

[0002] Ussuriwavir is a plant belonging to the genus Ussuriwavir in the family Polypodiaceae. Lepisorus ussuriensis The whole herb of *Regelet Maack* Ching is widely distributed in many regions including Jilin, Heilongjiang, Anhui, Henan, and Liaoning. Ussurivavir is a plant with significant medicinal value, effective for various ailments such as urinary retention and edema, hematuria, asthma, sore throat, and bleeding from cuts. Ussurivavir contains a variety of chemical components, including flavonoids, steroids, and phenols, exhibiting strong anti-inflammatory, analgesic, antibacterial, and antioxidant pharmacological effects.

[0003] Sepsis is a systemic infection syndrome caused by pathogenic microorganisms invading the bloodstream, multiplying, and producing large amounts of toxins and metabolic products, leading to severe toxemia symptoms. The liver, as the body's most important metabolic organ, has functions such as detoxification, blood clotting, immunity, and bile secretion. The liver is not only one of the most vulnerable organs in sepsis but also plays a crucial role in its occurrence and development. Sepsis-induced acute liver injury plays a significant role in the occurrence and development of multiple organ dysfunction syndrome and is one of the most important causes of death from sepsis. In recent years, the anti-sepsis effects discovered in some medicinal plants warrant further investigation. Summary of the Invention

[0004] The purpose of this invention is to provide an extract of Ussurivavir with high extraction rate, simple process and fast extraction speed, and also includes a method for preparing Ussurivavir extract and its application.

[0005] The technical solution adopted to achieve the above objectives is as follows: An extract of ursurivavir is prepared according to the following steps: (1) Take the dried whole plant of Ussuriwave and weigh it; (2) Add 85% ethanol at a ratio of 1g:10mL and soak for 24 hours; (3) Heating and ultrasonic extraction at a constant temperature of 70℃~90℃, extracting 3 times in 3h extraction time, filtering while hot, discarding the filter residue, and combining the filtrates; (4) Concentrate the filtrate under reduced pressure to obtain an extract, and then dry it in an oven; (5) Take the dried extract to obtain the product.

[0006] Furthermore, in step (3) above, ultrasonic extraction is preferably performed at a constant temperature of 90°C.

[0007] A method for preparing an extract of ursurivavir includes the following steps: (1) Take the dried whole plant of Ussuriwave and weigh it; (2) Add 85% ethanol at a ratio of 1g:10mL and soak for 24 hours; (3) Heating and ultrasonic extraction at a constant temperature of 70℃~90℃, extracting 3 times in 3h extraction time, filtering while hot, discarding the filter residue, and combining the filtrates; (4) Concentrate the filtrate under reduced pressure to obtain an extract, and then dry it in an oven; (5) Take the dried extract to obtain the product.

[0008] Furthermore, the above-mentioned Ussurivavir extract is used in the preparation of drugs for treating sepsis.

[0009] This invention provides an optimal extraction process for ursurivavir, using ethanol for heated ultrasonic extraction, which can achieve a high extraction efficiency in a short time and effectively improve the utilization rate of ursurivavir. In the experiment of this invention, a liver injury model was established in mice by intraperitoneal injection of LPS solution, and then the liver indicators of the liver injury model mice, the ursurivavir administration group and the blank group were compared to confirm the anti-septic effect of ursurivavir on the liver.

[0010] The extraction process disclosed in this invention has advantages such as simplicity, speed, high extraction rate, and strong activity, resulting in good extraction effects. The extract has anti-septic activity and can be used in research such as the preparation of anti-septic drugs. Attached Figure Description

[0011] Figure 1 The cell survival rate of RAW 264.7 macrophages was determined in this experiment.

[0012] Figure 2 The extract of this invention is effective against TNF-α. α Effects of induced macrophage NF-κB luciferase activity.

[0013] Figure 3 These are the liver function test values ​​for each experimental group in this invention.

[0014] Figure 4 These are images of HE-stained liver sections from various experimental groups in this invention.

[0015] Among them, Figures 1-4 In the diagram, A: blank group; B: control group; C: low-dose group; D: high-dose group. Detailed Implementation

[0016] An extract of ursurivavir is prepared according to the following steps: (1) Take the dried whole plant of Ussuriwave and weigh it; (2) Add 85% ethanol at a ratio of 1g:10mL and soak for 24 hours; (3) Heating and ultrasonic extraction at a constant temperature of 70℃~90℃, preferably 90℃, extracting 3 times in 3h extraction time, filtering while hot, discarding the filter residue, and combining the filtrates; (4) Concentrate the filtrate under reduced pressure to obtain an extract, and then dry it in an oven; (5) Take the dried extract, weigh it, and calculate the extraction rate.

[0017] A method for preparing an extract of ursurivavir includes the following steps: (1) Take the dried whole plant of Ussuriwave and weigh it; (2) Add 85% ethanol at a ratio of 1g:10mL and soak for 24 hours; (3) Heating and ultrasonic extraction at a constant temperature of 70℃~90℃, extracting 3 times in 3h extraction time, filtering while hot, discarding the filter residue, and combining the filtrates; (4) Concentrate the filtrate under reduced pressure to obtain an extract, and then dry it in an oven; (5) Take the dried extract, weigh it, and calculate the extraction rate.

[0018] The above-mentioned Usurivavir extract can be used in research such as the preparation of antiseptic drugs.

[0019] The extraction process of Ussurivavir involves three key conditions: the solid-liquid ratio, the ethanol volume fraction, and the ultrasonic extraction time. Single-factor experiments were conducted on these three aspects below.

[0020] Effect of feed-liquid ratio Table 1 shows the single-factor experiment of the material-liquid ratio when the ethanol volume fraction is 85% and the ultrasonic extraction time is 3h. The results show that the optimal material-liquid ratio is 1g:10mL.

[0021] Table 1. Effect of solid-liquid ratio on extraction rate .

[0022] Effect of ethanol volume fraction Table 2 shows the single-factor experiments on extraction temperature when the material-to-liquid ratio is 1g:10mL and the ultrasonic extraction time is 3h. The results show that 85% ethanol volume fraction is the optimal value.

[0023] Table 2 Effect of ethanol volume fraction on extraction rate .

[0024] Effect of extraction time Table 3 shows the single-factor experiment of ultrasonic extraction time when the material-to-liquid ratio is 1g:10mL and the ethanol volume fraction is 85%. The results show that the ultrasonic extraction time is 3h.

[0025] Table 3. Effect of ultrasonic extraction time on extraction rate .

[0026] Orthogonal experiment on extraction process conditions of Ussurivavir Table 4 Results of the orthogonal experiment .

[0027] Conclusion: Through single-factor experiments and orthogonal experiments, the extraction rates under different extraction process conditions were obtained. The optimal process conditions were found to be a material-to-liquid ratio of 1g:10mL, an ethanol volume fraction of 85%, and an ultrasonic extraction time of 3h.

[0028] The antiseptic cell assay of ursurivavir extract included the following steps: (a) Cytotoxicity assessment: The cytotoxicity of the compound was determined using the MTT assay.

[0029] (1) RAW264.7 macrophages were thawed at a concentration of 1×10⁻⁶. 4 The cells were seeded at a density of 100 μL / well in 96-well plates and incubated for 24 h.

[0030] (2) The drug treatment groups were given different concentrations of urivavir extract (final concentrations of 30 and 100 μg / mL, respectively) (50 μL / well), while the control group was given 50 μL of culture medium and cultured for 30 min. At the same time, both groups were stimulated with LPS (1 mg / L) for 24 h.

[0031] (3) After incubation, add MTT reagent to each well with a final concentration of 0.5 mg / mL and incubate the cells again for 4 h. After incubation, remove the supernatant and add 150 μL DMSO to each well to dissolve formazan. Shake vigorously at room temperature in the dark for 10 min to ensure that the formazan is fully dissolved.

[0032] (4) Use an ELISA reader to read the absorbance (OD) at a wavelength of 570 nm and perform data analysis on the results.

[0033] (II) NF-κB luciferase reporter gene assay: (1) Macrophages (1 × 10 5 Cells were seeded in 96-well plates for 24 h, and each well was transfected with pNF-κB-Luc plasmid, followed by incubation for 48 h.

[0034] (2) Remove the cell culture medium and replace it with fresh culture medium containing the sample concentrations (30 μg / mL and 100 μg / mL) for 8 h, and then treat with 10 mg / mL TNF-α for 8 h.

[0035] (3) Inject 100 μL of assay buffer containing luciferase into a microluma luminometer and measure luciferase activity for 10 s. Co-transfect with pRL-CMV and normalize the data to improve transfection efficiency.

[0036] The cytotoxic activity of ursurivavir ethanol extract at concentrations of 30 μg / mL and 100 μg / mL was determined. It showed no significant cytotoxic activity against RAW264.7 macrophages, and the relative cell viability of the extract was greater than that of the LPS control group. The effect of ursurivavir extract on TNF-α-induced NF-κB activation was investigated. The extract inhibited TNF-α-induced NF-κB reporter gene expression activity in macrophages at concentrations of 30 μg / mL and 100 μg / mL.

[0037] For RAW 264.7 macrophage cell survival rate, please refer to [reference needed]. Figure 1 .

[0038] Extracts against TNF-α α The effect of induced macrophage NF-κB luciferase activity is shown in [reference]. Figure 2 .

[0039] The study on the antiseptic effect of ursurivavir included the following steps: (1) Male ICR mice weighing 20-22g were divided into 4 groups (blank group, model group, high-dose group and low-dose group), with five mice in each group; light and dark were alternated for 12 hours, and food and water were cut off; (2) Prepare a solution of LPS (30 μg / Kg) for later use; (3) Prepare two doses of ursurivavir extract, 300 mg / kg and 100 mg / kg, for later use; (4) 300 mg / Kg and 100 mg / Kg of ursurivavir were administered by gavage to mice in the high-dose and low-dose groups, respectively; the remaining groups were administered an equal volume of physiological saline by gavage. (5) After 30 minutes, except for the blank group, the other groups were injected with LPS solution intraperitoneally, and the blank group was injected with an equal volume of normal saline. (6) Eight hours later, blood was collected from the orbital venous plexus of mice to detect TNF-α and IL-6; MDA level and SOD activity in liver tissue were detected to examine liver function in mice and statistical analysis was performed. (7) The livers of mice in each group were embedded in paraffin blocks and prepared into tissue sections for HE staining and observation.

[0040] The study on the antiseptic effect of ursurivavir included the success of model establishment and whether different doses of ursurivavir inhibited liver damage in mice. The experiments will focus on these two aspects.

[0041] Mice were injected intraperitoneally with LPS solution and observed for 48 hours without any deaths. The livers of the model group mice were removed and compared with those of the normal group mice. The livers of the model group mice were found to be darker in color, and the liver cells of the model group were deformed under an electron microscope, which proved that the livers of the mice in this group had certain damage, thus proving that the model was successfully created.

[0042] Mice were administered the drug via gavage at both high and low doses. The results showed that both the high- and low-dose groups improved liver damage compared to the model group, as evidenced by decreased TNF-α, decreased IL-6, decreased MDA, and increased SOD. The high-dose group showed a greater improvement. All experimental data are expressed as mean ± standard deviation (x±s). One-way ANOVA was performed using GraphPadPrism 5.0 software, with P < 0.05 indicating statistical significance. Liver samples from all four groups were collected and subjected to HE staining for comparison. Compared to the model group, the drug-treated groups showed reduced liver damage, with the high-dose group showing a more significant effect. This demonstrates that ursurivavir has a certain anti-septic effect without toxic side effects.

[0043] See liver function test values ​​for each experimental group. Figure 3 .

[0044] See the images of liver HE-stained sections from each experimental group. Figure 4 .

[0045] Conclusion: Experimental studies have shown that urivavir has antiseptic effects and no toxic side effects, and can be used for the preparation of subsequent antiseptic drugs and other research.

[0046] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the examples given above. Modifications, variations, or substitutions made by those skilled in the art within the scope of the present invention should be included within the protection scope of the present invention.

Claims

1. An antiseptic extract of ursurivavir, prepared according to the following steps: (1) Take the dried whole plant of Ussuriwave and weigh it; (2) Add 85% ethanol at a ratio of 1g:10mL and soak for 24 hours; (3) Heating and ultrasonic extraction at a constant temperature of 90℃, extracting 3 times in 3 hours, filtering while hot, discarding the filter residue, and combining the filtrates; (4) Concentrate the filtrate under reduced pressure to obtain an extract, and then dry it in an oven; (5) Take the dried extract to obtain the product.

2. A method for preparing an antiseptic extract of ursurivavir, comprising the following steps: (1) Take the dried whole plant of Ussuriwave and weigh it; (2) Add 85% ethanol at a ratio of 1g:10mL and soak for 24 hours; (3) Heating and ultrasonic extraction at a constant temperature of 90℃, extracting 3 times in 3 hours, filtering while hot, discarding the filter residue, and combining the filtrates; (4) Concentrate the filtrate under reduced pressure to obtain an extract, and then dry it in an oven; (5) Take the dried extract to obtain the product.

3. The use of the ursurivavir extract as described in claim 1 in the preparation of a medicament for treating sepsis.

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