Ephedraone composition with anti-hypoxia activity

Abstract

The invention provides a fern ephedra ketone composition with anti-hypoxia activity, and a fern ephedra ketone extract is obtained by taking an ethanol solution as an extracting solution, performing high-speed shearing, performing boiling reflux extraction, centrifuging the extracting solution, taking supernatant, and performing vacuum concentration. The amino acid sequence of the polypeptide with the hypoxia tolerance activity is SEQ ID NO: 1-7. The composition prepared by compounding the fern ephedra ketone and the eel bone polypeptide has remarkable advantages in an anti-hypoxia activity test. The anti-hypoxia activity of the composition is obviously superior to the effect of singly using pteridin-ephedra ketone or eel bone polypeptide, so that the pteridin-ephedra ketone and the eel bone polypeptide have a remarkable synergistic interaction effect in a specific proportion. The beneficial effect not only improves the biological activity of the composition, but also provides a new technical path for developing efficient anti-hypoxia related products, and has significant application value and industrialization potential.

Description

Technical Field

[0001] This invention belongs to the field of bioactive products technology, specifically relating to a fern flavonoid composition with hypoxia-resistant activity. Background Technology

[0002] The development of hypoxia-tolerant drugs is an important direction for treating hypoxia-related diseases such as altitude sickness, cardiovascular disease, stroke, and trauma. Hypoxia-tolerant drugs are mainly classified into the following categories: HIF (hypoxia-inducible factor) modulators, antioxidants, artificial oxygen carriers, and natural plant extracts. Chemically synthesized drugs (such as HIF modulators) may cause side effects (such as tumor risk); artificial oxygen carriers can temporarily replace red blood cell oxygen supply, but they carry metabolic burdens and potential toxicity; while natural drugs have the advantages of natural components and fewer toxic side effects, monotherapy often has limitations such as low bioavailability and single target. To improve clinical efficacy, modern research often employs compound formulations and other techniques to achieve synergistic therapeutic effects.

[0003] Currently, the development and utilization of natural medicines face several bottlenecks, including: inefficient traditional processing methods leading to significant loss of active ingredients, low extraction efficiency, and high solvent consumption; low utilization rate of functional components resulting in insufficient added value; existing technologies largely limited to primary processing and lacking high-purity component separation; and low standardization leading to large quality fluctuations. Therefore, to address these issues, it is necessary to optimize processes and employ scientific compounding to improve the development efficiency and application value of natural medicines. Summary of the Invention

[0004] The purpose of this invention is to provide a pterostilbene composition with hypoxia resistance activity, thereby achieving synergistic effects and enhancing the application value of natural medicines.

[0005] The present invention first provides one use of the fern rhubarb extract in the preparation of products for improving hypoxia tolerance; The aforementioned fern ephedrine extract is obtained by using an ethanol solution as the extraction solvent, followed by high-speed shearing, boiling and reflux extraction, centrifuging the extract to collect the supernatant, and concentrating under reduced pressure. As a specific example, the ethanol solution is 50% ethanol; More specifically, the fern root extract is prepared by weighing a certain amount of fern root powder into a round-bottom flask, adding 50% ethanol at a material-to-liquid ratio of 1:40, shearing at 14500 r / min for 50 s, boiling and refluxing for 0.5 h, centrifuging at 19000 r / min, collecting the supernatant and concentrating under reduced pressure, and drying in an oven at 80℃ to obtain crude fern root flavonoids.

[0006] The present invention also provides a polypeptide with hypoxia tolerance activity, wherein the amino acid sequence of the polypeptide is as follows: KIWHHTFYNEL (SEQ ID NO:1), GESGAGKTVNTK (SEQ ID NO:2), PMNPPKFD (SEQ ID NO:3), FTIIDQNRDGIISK (SEQ ID NO:4), GPVGNTGPK (SEQ ID NO:5), PDGDHDLK (SEQ ID NO:6), AGLQFPVGRVH (SEQ ID NO:7).

[0007] The present invention also provides one use of the aforementioned polypeptide for preparing products that improve hypoxia tolerance.

[0008] The present invention also provides a product for improving hypoxia tolerance, comprising the above-mentioned fern ephedrine extract; Furthermore, the product also contains one or more of the aforementioned polypeptides.

[0009] This invention utilizes a compound of pterostilbene and eel bone polypeptides to prepare a composition that exhibits significant advantages in hypoxia tolerance tests. The hypoxia tolerance activity of this composition is significantly superior to that of pterostilbene or eel bone polypeptides used alone, indicating a significant synergistic effect between the two at a specific ratio. This beneficial effect not only enhances the bioactivity of the composition but also provides a new technical pathway for developing highly efficient hypoxia tolerance-related products, demonstrating significant application value and industrialization potential. Attached Figure Description

[0010] Figure 1 : Rutin standard curve graph; Figure 2 Effects of different extraction methods on flavonoid extraction rate and hypoxia tolerance activity: 0: Control; 1: Extraction for 0.5 h; 2: Extraction for 1 h; 3: Extraction for 0.5 h after high-speed shearing for 30 s; Figure 3 : Effect of solid-liquid ratio on flavonoid extraction rate; Figure 4 : Effect of rotation speed on flavonoid extraction rate; Figure 5 : Effect of shear time on flavonoid extraction rate; Figure 6 : Effect of shearing number on flavonoid extraction rate; Figure 7 : Hypoxia tolerance activity of pterostilbene, where 1: control, 2: pterostilbene (low dose), 3: pterostilbene (medium dose), 4: pterostilbene (high dose). Figure 8: Hypoxia tolerance activity of different components of eel bone polypeptide, where 1: control; 2: eel bone polypeptide stock solution; 3: component F1; 4: component F2; 5: component F3; 6: component F4; 7: component F5; 8: component F6.

[0011] Figure 9 : Hypoxia tolerance activity of eel bone polypeptide (KIWHHTFYNEL), where 1: control; 2: eel bone polypeptide (low dose); 3: eel bone polypeptide (medium dose); 4: eel bone polypeptide (high dose).

[0012] Figure 10 : Hypoxia tolerance activity of the combination of fern ephedrine and eel bone polypeptide, where 1: control, 2: fern ephedrine, 3: eel bone polypeptide, 4: fern ephedrine + eel bone polypeptide. Detailed Implementation

[0013] The *Fern Root* used in this embodiment of the invention was harvested from Haibei Prefecture, Qinghai Province. Anhydrous ethanol (analytical grade) was from Tianjin Fine Chemical Co., Ltd.; rutin (analytical grade) was from Lanzhou Zhichun Biotechnology Co., Ltd.; sodium nitrite (analytical grade) was from Shanghai Yi'en Chemical Technology Co., Ltd.; aluminum nitrate nonahydrate (analytical grade) was from Shanghai Yi'en Chemical Technology Co., Ltd.; and sodium hydroxide (analytical grade) was from Sinopharm Chemical Reagent Co., Ltd.

[0014] D500 High-Speed ​​Shearing Machine, Wiegands GmbH, Germany; 5810R High-Speed ​​Centrifuge, Eppendorf GmbH, Germany; DHG-9070 Single-Fan Dryer, Shanghai Yiheng Scientific Instruments Co., Ltd.; AX224ZH Electronic Balance, Ohaus International Trading Co., Ltd., USA; SpectraMax Plus 384 Full-Wavelength Microplate Reader, Shanghai Meigu Molecular Instruments Co., Ltd.

[0015] The specific steps for plotting the standard curve in this embodiment of the invention are as follows: Weigh 10 mg of rutin standard dried to constant weight at 120℃, dissolve it in 70% ethanol and dilute to 100 mL to obtain a concentration of 100 μg / mL. Dilute the rutin solution with 70% ethanol to prepare concentrations of 0, 20, 40, 60, 80, and 100 μg / mL. Take 1 mL of each concentration and place it in a test tube. Add 1 mL of 70% ethanol and 0.3 mL of 5% NaNO2 solution, shake well, and let stand for 6 min. Then add 0.3 mL of 10% Al(NO3)3 solution, shake well, and let stand for 6 min. Finally, add 2 mL of 4% NaOH solution, shake well, and let stand for 15 min. Measure the absorbance at a wavelength of 510 nm.

[0016] As a result, a standard curve for rutin was plotted with the concentration of the rutin standard on the x-axis and the absorbance on the y-axis. The equation was y = 0.0013x + 0.0471, R0.2 =0.9936. This indicates that rutin exhibits a good linear relationship with absorbance in the concentration range of 20~100μg / mL. Figure 1 ).

[0017] The method for determining the flavonoid content in *Pteris vittata* extract is as follows: Weigh 10 mg of sample and prepare a 5 mg / mL solution with 70% ethanol. Take 1 mL of the solution into a test tube, add 1 mL of 70% ethanol, then add 0.3 mL of 5% NaNO2 solution, shake well, and let stand for 6 min. Then add 0.3 mL of 10% Al(NO3)3 solution, shake well, and let stand for 6 min. Finally, add 2 mL of 4% NaOH solution, shake well, and let stand for 15 min. Measure the absorbance at 510 nm and calculate the flavonoid concentration from the standard curve.

[0018] Calculate the flavonoid extraction rate.

[0019] (1) Wherein, c0 is the flavonoid concentration, c is the sample concentration, m0 is the sample mass, and m is the mass of fern root powder.

[0020] The method for detecting hypoxia tolerance in this invention embodiment is as follows: Adult inbred mice of single sex, weighing 18-22g, were selected for the experiment, with 10-15 mice per group. Three dosage groups and one control group were established. The three dosage groups were 400mg / kg, 800mg / kg, and 1200mg / kg, respectively. A negative control was physiological saline, and a positive control group was provided if necessary. The test sample was administered for 30 days. Each dosage group was continuously administered the test sample at different concentrations orally, while the control group received the same volume of solvent. One hour after the last gavage, each mouse was placed in a 250mL ground-glass stoppered bottle containing 5g of soda lime (one mouse per bottle). The bottle was sealed with Vaseline to prevent air leakage, and the time to death due to hypoxia was immediately recorded, with respiratory arrest as the indicator.

[0021] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0022] Example 1: Comparison of hypoxia tolerance activity of extracts from *Pteris vittata* obtained by different extraction methods Traditional method: Weigh a certain amount of fern root powder into a round-bottom flask, add 50% ethanol at a material-to-liquid ratio of 1:40, boil and reflux for a certain time, centrifuge, take the supernatant and concentrate under reduced pressure, and dry in an oven at 80℃ to obtain fern root crude flavonoids.

[0023] High-speed shearing method: Weigh a certain amount of fern root powder into a round-bottom flask, add 50% ethanol at a material-to-liquid ratio of 1:40, shear at 14500 r / min for 30 s, boil and reflux for 0.5 h, centrifuge, take the supernatant and concentrate under reduced pressure, and dry in an oven at 80℃ to obtain fern root crude flavonoids.

[0024] The extraction of pterostilbene by conventional methods and high-speed shearing methods were investigated separately, and the results are as follows: Figure 2 As shown, the extraction rates of pterostilbene were 1.48% and 2.67%, respectively, indicating that the high-speed shear method significantly increased the extraction rate of pterostilbene than the traditional method. The results of hypoxia tolerance tests showed that, under the same extraction time conditions, the pterostilbene obtained by the high-speed shear method exhibited significantly better hypoxia tolerance than that obtained by the traditional extraction method. Therefore, the hypoxia tolerance activity of pterostilbene is positively correlated with its content.

[0025] Example 2: Determination of Optimal Process Conditions 1. Single-factor experiment The factors and levels of the single-factor experiment are shown in Table 1. Table 1: Factors and Levels in Single-Factor Experiments

[0026] 1) Effect of material-to-liquid ratio on the extraction rate of pterostilbene The effect of the material-to-liquid ratio on the flavonoid extraction rate was investigated under the conditions of a rotation speed of 19000 r / min, a shearing time of 40 s, and two shearing cycles. The results showed that... Figure 3 It can be seen that the extraction rate of flavonoids from *Ephedra sinica* gradually increases with the increase of the solid-liquid ratio. When the solid-liquid ratio is between 1:10 and 1:30, the extraction rate of flavonoids increases rapidly. When the solid-liquid ratio is greater than 1:30, the increase in the extraction rate of flavonoids tends to slow down. Increasing the solid-liquid ratio will increase the solubility of *Ephedra sinica* in the solvent and increase the extraction rate of flavonoids. Therefore, a solid-liquid ratio of 1:20 to 1:40 is selected as the orthogonal solid-liquid ratio condition.

[0027] 2) Effect of rotation speed on the extraction rate of pterostilbene ephedrine The effect of rotational speed on flavonoid extraction rate was investigated under the conditions of a material-to-liquid ratio of 1:40, a shearing time of 40 s, and two shearing passes. Figure 4 It was found that the extraction rate of pterostilbene gradually increased with increasing rotational speed. Above 16750 r / min, although the extraction rate increased with increasing rotational speed, the increase was slow. This may be because the high-speed shearing caused the pterostilbene cells to rupture almost completely, resulting in almost complete dissolution of pterostilbene in the extract. When the cells are broken to a certain extent, the effect of rotational speed becomes smaller. Therefore, 14500 r / min to 19000 r / min was selected as the rotational speed conditions for the orthogonal experiment.

[0028] 3) Effect of shearing time on the extraction rate of ephedrine from pterostilbene The effect of shearing time on flavonoid extraction rate was investigated under the conditions of a material-to-liquid ratio of 1:40, a rotation speed of 19000 r / min, and two shearing cycles. Shearing time affects the cell wall disruption effect; therefore, shearing time can influence the flavonoid extraction rate. Figure 5 It can be seen that the longer the shearing time, the higher the extraction rate of flavonoids. The shearing time has a significant impact on the flavonoid extraction rate within the range of 10-40 seconds. After 40 seconds, the increase in flavonoid extraction rate slows down. When the time increases to a certain value, the effect of time becomes smaller. Therefore, 30-50 seconds was selected as the orthogonal shearing time condition.

[0029] 4) Effect of shearing times on the extraction rate of pterostilbene The effect of the number of shearing operations on the flavonoid extraction rate was investigated under the conditions of a liquid-to-transfer ratio of 1:40, a feed rate of 19000 r / min, and a shearing time of 40 s. Figure 6 It was found that increasing the number of shearing cycles from 1 to 2 rapidly increased the flavonoid extraction rate. However, after 2 cycles, the increase in flavonoid extraction rate slowed down with further increases in the number of cycles. Increasing the number of cycles is equivalent to increasing the shearing time, which is beneficial for the continuous fragmentation of large molecular particles in the *Pteris vittata* aqueous solution, resulting in more complete dissolution of flavonoids. However, once the flavonoids are completely dissolved, the effect of the number of cycles on the extraction rate becomes smaller. Therefore, 1 to 3 cycles were selected as the orthogonal shearing cycle condition.

[0030] 2. Optimization of the extraction process of pterostilbene ephedrine Using Design-Expert 13.0.1.0 software, based on single-factor experiments, four influencing factors—material-liquid ratio, rotation speed, shearing time, and shearing times—were combined, with the extraction rate of *Pteris vittata* ethanol extract as the evaluation index, in an L9(3) experiment. 4 Orthogonal experiment. The factor level table is shown in Table 2.

[0031] Table 2: Factor Level Table for Orthogonal Experiment

[0032] 3. Data Processing The graphs were plotted using Microsoft Excel 2019; the variance analysis was performed using IBM SPSS Statistics 27.

[0033] The results of the orthogonal experiment are shown in Table 3, and the analysis of variance is shown in Table 4. Table 3 shows that the influence of each factor on the extraction rate of fern ephedrine is: B>A>D>C, that is, the number of shearing times has the greatest influence, and the other factors are, in descending order, the material-liquid ratio, shearing time, and rotation speed.

[0034] Table 3: Results of Orthogonal Experiments

[0035] Table 4: Analysis of Variance Table

[0036] The optimal process conditions, determined by the range, are A3B3C3D3, which means 3 shearing cycles, a material-to-liquid ratio of 1:40, a time of 50 seconds, and a rotation speed of 19000 r / min. Under these optimal conditions, the extraction rate of pterostilbene was 2.90% ± 0.07%, indicating that the process is stable and reliable.

[0037] Example 3: Determination of hypoxia tolerance activity 1) Experimental materials: 250mL ground glass bottle, stopwatch, petroleum jelly, soda lime (or equal amounts of sodium hydroxide and calcium carbonate).

[0038] 2) Experimental animals: Inbred adult mice, single sex, 18-22g, 10-15 mice per group.

[0039] 3) Experimental sample: The extraction rate of pterostilbene was 2.92% under the optimal process conditions of 3 shearing times, material-liquid ratio of 1:40, time of 50s, rotation speed of 19000r / min.

[0040] 4) Dosage grouping and administration time of test samples: The experiment consisted of three dosage groups and one control group. The three dosage groups were 400 mg / kg, 800 mg / kg, and 1200 mg / kg, respectively. The negative control was physiological saline. The administration time of the test samples was 30 days.

[0041] (5) Experimental procedure: Each dose group was given different concentrations of the test sample orally, and the control group was given the same volume of solvent. One hour after the last gavage, each group of mice was placed in a 250mL ground glass bottle containing 5g of soda lime (one mouse per bottle). The bottle mouth was sealed with Vaseline to prevent air leakage. The time was immediately started, and the time of death due to hypoxia was observed with respiratory arrest as the indicator.

[0042] Result: From Figure 7 As shown, compared with the control group, the hypoxia tolerance time of mice in each dose group of fern ephedrine exceeded 20 min, showing significant hypoxia tolerance activity. Among them, the high dose group had the best effect, with a hypoxia tolerance time of 23.78 min.

[0043] Example 4: Preparation of eel bone polypeptides with hypoxia resistance activity After drying and pulverizing the eel bones, add papain and alkaline protease at a ratio of 3% of dry weight (enzyme activity ratio of 2:1), and enzymatically hydrolyze at 50℃ for 3-5 hours. After enzymatic hydrolysis, inactivate the enzyme at 90℃ for 15 minutes, centrifuge at 12000 rpm for 20 minutes, and then take the supernatant after centrifugation and dry it to obtain eel bone polypeptide.

[0044] 10 g of eel bone polypeptide powder was dissolved in 100 mL of pure water to prepare a 100 mg / mL eel bone polypeptide stock solution. The eel bone polypeptide was separated using preparative liquid chromatography (Dubhe C18 preparative column (250 × 20 mm, 10 μm), flow rate 8 mL / min, injection volume 1 mL, 100 mg / mL, column temperature room temperature, detection wavelength 220 nm). The fractions eluted with 5% methanol were collected as F1, 15% methanol as F2, 40% methanol as F3, 50% methanol as F4, 65% methanol as F5, and 80% methanol as F6.

[0045] Using mouse experiments, the hypoxia tolerance activity of different components of eel bone polypeptides was detected. It was found that the hypoxia tolerance time in the F3 group of mice was higher than that in the control and other experimental groups, reaching 24.23 min (…). Figure 8 It exhibits significant hypoxia tolerance activity, and the obtained sample is eel bone hypoxia tolerance peptide.

[0046] The structure of hypoxia-resistant peptides from eel bone was identified under the following chromatographic conditions: Instrument type: LCMS-9030 Q-TOF quadrupole-time-of-flight mass spectrometer; Column: Inertsil HILIC C18 reversed-phase column (150 mm × 3.0 mm, 3 μm, Shimadzu, Japan); Column temperature: 30 ℃; Injection volume: 1.0 μL; Mobile phase: A was 0.1% (v / v) formic acid aqueous solution, Mobile phase B was 0.1% (v / v) formic acid acetonitrile solution; Gradient elution program: 0-5.0 min, 5%, 5.0-10.0 min, 10%, 10.0-15.0 min, 20%, 15.0-20.0 min, 40%; Flow rate: 0.3 mL / min; Mass spectrometry ion mode: ESI source, positive ion mode; Ion source interface voltage: 4.0 kV; Gas flow rate: 10.0 kV. L / min, Drying gas flow rate: 10.0 L / min, Heating gas flow rate: 10.0 L / min; Heating module temperature: 400℃, Interface temperature: 300℃, Scanning mode: Full scan mode (m / z 50-2000).

[0047] The obtained mass spectrometry information was identified using proteomics technology, yielding 263 effective peptides.

[0048] Molecular docking was performed using AutoDockFR + Rosetta FlexPepDock, where AutoDockFR supports receptor flexibility and Rosetta FlexPepDock provides efficient scoring. The combined score was used to predict the hypoxia tolerance of eel bone peptides.

[0049] Hypoxia-inducible factor-1 (HIF-1) was first discovered by Semenza and Wang in 1992. HIF-1 is ubiquitous in human and mammalian cells. Under normoxic conditions (21% O2), HIF-1 protein is rapidly degraded by intracellular oxygen-dependent ubiquitin proteases, and can only be stably expressed under hypoxic conditions. The main function of HIF-1 is to regulate the cellular response to hypoxia, increasing ATP production by increasing glycolysis and reducing oxidative phosphorylation. Simultaneously, HIF-1 also regulates the expression of a series of target genes, such as erythropoietin and vascular endothelial growth factor, which play important roles in angiogenesis and cellular adaptation to hypoxia. The prediction results are shown in Table 2. Seven peptides had high overall scores, suggesting hypoxia tolerance activity, with peptide 5 (KIWHHTFYNEL) having the highest overall score, suggesting the best hypoxia tolerance activity.

[0050] Table 5: Comprehensive Score Table for Eel Bone Peptide Molecular Docking

[0051] The peptide 5KIWHHTFYNEL (SEQ ID NO:1) was synthesized using a solid-phase peptide synthesis method. Its hypoxia tolerance activity was verified using mouse experiments, and the results are as follows: Figure 9 As shown, compared with the control group, the experimental group mice had a significantly longer hypoxia tolerance time, and the effect was positively correlated with the dose. The high-dose group had a hypoxia tolerance time of 23.89 min, indicating that the peptide has significant hypoxia tolerance activity.

[0052] After combining fern ephedrine and eel bone polypeptide at a 1:1 mass ratio, their hypoxia tolerance activity was tested. Figure 10 As shown, the hypoxia tolerance time of mice in the control group was 18.83 min, that in the fern fern ephedra group was 21.97 min, that in the eel bone polypeptide group was 21.47 min, and that in the combined group was 24.63 min, which was significantly higher than that of other experimental groups. This indicates that the combination of fern fern ephedra and eel bone polypeptide can significantly improve the hypoxia tolerance activity of mice compared with the use of fern fern ephedra and eel bone polypeptide alone.

Claims

1. Use of a Potentilla Frigida flavone extract in the preparation of a product for improving hypoxia tolerance.

2. Use according to claim 1, wherein The Potentilla Frigida flavone extract is obtained by using an ethanol solution as an extraction liquid, high-speed shearing, boiling reflux extraction, centrifugation of the extraction liquid to obtain supernatant, and vacuum concentration.

3. Use according to claim 2, wherein the compound is ###0002### The ethanol solution is a 50% ethanol solution.

4. The use according to claim 2, wherein the compound is ###0002### The Potentilla Frigida flavone extract is obtained by adding a 50% ethanol solution to Potentilla Frigida powder at a solid-liquid ratio of 1:40, high-speed shearing at 14500 r / min for 50 s, boiling reflux extraction for 0.5 h, centrifugation at 19000 r / min to obtain supernatant, vacuum concentration, and drying.

5. A polypeptide having hypoxia tolerance activity, characterized by, The polypeptide comprises any one or several of the following sequences: KIWHHTFYNEL (SEQ ID NO: 1), GESGAGKTVNTK (SEQ ID NO: 2), PMNPPKFD (SEQ ID NO: 3), FTIIDQNRDGIISK (SEQ ID NO: 4), GPVGNTGPK (SEQ ID NO: 5), PDGDHDLK (SEQ ID NO: 6), and AGLQFPVGRVH (SEQ ID NO: 7).

6. Use of the polypeptide of claim 5 in the preparation of a product for improving hypoxia tolerance.

7. An article of manufacture for increasing hypoxia tolerance, comprising: The product contains the Potentilla Frigida flavone extract, which is obtained by adding a 50% ethanol solution to Potentilla Frigida powder at a solid-liquid ratio of 1:40, high-speed shearing at 14500 r / min for 50 s, boiling reflux extraction for 0.5 h, centrifugation at 19000 r / min to obtain supernatant, vacuum concentration, and drying.

8. The article of claim 7, wherein, The product also contains the polypeptide of claim 5.

9. The article of claim 7, wherein, The product, wherein the mass ratio of the Potentilla Frigida flavone to the polypeptide of the Chinese eel bone is 1:1.

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