A composition of polysaccharide of cistanche and polypeptide of sea-buckthorn with hypoxia tolerance activity

Abstract

The present application provides a Cistanche deserticola polysaccharide and Hippophae rhamnoides polypeptide composition with hypoxia tolerance activity, and the amino acid sequence of the Hippophae rhamnoides polypeptide is SEQ ID NO:1. The composition provided by the present application can significantly prolong the survival time of mice, is superior to single components and positive controls, and exhibits synergistic effect. Molecular docking analysis shows that the used polypeptide has high affinity with SIRT1, supporting its mechanism in optimizing cell energy metabolism and antioxidant stress. The composition is suitable for highland medical protection and adjuvant treatment of hypoxic diseases, and has the advantages of clear components and stable biological activity.

Description

Technical Field

[0001] This invention belongs to the field of bioactive product preparation and application technology, specifically relating to a composition of Cistanche deserticola polysaccharide and Hippophae rhamnoides polypeptide with hypoxia resistance activity. Background Technology

[0002] Hypoxic stress is a key pathophysiological process leading to tissue and cell damage and bodily dysfunction. Under hypoxic conditions, imbalances in cellular energy metabolism and excessive accumulation of reactive oxygen species (ROS) often induce severe oxidative stress damage. Therefore, screening and developing substances with proven hypoxia-resistant bioactivity has significant academic value and application prospects for high-altitude medical protection and adjunctive treatment of clinical hypoxic diseases.

[0003] In current technologies, research on hypoxia-tolerant substances largely focuses on crude extracts of natural products or complex mixtures (such as plant polysaccharides and flavonoid mixtures). Although these components exhibit certain biological activities, their extremely complex composition and unknown core active monomers make it difficult to control the quality stability of the preparation process, and it is impossible to clarify their dose-response relationship through precise pharmacodynamic studies. This greatly limits their application in precision medicine and high-reproducibility industrial applications.

[0004] In contrast, peptides with well-defined structures, known sequences, and clearly defined biological targets have become a key focus in the development of hypoxia-tolerant drugs due to their high bioavailability and low immunogenicity. However, this field still faces challenges such as a scarcity of bioactive peptide sequences and insufficient depth of resource exploration. This is especially true for sea buckthorn (Hippophae rhamnoides). Hippophae rhamnoides L.) is a plant resource with a natural background of hypoxia resistance. Existing research has mostly stayed at the level of verifying the apparent efficacy, and no research has yet gone deep into the protein degradation level to discover short peptides with specific primary structures that are hypoxia resistant.

[0005] Therefore, how to isolate, identify and synthesize novel polypeptide sequences with significant hypoxia tolerance from sea buckthorn, and elucidate the correspondence between their structure and function, is a common technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to develop a combination of Cistanche deserticola polysaccharide and Hippophae rhamnoides polypeptide with hypoxia resistance activity, thereby achieving synergistic effects and enhancing the application value of natural medicines.

[0007] The present invention first provides a seabuckthorn polypeptide, which is prepared by drying and pulverizing seabuckthorn fruit residue, adding distilled water, adding a compound protease preparation, enzymatically hydrolyzing at 55°C, centrifuging the enzymatic hydrolysate after hydrolysis, and freeze-drying the supernatant. The aforementioned compound protease preparation is a complex system composed of neutral protease, alkaline protease, and flavor protease in a ratio of 2:2:1.

[0008] Furthermore, the sea buckthorn polypeptide has the amino acid sequence AGFQKVRAWGPGLKTGMVGK (SEQ ID NO:1).

[0009] The present invention also provides the application of the seabuckthorn polypeptide in the preparation of products with hypoxia resistance activity.

[0010] In another aspect, the present invention provides an article with hypoxia-resistant activity, comprising the above-mentioned sea buckthorn polypeptide; Furthermore, the product also contains Cistanche deserticola polysaccharide; The aforementioned Cistanche deserticola polysaccharide is obtained by adding Cistanche deserticola powder to distilled water, extracting it by high-speed shearing and reflux at 100°C, concentrating the extract by centrifugation, adding 95% anhydrous ethanol, allowing it to stand at 4°C, collecting the precipitate by centrifugation, and washing it with anhydrous ethanol.

[0011] This invention provides a composition of Cistanche deserticola polysaccharide and Hippophae rhamnoides polypeptide with hypoxia-resistance activity. This composition significantly prolongs the survival time of mice, superior to single-component and positive controls, demonstrating a synergistic effect. Molecular docking analysis shows that the polypeptides used have a high affinity for SIRT1, supporting its mechanism in optimizing cellular energy metabolism and anti-oxidative stress. This composition is suitable for high-altitude medical protection and adjunctive treatment of hypoxic diseases, with the advantages of well-defined components and stable biological activity. Detailed Implementation

[0012] This invention utilizes a bioactivity tracking and evaluation system to directionally screen and identify a specific polypeptide sequence with significant hypoxia tolerance effects from sea buckthorn protein hydrolysate. This polypeptide possesses a well-defined bioavailability, overcoming the limitations of traditional extracts with vague components and uncontrollable efficacy, thus laying the molecular foundation for developing highly reproducible hypoxia tolerance agents.

[0013] Furthermore, this invention reveals that the sea buckthorn-derived active peptide and Cistanche deserticola polysaccharide, which possesses anti-hypoxia activity, exhibit a significant synergistic effect. When used in combination, they can construct a complex protective mechanism from multiple dimensions, including optimization of cellular energy metabolism and defense against oxidative stress.

[0014] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0015] Example 1: Extraction of polysaccharides from Cistanche deserticola High-speed shearing method: Weigh 10g of Cistanche deserticola powder, add distilled water at a material-to-liquid ratio of 1:30, shear at 10000r / min for 30s, then reflux at 100℃ for 30min. After centrifugation, concentrate the extract to 1 / 10 of its original volume, add 3 times the volume of 95% anhydrous ethanol, let stand overnight at 4℃, collect the precipitate by centrifugation, and wash with anhydrous ethanol to obtain crude Cistanche deserticola polysaccharide.

[0016] The polysaccharide content was determined by the sulfuric acid-phenol method. After three parallel experiments, the polysaccharide yield of Cistanche deserticola was determined to be 2.31%.

[0017] The hypoxia tolerance activity of the test substances was evaluated using a sodium nitrite poisoning hypoxia model. Healthy male Kunming mice (6-8 weeks old, weighing 18-22g) were used as experimental subjects and placed in a standard laboratory environment with a temperature of 22±2°C and a humidity of 50±10% for 12 hours, during which time the animals were ensured to have free access to food and water.

[0018] During the experimental grouping and administration phase, mice were randomly divided into five groups of 10 mice each: a model control group, a low-dose group (400 mg / kg), a medium-dose group (600 mg / kg), a high-dose group (800 mg / kg), and a positive control group (rhodioloside, 300 mg / kg). Samples in each administration group were prepared to the required concentration using physiological saline, while the model control group received an equal volume of physiological saline. The experiment involved daily gavage administration for 30 consecutive days.

[0019] Hypoxia tolerance was assessed one hour after the last administration. First, all mice were intraperitoneally injected with sodium nitrite solution (240 mg / kg) at a rate of 0.1 mL / 10 g body weight, following the effective dose determined in the preliminary experiment, to induce methemoglobinemia and simulate acute tissue hypoxia. Immediately after injection, the mice were placed in individual observation cages lined with clean bedding, and a timing program was started simultaneously.

[0020] During the experiment, complete respiratory arrest in mice was used as the endpoint for determining death. Survival time (in minutes) from the moment of sodium nitrite injection to respiratory cessation was precisely recorded. Data from each group were collected, and the average survival time and survival extension rate were calculated (the formula is: Survival extension rate = [Average survival time of the treatment group - Average survival time of the model group] / Average survival time of the model group × 100%). This indicator was used to quantitatively evaluate the biological effects of the combination of sea buckthorn peptides and Cistanche deserticola polysaccharides in alleviating hypoxic injury and prolonging survival.

[0021] As shown in Table 1, intervention with high concentration (800 mg / kg) of Cistanche deserticola polysaccharide significantly prolonged the average survival time of mice to 15.00 min, with a survival extension rate of 32.2%. This indicates that within the dosage range of this experiment, the hypoxia resistance activity of Cistanche deserticola polysaccharide is positively correlated with its content (dose), meaning that its hypoxia resistance protective effect increases with increasing polysaccharide dosage.

[0022] Table 1: Effects of different concentrations of Cistanche deserticola polysaccharide on the hypoxia tolerance of mice poisoned by sodium nitrite.

[0023] Example 2: Extraction and preparation of sea buckthorn polypeptides

[0024] After drying and pulverizing the sea buckthorn pomace, distilled water was added at a material-to-liquid ratio of 1:40 using water as the extraction solvent. A complex protease preparation (a composite system of neutral protease, alkaline protease, and flavor protease in a 2:2:1 ratio) was added at 2.5% of the sea buckthorn dry powder mass. Enzymatic hydrolysis was carried out in a constant-temperature water bath at 55℃ for 4 hours with shaking. After hydrolysis, the enzyme was inactivated at 95℃ for 10 minutes. The hydrolysate was centrifuged at 12000 rpm for 15 minutes, and the supernatant was freeze-dried to obtain sea buckthorn polypeptide. 5.00 g of sea buckthorn polypeptide powder was accurately weighed and diluted to 50 mL with ultrapure water to prepare a 100 mg / mL stock solution. Separation and purification were performed using a liquid chromatography system equipped with an Agilent ZORBAX SB-C18 preparative column (250 × 21.2 mm, 5 μm) at a flow rate of 10 mL / min, monitored at room temperature with a detection wavelength of 214 nm. Different polarity elution fractions were collected using a gradient elution program: the 10% ethanol elution fraction was designated as fraction S1, the 30% ethanol fraction as fraction S2, the 50% ethanol fraction as fraction S3, and the 90% ethanol fraction as fraction S4.

[0025] In the activity screening based on a mouse hypoxia tolerance model, different components of sea buckthorn peptides exhibited different bioactivity characteristics. Among them, the S3 component, obtained by elution with 50% ethanol, showed the most significant activity, with the average survival time of mice in the intervention group reaching 16.17 minutes. Statistical analysis showed that this result was not only significantly better than the negative control group (P < 0.01), but also significantly higher than other eluted components (P < 0.05). This finding suggests that the S3 component may be enriched with key sea buckthorn peptide sequences that enhance the body's hypoxia tolerance. Detailed experimental data are shown in Table 2.

[0026] Table 2: Effects of different sea buckthorn polypeptide eluents on the hypoxia tolerance of mice poisoned with sodium nitrite.

[0027] The structure and sequence of the sea buckthorn polypeptide S3 fraction were characterized using an LCMS-9060 Q-TOF liquid chromatography-mass spectrometry system. Chromatographic separation was performed using a ZORBAX HILIC Plus C18 reversed-phase column at 35°C with gradient elution at a flow rate of 0.25 mL / min. Mobile phase A was an aqueous solution containing 0.1% formic acid and 5 mM ammonium formate, and mobile phase B was an acetonitrile solution containing 0.1% formic acid. The elution gradient was set as follows: 0–6 min to maintain 10% B, 6–12 min to increase to 25% B, 12–18 min to increase to 45% B, and 18–24 min to increase to 80% B. Mass spectrometry was performed using an alternating positive and negative electrospray ionization source with an ionization voltage of ±3.5 kV. The flow rates of the drying gas, heating gas, and collision gas were set to 8.0 L / min, 10.0 L / min, and 7.0 L / min, respectively. The interface temperature was 350°C, the desolvation tube temperature was 250°C, and the scan mass range was set to m / z 100–1500. The acquired mass spectrometry data were analyzed through database retrieval and de novo sequencing, identifying 52 bioactive peptides with well-defined sequences derived from sea buckthorn.

[0028] SIRT1 is a key metabolic and epigenetic regulator in the body's response to hypoxic stress. In a hypoxic microenvironment, changes in the intracellular NAD⁺ / NADH ratio can activate SIRT1, which then modifies various downstream target proteins (such as PGC-1α, FOXO1, and HIF-1α) through deacetylation. SIRT1 synergistically regulates processes such as glucose and lipid metabolism, mitochondrial biosynthesis, antioxidant defense, and autophagy, thereby enhancing the metabolic adaptability and survival ability of cells under hypoxic conditions.

[0029] To investigate whether sea buckthorn peptides regulate hypoxia response by acting on SIRT1, a molecular docking strategy combining AutoDockFR and RosettaFlexPepDock was employed. First, AutoDockFR was used to assess the flexible conformational space of the SIRT1 protein. Then, RosettaFlexPepDock was used to optimize the conformation of the peptide-receptor complex and accurately calculate the binding free energy, thereby predicting the binding mode and affinity between sea buckthorn peptides and SIRT1. The docking results are shown in Table 3. Several sea buckthorn peptides exhibited stable binding in the active pocket or regulatory region of SIRT1. Among them, the peptide with the sequence AGFQKVRAWGPGLKTGMVGK had the highest overall score, suggesting that it may have strong SIRT1 binding and regulatory potential, providing molecular-level evidence for elucidating the hypoxia tolerance mechanism of sea buckthorn peptides.

[0030] Table 3: Overall Score Table for Seabuckthorn Peptide Molecular Docking serial number polypeptide sequence AutoDock Vina (kcal / mol) Rosetta I_score 1 AGFQKVRAWGPGLKTGMVGK -9.6 -12.9 2 KDSLDFPEYDGKDRVHDL -8.5 -10.7 3 RGLFIIDDKGILR -7.2 -8.1 4 SVGDKVPADIRIVSIK -6.8 -7.5 5 VDTSGVHVYGPGVEPRGVLREVTTH -6.5 -6.7 6 VGDKVPADIRIVSIK -6.1 -5.8 Example 3: Determination of the hypoxia tolerance activity of sea buckthorn polypeptide (SEQ ID NO.1)

[0031] The target polypeptide sequence (SEQ ID NO.1) was successfully prepared using solid-phase synthesis technology. As shown in Table 4, the sea buckthorn polypeptide exhibited a significant dose-dependent protective effect. With increasing dosage (200→400→600 mg / kg), the average survival time of mice gradually increased from 13.2 minutes to 18.3 minutes, and the survival extension rate increased from 14.8% to 59.1%, indicating that the sea buckthorn polypeptide has strong hypoxia resistance activity.

[0032] Table 4: Data on the effect of sea buckthorn peptides on the average survival time and survival extension rate of mice poisoned with sodium nitrite.

[0033] Example 4: Determination of the hypoxia tolerance activity of a composition of Cistanche deserticola polysaccharide and Hippophae rhamnoides polypeptide (SEQ ID NO.1)

[0034] As shown in Table 5, the combination group (Cistanche deserticola polysaccharide 300 mg / kg + Hippophae rhamnoides polypeptide 300 mg / kg, total dose 600 mg / kg) exhibited significant hypoxia resistance activity, with an average survival time of 18.50 min and a survival extension rate of 60.9%. This effect was not only significantly better than the model control group, but also significantly better than the positive control drug rhodioloside (300 mg / kg, survival extension rate 34.8%), indicating that the combination has a significant synergistic effect.

[0035] Table 5: Data on the effect of the composition on the hypoxia tolerance of mice poisoned with sodium nitrite.

[0036] The above results indicate that gavage administration of the combination of sea buckthorn polypeptide and Cistanche deserticola polysaccharide significantly prolongs the survival time of mice under acute hypoxia. Experimental data show that the survival prolongation rate in each treatment group exhibits a good dose-response relationship, and the high-dose group shows a superior protective effect against hypoxia compared to the single component. The sea buckthorn polypeptide and its composition provided by this invention can effectively enhance the body's tolerance to hypoxic stress and have significant potential for development into drugs or functional foods for the prevention and relief of hypoxic injury.

Claims

1. A sea buckthorn polypeptide, characterized in that, The seabuckthorn polypeptide is prepared by drying and pulverizing seabuckthorn fruit residue, adding distilled water, adding a compound protease preparation, enzymatically hydrolyzing at 55°C, centrifuging the hydrolysate after hydrolysis, and then freeze-drying the supernatant.

2. The sea buckthorn polypeptide as described in claim 1, characterized in that, The aforementioned compound protease preparation is a complex system composed of neutral protease, alkaline protease, and flavor protease in a ratio of 2:2:

1.

3. The sea buckthorn polypeptide as described in claim 1, characterized in that, The seabuckthorn polypeptide has the amino acid sequence SEQ ID NO:

1.

4. The use of the sea buckthorn polypeptide according to claim 1 in the preparation of products with hypoxia resistance activity.

5. A product with oxygen deficiency resistance, characterized in that, The product with hypoxia resistance activity contains the sea buckthorn polypeptide as described in claim 1.

6. The article of claim 5, characterized in that, The product also contains Cistanche deserticola polysaccharide.

7. The article of claim 6, characterized in that, The aforementioned Cistanche deserticola polysaccharide is obtained by adding Cistanche deserticola powder to distilled water, extracting it by high-speed shearing and reflux at 100°C, concentrating the extract by centrifugation, adding 95% anhydrous ethanol, allowing it to stand at 4°C, collecting the precipitate by centrifugation, and washing it with anhydrous ethanol.

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