A biological product of Cistanche deserticola bioactive polypeptide with anti-fatigue effect

Abstract

The object of the present invention is to provide a biological product of Cistanche deserticola bioactive polypeptide with anti-fatigue effect, wherein the Cistanche deserticola polypeptide is enzymatically hydrolyzed by a hydrolase, and the enzymatic hydrolyzate is then prepared by S-8 type macroporous adsorption resin, Sephadex G-15 gel chromatography and high performance liquid chromatography purification. The polypeptide, one of which has an amino acid sequence of SEQ ID NO: 1. The polypeptide provided by the present invention is extracted from Cistanche deserticola, which can reduce the serum BUN and LA levels of mice after exercise, increase the content of liver glycogen and muscle glycogen, and significantly improve the swimming exhaustion time of weight-bearing mice. In addition, it has good antioxidant properties and can be used as a functional beverage.

Description

Technical Field

[0001] The invention belongs to the technical field of bioactive products, and specifically relates to a Cistanche deserticola bioactive polypeptide biological product with anti-fatigue effect. Background Art

[0002] Fatigue is defined as the state that occurs when the body's physiological functions are unable to maintain a certain level of efficiency or when it is unable to maintain a preset exercise intensity. The academic community has refined this phenomenon into sports fatigue, which specifically refers to the temporary decline in the body's athletic ability and overall physiological function during continuous exercise. This type of fatigue is physiological in nature, indicating that it can be naturally reversed through proper rest and recovery measures. It deeply reveals a series of complex biochemical processes and reactions in the body.

[0003] Regarding the root causes of fatigue, there are the following views:

[0004] 1) Gradual accumulation of fatigue-related products during metabolism (accumulation hypothesis);

[0005] 2) The key biological substrates that support motor activity are gradually depleted (exhaustion theory);

[0006] 3) Subtle changes in the matrix at the physiological and chemical level (physiological and chemical state change theory);

[0007] 4) Dysfunction within the regulatory and coordination system (dysregulation hypothesis).

[0008] In addition, the mutation theory that has emerged in recent years has introduced a new dimension to fatigue research, which believes that fatigue is a self-protective reaction under the synergistic action of multiple factors in the body.

[0009] With the vigorous development of social economy and the significant improvement of people's quality of life, people pay more and more attention to health maintenance, disease prevention and rehabilitation than ever before. Especially for those occupations with extremely high physical requirements, such as athletes, heavy manual laborers and astronauts, maintaining a strong physique has become the key to their careers. Therefore, the market demand for efficient and safe anti-fatigue nutritional health products is growing.

[0010] my country has rich and diverse Chinese herbal medicine resources and a series of unique advantages, which has laid a solid foundation for the development of health foods with distinctive Chinese characteristics. Cistanche deserticola is one of the most precious medicinal materials in traditional Chinese medicine. Its medicinal history is long and can be traced back to the Shennong Herbal Classic.

[0011] The reason why Cistanche deserticola is regarded as a treasure is that it is rich in a variety of active ingredients, such as phenylethanol glycosides, lignin and sugars, which give it multiple pharmacological effects such as tonifying the kidney and strengthening yang, moistening the intestines and promoting bowel movements, and anti-oxidation and anti-aging. In recent years, domestic and foreign academic circles have shown increasing interest in the research of Cistanche deserticola, especially in the analysis of its chemical components, exploration of its pharmacological effects, and optimization of its cultivation technology. However, there are currently no reports on the use of Cistanche deserticola peptides in the preparation of anti-fatigue products. Summary of the invention

[0012] The purpose of the present invention is to provide a Cistanche deserticola bioactive polypeptide biological product with anti-fatigue effect, thereby making up for the shortcomings of the prior art.

[0013] The present invention firstly provides a Cistanche deserticola polypeptide with anti-fatigue effect, wherein the Cistanche deserticola polypeptide is enzymatically hydrolyzed by a hydrolase, and the enzymatic hydrolysis product is then purified by S-8 macroporous adsorption resin, Sephadex G-15 gel chromatography and high performance liquid chromatography;

[0014] Furthermore, the hydrolase is trypsin;

[0015] The enzymatic hydrolysis is carried out at a pH of 8.0 and at 37° C. for 3 h;

[0016] Furthermore, the amino acid sequence of the Cistanche deserticola polypeptide having anti-fatigue effect is

[0017] QKNSRREM (SEQ ID NO: 1).

[0018] The present invention also provides an application of the Cistanche deserticola polypeptide with anti-fatigue effect, which is application in the preparation of anti-fatigue products;

[0019] The product is a functional beverage or a health product.

[0020] The polypeptide provided by the present invention is extracted from Cistanche deserticola, which can reduce the serum BUN and LA levels of mice after exercise, increase the content of liver glycogen and muscle glycogen, and significantly improve the swimming exhaustion time of weight-bearing mice. In addition, it has good antioxidant properties and can be developed as a functional drink or health product. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 :Sephadex G-15 method peptide separation peak diagram;

[0022] Figure 2 : Chromatogram of peptide separation and purification by liquid chromatography;

[0023] Figure 3 : Serum BUN and LA levels of mice in each group;

[0024] Figure 4 :Graphs showing liver glycogen and muscle glycogen contents in each group of mice. DETAILED DESCRIPTION

[0025] The present invention is described in detail below in conjunction with embodiments and drawings.

[0026] Example 1: Preparation of Cistanche deserticola polypeptide

[0027] 1. Preparation of Cistanche deserticola crude polypeptide solution

[0028] Cistanche deserticola was crushed through a 50-mesh sieve, and then deionized water was added at a solid-liquid ratio of 1:15 (m / m), and the pH was adjusted to 9.0 using a 10% (w / %) NaOH solution. Ultrasonic assisted extraction was performed for 20 minutes at a temperature of 40°C. The mixture was cooled to room temperature and centrifuged (5,000r / min for 10 minutes), and the supernatant was taken. The pH of the supernatant was adjusted to 4.5 using 10% (w / %) citric acid, centrifuged again, the precipitate was collected and repeatedly washed with water, and the protein freeze-dried powder was obtained by freeze-drying.

[0029] Weigh an appropriate amount of protein freeze-dried powder and prepare a protein solution with a mass fraction of 5.0%. Use 10% (w / %) sodium hydroxide solution and 10% (w / %) citric acid solution to adjust the pH of the protein solution to an appropriate pH of 8.0, keep it in a water bath at 37°C for 20 minutes, then add trypsin to the protein solution and keep it warm for enzymatic hydrolysis for 3 hours, take out the mixture and place it in boiling water for 15 minutes to kill the enzyme, after the hydrolysis is completed, centrifuge it (5,000r / min, for 10 minutes), and take the supernatant to obtain the polypeptide solution.

[0030] 2. Separation and purification of active peptides from Cistanche deserticola using S-8 macroporous adsorption resin

[0031] Soak the S-8 macroporous adsorption resin in a 95% ethanol solution for 20 hours, then wash it with double water until there is no ethanol smell. Next, wash the resin with a 5% HCl solution for 3 hours, filter and retain the resin, and wash the precipitate until the solution is neutral. After that, pass a 6% NaOH solution 3 to 5 times the volume of the macroporous adsorption resin through the column for 3 hours, filter and retain the resin again, and wash the precipitate until the pH value of the macroporous adsorption resin is about 7 for later use. Take 10g of the resin treated as above, add an appropriate amount of double water to keep it moist, and then fill it into a glass column. Rinse the column with double water for 1 hour, adjust the pH value of the loading solution to 5, and start loading at a rate of 1.5mL / min. After loading, rinse with double water until the outflowing solution is colorless, and finally use ethanol solution for elution.

[0032] 3. Separation and purification of active peptides from Cistanche deserticola by dextran gel

[0033] Accurately measure 20g of Sephadex G-15, a dextran gel, and fill it into a 2cm×40cm gel column using a wet method. Subsequently, the Cistanche polypeptide solution that has been initially purified by a macroporous adsorption resin is diluted to a concentration of 5mg / mL as the loading solution. After the liquid is filtered through a 0.45-micron fine filter membrane, 5mL of the clear filtrate is injected into the gel chromatography column. The elution rate is set to 0.5mL per minute, and the elution process is monitored under ultraviolet light at 220 nanometers. The eluate is tested, and the corresponding components are collected and combined according to their antioxidant activity. Sephadex G-15 is known for its mild separation conditions, which has little effect on the biological activity of the sample and has good regeneration performance. Therefore, it has been widely used in the separation of active substances such as proteins and peptides. Figure 1 As shown in the figure, after enrichment and preliminary purification by macroporous adsorption resin, Sephadex G-15 further separated the Cistanche deserticola polypeptide liquid into three components with similar molecular weights. The retention time of these components increased accordingly with the decrease of molecular weight, but the elution process was completed within 80 minutes, indicating that the molecular weight differences of these components were not significant.

[0034] 4. Liquid chromatography separation and purification of Cistanche deserticola active peptides

[0035] The component T2 with the highest activity after separation by glucose gel Sephadex G-15 was selected for further purification, and a ZORBAX Eclipse XDB-C column (specifications: 4.6 mm x 250 mm, 5 um) was used, and water (containing 0.1% TFA) was used as mobile phase A, and acetonitrile (containing 0.1% TFA) was used as mobile phase B, and linear gradient elution was performed. The specific conditions were that the acetonitrile concentration gradually increased from 0% to 75% over a period of 0 to 30 minutes, the flow rate was set at 0.8 mL / min, and the detection wavelength was set at 214 nm. Figure 2 It can be seen that a substance peak (F4) with a higher response value appeared in the ultraviolet absorption graph of the T2 component obtained by liquid chromatography separation. After collection and concentration, the peptide sequence was identified.

[0036] Table 1: Peptide mass spectrometry results

[0037] Serial number sequence Activity (U / μg) a PLASRDDN 3.65 b PPDSRGFNCKM 0.87 c QKNSRREM 4.62 d KBVGFFTYIPP 1.24 e KERDAAFGHK 1.33 f GGKTKMDFNB 0.90 g SGHTIFRFCF 1.01 h WHYSDFASE 0.76 i QERHPDCLK 0.82

[0038] As shown in Table 1, 9 peptides with high activity were isolated from the F4 fraction, among which peptide a and peptide c had the highest activity, and the sequences were PLASRDDN and QKNSRREM. Peptide a and peptide c were selected, and the sequences were sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis and then further research was carried out.

[0039] Example 2: Determination of the anti-fatigue property of polypeptides

[0040] 1. Screening of anti-fatigue peptides

[0041] Thirty healthy male mice with symmetrical body were selected and randomly divided into 4 groups, 10 mice in each group. The blank control group was gavaged with normal saline, and the experimental groups were gavaged with 0.05 mg / g of polypeptide a and polypeptide c purified products, once a day, for 30 consecutive days. Each group of mice was given swimming training in a constant temperature swimming pool 1 hour after gavage, with a training cycle of 5 days, and a total of 4 weeks of training.

[0042] 30 minutes after the last oral gavage, each group of mice had a lead wire equivalent to 10% of their body weight fixed to their tails as an additional weight. Subsequently, the mice were introduced one by one into a special constant temperature swimming box, the water depth of which was strictly set to 30 cm, while ensuring that the water temperature was kept constant within the precise range of (30.0±0.5)°C. Using a stopwatch tool, the time from the moment the mouse started swimming until it reached exhaustion, that is, the head was completely submerged in the water and could not float up on its own for more than 10 seconds, and this period of time was accurately recorded, which was defined as the weighted swimming endurance duration of the mouse. This standard is a key indicator for evaluating the physical exhaustion and swimming endurance of mice.

[0043] Table 2: Effects of Cistanche deserticola polypeptide on swimming time of mice

[0044]

[0045] As can be seen from Table 2, at the same dose, polypeptide a had no significant effect on the swimming time of mice, while the swimming exhaustion time of mice in the polypeptide c group was significantly higher than that of the blank control group and the polypeptide a group, confirming that polypeptide c can improve the anti-fatigue performance of mice, and its amino acid sequence is QKNSRREM (SEQ ID NO: 1).

[0046] 2. Study on the anti-fatigue properties of Cistanche deserticola polypeptide

[0047] 1) Model establishment

[0048] Fifty healthy male mice were selected and randomly divided into five groups (n=10 / group). The blank control group was given normal saline by gavage, the positive control group was given 0.1 mg / g American ginseng by gavage according to body weight, and the low, medium and high dose groups received 0.05, 0.10 and 0.20 mg / g purified polypeptide c by gavage, respectively, once a day for 30 days. One hour after gavage, each group of mice was trained in swimming in a constant temperature (30.0±0.5)℃ swimming pool, with a cycle of 5 days for a total of 4 weeks. The effect of the drug on the physical fitness of mice was evaluated by measuring the time from the start of swimming to physical exhaustion (head submerged in water for more than 10 seconds and unable to float up), that is, the weighted swimming time.

[0049] 2) Weighted swimming test

[0050] 30 minutes after the last gavage, a lead wire of 10% of the body weight was attached to the tail of each group of mice. Then, the mice were introduced into a specially made swimming box with a length of 30 cm and a constant temperature of (30.0±0.5)°C.

[0051] During the experiment, a stopwatch was used to record the time period from the start of swimming to exhaustion of each mouse, which was defined as the weighted swimming time. The criterion for exhaustion was that the mouse's head was completely submerged in water and failed to float to the surface on its own for more than 10 seconds.

[0052] Table 3: Effects of different doses of Cistanche polypeptide C on the swimming time of mice

[0053]

[0054] The results in Table 3 show that compared with the blank control group, each group of Cistanche polypeptide C increased the swimming time of mice to varying degrees, and the high-dose group had the strongest promoting effect, which was significantly different from the positive control (American ginseng) group, confirming that the high-dose Cistanche polypeptide C had the best anti-fatigue effect.

[0055] 3) Serum BUN and LA determination

[0056] After the weighted swimming experiment, blood was collected from the eyeballs. The blood sample volume was 2 mL, and heparin was used as an anticoagulant. The processed blood sample was placed in a centrifuge and centrifuged at 3000 r / min for 10 min, and the supernatant was collected. The colorimetric method was used according to the operating instructions and instructions provided by the kit to determine the content of blood urea nitrogen (BUN) and lactic acid (LA).

[0057] Serum urea nitrogen (BUN) is a key indicator for evaluating protein catabolism and reflects the metabolic adaptation of the body during exercise. Studies have shown that with the increase in exercise intensity, serum BUN levels show an upward trend, and this upward trend is particularly significant in individuals with weaker adaptability to exercise load, further highlighting the importance of BUN as an indicator for monitoring exercise adaptation. Lactic acid (LA), as a direct product of the glycolysis process, is produced and accumulated in large quantities in muscles during strenuous exercise due to the rapid anaerobic metabolism of glucose, resulting in local H + The concentration rises sharply and the pH value drops accordingly. This change triggers a complex biochemical cascade reaction, interferes with the body's normal metabolic process, and ultimately promotes the production of fatigue. Therefore, the detection of serum LA concentration after exercise has become an effective means to evaluate anaerobic energy supply efficiency, body fatigue level and potential muscle damage.

[0058] This experiment found that ( Figure 3 ), compared with the positive control group, the serum BUN and LA levels of the blank control group mice were significantly increased (P<0.05), indicating that the blank control group mice had a higher protein catabolism rate and anaerobic metabolic pressure. However, after the intervention of Cistanche polypeptide c, the serum BUN and LA levels of mice in each dose group were significantly reduced (P<0.05), and the effect of the high-dose group was similar to that of American ginseng. This result strongly proves that Cistanche polypeptide c can improve the tolerance of mice to exercise load and lactic acid accumulation, effectively restore the body's internal environment homeostasis damaged by lactic acid accumulation, and promote the body to restore normal metabolic function, thereby showing a significant anti-fatigue effect.

[0059] 4) Determination of liver glycogen and muscle glycogen content

[0060] After killing the mice in each group, the mice were dissected, the abdominal cavity of the mice was opened, and about 200 mg of liver tissue was taken out. The quadriceps femoris of the mice was peeled off and about 200 mg was weighed. They were added to a container containing 0.9% sodium chloride injection, and fully ground at a speed of 15,000 rpm using a high-speed homogenizer until a uniform 10% tissue homogenate was formed, and then centrifuged at a speed of 6,000 rpm for 15 minutes to separate the supernatant. The determination of liver glycogen and quadriceps femoris glycogen content was carried out strictly in accordance with the operating instructions provided by the kit.

[0061] Glycogen is an important energy source during exercise, and its reserve directly affects the endurance of endurance exercise. When the reserve is sufficient, exercise endurance is enhanced; conversely, the body is prone to fatigue after a large amount of consumption. This experiment found that ( Figure 4 ), the liver glycogen and muscle glycogen content of the positive control group mice were significantly lower than those of the blank control group, while the supplementation of Cistanche deserticola polypeptide C significantly increased the content of these glycogens in each dose group (P<0.05). This shows that Cistanche deserticola polypeptide C can effectively increase the glycogen reserves of mice, thereby possibly prolonging their exercise endurance time.

[0062] 5) Determination of SOD, MDA and GSH-PX activities in liver tissue

[0063] About 200 mg of liver tissue samples were selected and finely ground using liquid nitrogen freezing technology. The ground liver tissue was prepared into a uniform slurry with a concentration of 10% using a specific homogenate solution provided in the kit. In order to evaluate the activity levels of superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-PX) in the tissue samples, three scientific methods, xanthine oxidase assay, spectrophotometry and colorimetric detection, were used respectively. During the test process, the operating instructions provided by the kit were strictly followed.

[0064] Table 4: Comparison of SOD, MDA and GSH-PX activities in liver tissues of mice in each group

[0065]

[0066] SOD (superoxide dismutase) and GSH-Px (glutathione peroxidase) are vital antioxidant defense lines in the body, and their activity levels are important indicators of the body's ability to resist free radical damage. When the activity of these enzymes increases, it indicates that the body has a stronger antioxidant capacity. On the contrary, MDA (malondialdehyde), as the final product of unsaturated fatty acid peroxidation, its accumulation will seriously damage the cell membrane structure, increase membrane permeability, and then cause cell swelling and even necrosis. Therefore, changes in the content of MDA can indirectly reflect the degree of cell attack and peroxidation damage.

[0067] The results of this experiment show that compared with the positive control group, the activities of SOD and GSH-Px in the liver tissue of the blank control group mice were significantly reduced (P < 0.05), while the activity of MDA was significantly increased (P < 0.05), which indicates that the antioxidant capacity of the blank control group mice was weakened, resulting in more serious free radical damage. However, after the mice were treated with various doses of Cistanche polypeptide c, the activities of SOD and GSH-Px in their liver tissues were significantly increased (P < 0.05), while the activity of MDA was significantly decreased (P < 0.05). This shows that Cistanche polypeptide c has the potential to enhance the resistance of aging mice to free radical damage and improve antioxidant capacity, which helps to promote the effective removal of free radicals in the body, thereby maintaining cell health.

Claims

1. A Cistanche polypeptide having an anti-fatigue effect, characterized in that: The amino acid sequence of the Cistanche deserticola polypeptide is SEQ ID NO:

1.

2. Use of the Cistanche deserticola polypeptide according to claim 1 in the preparation of anti-fatigue products.

3. Use of the Cistanche deserticola polypeptide according to claim 1 in the preparation of a product for resisting free radical damage and improving antioxidant capacity.

4. The use according to claim 2 or 3, characterized in that: The product is a liquid beverage or a solid health product.

5. A product for anti-fatigue, characterized in that: The product contains the Cistanche deserticola polypeptide according to claim 1 at a pharmacologically effective concentration.

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