A lycium barbarum glycopeptide composition for anti-fatigue

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a wolfberry glycopeptide composition for anti-fatigue and its uses. The composition contains wolfberry glycopeptide as a single active ingredient, or the composition consists of wolfberry glycopeptide and scutellarin in a mass ratio of 1:5 to 5:1. The fatigue referred to is general fatigue or exercise-induced fatigue. Experimental results of this invention confirm that wolfberry glycopeptide, a characteristic active extract of the traditional Chinese medicinal herb wolfberry, possesses clear anti-fatigue activity on its own, and the composition combined with scutellarin can further enhance its synergistic effect. The composition provided by this invention has clear components and stable activity, possessing broad prospects for development into various types of anti-fatigue products such as pharmaceuticals, health foods, and special medical purpose formula foods, thus having significant social and economic value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a wolfberry glycopeptide composition for anti-fatigue and its uses. Background Technology

[0002] Fatigue is a complex physiological process involving the interaction of multiple factors. It can be a protective warning signal spontaneously generated by the body after a short period of mental or physical exertion exceeds the tolerance threshold, or it can be a persistent subjective feeling caused by the accumulation of long-term sub-health conditions or the progression of chronic diseases. When the body is continuously active, whether it is the accumulation of metabolic products from muscle labor or the depletion of neurotransmitters from continuous brain work, the body's working capacity will temporarily decrease. This is normal physiological fatigue, which can usually be quickly relieved with sufficient rest. However, if the state of fatigue persists and cannot be improved by regular rest, it is not only a manifestation of declining physical fitness, but may also be a precursor to the development of various diseases, requiring sufficient attention.

[0003] Although fatigue doesn't directly threaten life like cancer or acute cardiovascular disease, it is a hidden potential health hazard that is profoundly impacting the quality of life and work efficiency of tens of millions of people as modern life becomes faster-paced and work pressures increase. Contemporary young people commonly have unhealthy lifestyle habits such as staying up late, prolonged sitting, and irregular meals. Long-term sleep disturbances and energy imbalances make fatigue a "daily norm" for working professionals: many feel drowsy and weak during the day, unable to concentrate, and experience a significant drop in work efficiency; at night, they are too excited to fall asleep, falling into a vicious cycle of "tired but unable to sleep." This persistent low-intensity fatigue is constantly expanding the sub-healthy population. Epidemiological surveys of sub-health in my country show that the proportion of sub-healthy people in first-tier cities exceeds 70%, with persistent fatigue being the number one typical symptom.

[0004] Goji berries, a traditional Chinese medicine, are the mature fruit of the *Lycium barbarum* plant, belonging to the Solanaceae family. They have been revered as a precious traditional Chinese medicine since ancient times and are used in various tonic formulas. The *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) records that goji berries "treat internal evils, thirst due to heat, rheumatism, and with prolonged use, strengthen muscles and bones, promote longevity, and improve resistance to cold and heat." They are sweet and neutral in nature, and their pharmacological effects include nourishing the kidneys, moistening the lungs, tonifying the liver, dispelling wind, and improving eyesight. They are used to treat liver and kidney yin deficiency, lower back and knee weakness, dizziness, and vertigo. Since 2002, goji berries have been included in the list of dual-use medicinal and edible herbs by the former Ministry of Health of the People's Republic of China. This dual use of medicine and food is a significant advantage of goji berries, giving them excellent development prospects. Modern pharmacological results show that goji berries have neuroprotective, free radical scavenging, and anti-inflammatory effects, and are widely used in health preservation and other fields.

[0005] Lycium barbarum glycopeptide (LbGp) is a glycoconjugate considered the most promising monomeric component of Lycium barbarum fruit. It includes LbGp1 (88 kDa), LbGp2 (68.2 kDa), LbGp3 (92.5 kDa), LbGp4 (214.8 kDa), and LbGp5 (23.7 kDa). It is a polypeptide primarily composed of glycans linked by covalent bonds, with a molecular weight of 88 kDa. Its monosaccharide composition consists of arabinose (Ara), galactose (Gal), and glucose (Glc) in a molar ratio of 2.5:1.0:1.0. Its protein content is 30%, with O-linked connections between the glycosyl groups and the core protein backbone. It also contains 18 other natural amino acids, ensuring the quantification and reproducibility of experimental drug studies. Current research indicates that LbGp has effects such as lowering blood sugar and lipids, reducing depressive and anxiety behaviors, and preventing the occurrence and progression of acute colitis.

[0006] However, to date, there have been no reports on the anti-fatigue effects of goji glycopeptides, or combinations of goji glycopeptides with other natural product active ingredients. Therefore, the inventors have developed a new application for goji glycopeptides in anti-fatigue. Summary of the Invention

[0007] The purpose of this invention is to overcome the defects and deficiencies in the existing technology, realize the development and utilization of natural products using modern research methods, and provide a wolfberry glycopeptide composition for anti-fatigue and its uses.

[0008] Specifically, the present invention is achieved through the following technical solutions: In a first aspect, the present invention provides a wolfberry glycopeptide composition for anti-fatigue purposes, the composition comprising wolfberry glycopeptide as an active ingredient.

[0009] Alternatively, in the above composition, the preparation method of the wolfberry glycopeptide mainly includes the steps of washing, soaking, crushing and pulping, extraction, double-stage pulping, two-stage separation, membrane separation, single-effect concentration, and freeze-drying. Specifically, wolfberry seeds are separated in the double-stage pulping step, wolfberry pulp is separated in the two-stage centrifugation step, and oligosaccharides are separated in the membrane separation step, ultimately obtaining the wolfberry glycopeptide.

[0010] Alternatively, in the above composition, the wolfberry glycopeptide comprises one or more of the following: LbGp1, LbGp2, LbGp3, LbGp4, or LbGp5.

[0011] Alternatively, in the above composition, the wolfberry glycopeptide comprises LbGp1, LbGp2, LbGp3, LbGp4 and LbGp5, wherein the mass ratio of LbGp1, LbGp2, LbGp3, LbGp4 and LbGp5 is (0.01-0.02):(0.005-0.01):(0.01-0.02):(0.02-0.03):(0.01-0.02).

[0012] Preferably, in the wolfberry glycopeptide, the mass ratio of LbGp1, LbGp2, LbGp3, LbGp4 and LbGp5 is 0.018:0.0077:0.018:0.029:0.014.

[0013] Preferably, the "Lycium Barbarum Glycopeptide (LbGp)" powder used in this invention is provided by Ningxia Tianren Goji Berry Biotechnology Co., Ltd.

[0014] The preparation of the wolfberry glycopeptide of this invention includes the extraction and separation of a polysaccharide and peptide chain covalently bound by a process involving pure physical centrifugation, membrane filtration purification, and vacuum freeze-drying of an aqueous extract of wolfberry. It contains LbGp1, LbGp2, LbGp3, LbGp4, and LbGp5. Specifically, LbGp1 contains 28.87% protein, LbGp2 contains 9.25% protein, LbGp3 contains 5.19% protein, LbGp4 contains 10.75% protein, and LbGp5 contains 59.87% protein. The mass ratio of LbGp1, LbGp2, LbGp3, LbGp4, and LbGp5 in the wolfberry glycopeptide is 0.018:0.0077:0.018:0.029:0.014.

[0015] Alternatively, in the above composition, the composition may contain goji berry glycopeptide as a single active ingredient, or the composition may further contain one or more active ingredients that do not antagonize goji berry glycopeptide.

[0016] Alternatively, in the above composition, the composition comprises lycopene glycopeptide and scutellarin, wherein the mass ratio of lycopene glycopeptide to scutellarin is 1:5 to 5:1.

[0017] Alternatively, in the above composition, the composition consists of goji glycopeptides and scutellarin.

[0018] Alternatively, in the above composition, the mass ratio of the goji berry glycopeptide to the scutellarin is 2:1 to 5:1.

[0019] Preferably, the mass ratio of the goji berry glycopeptide to the scutellarin is 4:1.

[0020] Preferably, the scutellarin is extracted by those skilled in the art from plants containing natural sources of scutellarin (e.g., Scutellaria baicalensis, Smilax glabra, Smilax glabra, etc.) using conventional methods, or is obtained directly by purchase.

[0021] In a second aspect, the present invention provides the use of the composition described in the first aspect above in the preparation of anti-fatigue products.

[0022] Alternatively, in the above-described uses, the fatigue is general fatigue or fatigue caused by exercise.

[0023] Alternatively, in the above-described uses, the product may be a pharmaceutical, health supplement, or functional food.

[0024] Alternatively, in the above-described uses, the product may also contain a pharmaceutically, health-promoting, or functional food-grade carrier.

[0025] Alternatively, in the above-described uses, the dosage form of the product is tablets, pills, oral liquids, capsules, syrups, granules, injections, or suppositories.

[0026] Compared with the prior art, the present invention has the following beneficial effects: The inventors have discovered for the first time the anti-fatigue effect of Lycium barbarum glycopeptides. Furthermore, they found that combining Lycium barbarum glycopeptides with scutellarin at a specific mass ratio significantly enhances the anti-fatigue effect of Lycium barbarum glycopeptides. Experimental results from this invention confirm that Lycium barbarum glycopeptides, a characteristic active extract of the traditional Chinese medicinal herb Lycium barbarum, possesses clear anti-fatigue activity on their own, and the combination with scutellarin further enhances their synergistic effect. The composition provided by this invention has clear components and stable activity, possessing broad prospects for development into various types of anti-fatigue products, including pharmaceuticals, health foods, and special medical purpose formula foods, thus possessing significant social and economic value. Detailed Implementation

[0027] This invention utilizes modern research methods to develop and utilize natural products. Through extensive screening, it was discovered for the first time that a wolfberry glycopeptide composition possesses anti-fatigue properties. Based on this discovery, this invention was completed.

[0028] In the applications described above, the timing, frequency, and duration of administration of "goji berry glycopeptide" and other active ingredients need to be determined based on the specific diagnostic results of the condition, which is within the technical scope of those skilled in the art. For example, when applying a treatment regimen for mice or rats to humans, the effective dose of all drugs for humans can be converted from the effective dose of the drug for mice or rats, which is easily achievable by those skilled in the art.

[0029] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0030] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0031] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0032] Unless otherwise stated, all percentages and parts in this invention refer to weight percentages and weight parts.

[0033] Example: Example 1: Preparation method of Lycium barbarum glycopeptides The preparation process of wolfberry glycopeptides involves the following steps: raw material (dried wolfberries) - washing - soaking - crushing - extraction - filtration - concentration - freeze-drying - finished product. The main steps are summarized below: After harvesting, the wolfberries need to be dried, washed, and chopped. The fat-soluble and partially water-soluble pigments in the wolfberries are extracted sequentially using ether and acetone. Then, the berries are soaked in neutral water. After soaking, the liquid is filtered and centrifuged to extract the supernatant. The supernatant is precipitated using anhydrous ethanol, and the precipitate is then dissolved in water. The proteins in the solution are released using a mixture of chloroform and n-butanol. After multiple dialysis and centrifugation to remove the precipitate, the supernatant is freeze-dried to obtain wolfberry polysaccharide (LBP). Next, the extracted crude LBP polysaccharide was purified again. First, LBP was subjected to column chromatography using DEAE-Cellulose and eluted with 0.05 mol / L NaHCO3 solution to obtain LBP-1. Then, LBP-1 was dialyzed to desalt and concentrate, and then subjected to dextran gel (Sephadex G100) column chromatography and eluted with 0.10 M NaCl solution. The eluted solution was freeze-dried to obtain wolfberry glycopeptide (LbGp).

[0034] Example 2: The effect of wolfberry glycopeptides on improving fatigue in mice 2.1 Experimental Methods (1) Experimental design In this embodiment, 20 eight-week-old male C57BL / 6 mice were randomly divided into two groups using a random number table: a control group (FTG, n=10) and a Lycium barbarum glycopeptide (LbGp) intervention group (n=10). A chronic fatigue model was established through a 6-week exercise load.

[0035] Twenty-four hours after the last exercise session, behavioral and athletic performance tests were conducted sequentially. The behavioral tests included the open field test and the sucrose preference test, while the athletic performance test was the treadmill exhaustion test.

[0036] (2) Exercise program for fatigue model mice Mice were exercised for 6 weeks, 7 days a week, for 30 minutes each day. The treadmill incline was fixed at 10° for the first three weeks, with interval training involving progressively increasing speeds each week. Each week's training included four speed levels: 5, 10, 15, and 20 m / min, with the exercise time at each level systematically increased week by week. Week 1: The exercise time combinations at each speed level were 5 min (5 m / min), 5 min (10 m / min), 15 min (15 m / min), and 5 min (20 m / min). Week 2: The time combinations were adjusted to 5 min (5 m / min), 5 min (10 m / min), 10 min (15 m / min), and 10 min (20 m / min). Week 3: Further adjustments were made to 5 min (5 m / min), 5 min (10 m / min), 5 min (15 m / min), and 15 min (20 m / min). Weeks 4 to 6 maintained the same slope, focusing on extending the duration of medium- and high-speed exercise. The exercise time for 10 m / min and 15 m / min was 5 min, and the exercise time for 20 m / min was extended to 20 min.

[0037] (3) Intervention Program Starting from week 4, the group receiving the goji berry glycopeptide group was given a goji berry glycopeptide solution by gavage at a dose of 50 mg / kg 30 minutes after the end of daily exercise training. The control group received an equal volume of solvent (pure water) by gavage during the same period. This intervention continued until the end of the experiment, for a total of 3 weeks.

[0038] (4) Data representation methods and statistical analysis methods Experimental data are expressed as mean ± standard deviation (x±s), and independent samples t-tests were used for statistical analysis. A p-value < 0.05 was considered statistically significant between the two groups.

[0039] 2.2 Experimental Results (1) Results of athletic ability test As shown in Table 1, compared with the simple fatigue model group (control group), the exercise endurance of mice was significantly improved after LbGp intervention. The exhaustion distance and exhaustion time of mice in the LbGp group were significantly increased compared with those in the model group, indicating that LbGp can effectively enhance the body's sustained work capacity and fatigue resistance under fatigue. However, there was no significant difference in the maximum speed reached by the two groups of mice at exhaustion, suggesting that LbGp mainly improves the endurance of exercise, rather than the instantaneous output power under extreme conditions.

[0040] Table 1: Effects of Lycium barbarum glycopeptides on the motor function of mice (n=10, x±s) Note: Compared with the fatigue model group. p <0.05.

[0041] (2) Behavioral test results As shown in Table 2, in the open field experiment assessing anxiety-like behavior and spontaneous exploration, the total movement distance, the proportion of movement distance in the central region, and the proportion of time spent in the central region were all significantly increased in the LbGp intervention group compared to the fatigue model group (control group). The increase in total movement distance suggests that LbGp improved the overall willingness to move and the drive to explore in the mice; while the significant prolongation of activity time in the central region directly indicates that the avoidance anxiety-like behavior caused by fatigue was effectively alleviated.

[0042] Furthermore, the depressive mood was assessed using a sucrose preference test. As shown in Table 2, the LbGp intervention group showed a significantly higher sucrose intake rate than the fatigue model group (control group). The recovery of the sucrose preference rate indicates that LbGp intervention alleviated the anhedonia or loss of interest that may accompany chronic fatigue.

[0043] Table 2: Effects of Lycium barbarum glycopeptides on anxiety and depression in mice (n=10, x±s) Note: Compared with the fatigue model group. p <0.05, p <0.01, p <0.001.

[0044] Example 3: The effect of the combination of wolfberry glycopeptide and scutellarin on improving fatigue in mice. 3.1 Test Methods (1) Grouping and administration of experimental animals Male C57BL / 6 mice aged 6–8 weeks were randomly divided into 5 groups (n=8 per group): blank control group, solvent control group, Lycium barbarum glycopeptide group (50 mg / kg), Lycium barbarum glycoside group (12.5 mg / kg), and combination group (Lycium barbarum glycopeptide 50 mg / kg + Lycium barbarum glycoside 12.5 mg / kg). The experimental groups were administered the different test drugs via gavage. The blank control group received no treatment. The solvent control group received the same volume of 0.1% sodium carboxymethyl cellulose solution via gavage as the test drug groups. Administered once daily for 14 consecutive days.

[0045] (2) Mouse normobaric hypoxia tolerance test After the last administration of the drug, the mice in each group of the above-mentioned group were placed in a 250 mL ground glass bottle containing 10 g of soda lime and sealed with a lid. Their survival time was observed, with respiratory arrest as the indicator of death.

[0046] (3) Mouse weighted swimming experiment Another group of mice from each of the groups in Part (1) above were taken. The day after the last administration, the mice were placed in a swimming tank for swimming. The water depth was not less than 30 cm, the water temperature was (25±1.0)℃, and a lead plate with 10% of the mouse's body weight was tied to the base of the mouse's tail. The mice were forced to swim until exhaustion, and the swimming ended when the mouse's head was submerged in the water for 5 seconds and no longer floated up. The time was recorded as the weighted swimming time of the mouse. After the mice were removed, they were euthanized, blood was collected from the eyeballs, and the serum was separated by centrifugation. The levels of lactate dehydrogenase (LDH) and blood urea nitrogen (BUN) in the serum were measured.

[0047] (4) Statistical processing All experimental data are expressed as x±s, and one-way ANOVA analysis was used for statistical analysis. p The difference between the two groups was statistically significant when the inequality was less than 0.05.

[0048] 3.2 Results (1) Effects on normobaric hypoxia tolerance test in mice As shown in Table 3 below, the experimental data of the blank control group and the solvent control group are very similar, indicating that the drug administration solvent has no effect on the experimental results. The Lycium barbarum glycopeptide group significantly prolonged the survival time of mice under normobaric hypoxia, showing a significant difference compared to the solvent control group. p <0.05%. Compared with the solvent control group, the scutellarin group had no effect on the survival time of mice under normobaric hypoxia. However, the results of the combination group showed that when scutellarin was used in combination with Lycium barbarum glycopeptide, although scutellarin itself had no effect on the survival time of mice under normobaric hypoxia, it could further significantly enhance the effect of Lycium barbarum glycopeptide on prolonging the survival time of mice under normobaric hypoxia. p <0.001).

[0049] Table 3: Effects of the combination of Lycium barbarum glycopeptides and Lycopene on the normobaric hypoxia tolerance of mice (n=8, x±s) Note: Compared with the solvent control group p <0.05, p <0.001.

[0050] (2) Effects on the weighted swimming test in mice As shown in Table 4 below, the experimental data of the blank control group and the solvent control group are very similar, indicating that the drug administration solvent has no effect on the experimental results. The Lycium barbarum glycopeptide group significantly prolonged the weight-bearing swimming time of mice. p <0.01), enhancing serum LDH activity in mice ( p <0.05, reduced serum BUN levels in mice, showing a significant difference compared to the solvent control group ( p <0.05%. Compared with the solvent control group, the scutellarin group had no significant effect on these indicators. However, the results of the combination group show that when scutellarin is used in combination with Lycium barbarum glycopeptide, it can further significantly enhance the effect of Lycium barbarum glycopeptide on prolonging the swimming time of mice under load. p <0.001), significantly enhanced serum LDH activity in mice ( p <0.001) and significantly reduced serum BUN levels in mice ( p <0.01).

[0051] Table 4: Effects of the combination of Lycium barbarum glycopeptide and Lycopene on the weight-bearing swimming test in mice (n=8, x±s) Note: Compared with the solvent control group p <0.05, p <0.01, p <0.001.

[0052] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A wolfberry glycopeptide composition for anti-fatigue, characterized in that: The composition contains goji berry glycopeptide as an active ingredient.

2. The composition of claim 1, wherein: The goji berry glycopeptide comprises one or more of the following: LbGp1, LbGp2, LbGp3, LbGp4 or LbGp5. Preferably, the goji berry glycopeptide comprises LbGp1, LbGp2, LbGp3, LbGp4 and LbGp5, wherein the mass ratio of LbGp1, LbGp2, LbGp3, LbGp4 and LbGp5 is (0.01-0.02):(0.005-0.01):(0.01-0.02):(0.02-0.03):(0.01-0.02).

3. The composition of claim 1 or claim 2, wherein: The composition contains goji berry glycopeptide as a single active ingredient, or the composition further contains one or more active ingredients that do not antagonize goji berry glycopeptide.

4. The composition of claim 3, wherein: The composition comprises lycine glycopeptide and scutellarin, wherein the mass ratio of lycine glycopeptide to scutellarin is 1:5 to 5:

1.

5. The composition of claim 4, wherein: The mass ratio of the goji berry glycopeptide to the scutellarin is 2:1 to 5:

1.

6. Use of the composition according to any one of claims 1 to 5 in the preparation of an anti-fatigue product.

7. Use according to claim 6, characterized in that: The fatigue mentioned refers to general fatigue or fatigue caused by exercise.

8. Use according to claim 6, characterized in that: The product is a medicine, health product, or functional food.

9. Use according to claim 8, characterized in that: The product also contains a carrier that is pharmaceutically, health-promoting, or functional food acceptable.

10. Use according to claim 9, characterized in that: The dosage form of the product is tablets, pills, oral liquids, capsules, syrups, granules, injections, or suppositories.

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