An immunomodulatory and anti-fatigue agent and a preparation method thereof

Abstract

This invention provides an immunomodulatory and anti-fatigue agent and its preparation method, belonging to the field of traditional Chinese medicine technology. The invention provides an immunomodulatory and anti-fatigue agent composed of black goji berry polysaccharide and yellow-green mushroom polysaccharide, possessing immunomodulatory and anti-fatigue effects. The prepared compound drug shows regulatory effects on metabolites and oxidative enzymes in fatigue model mice, as well as immunomodulatory effects on LPS-induced RAW 264.7 macrophages. The black goji berry polysaccharide LRWP extracted by this method has a sugar content of 54.7%, a uronic acid content of 0.9%, and a protein content of 1.3%; the yellow-green mushroom crude polysaccharide HMGWP has a sugar content of 25.7%, a uronic acid content of 0.4%, and a protein content of 4.8%.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology and relates to an immune modulator and anti-fatigue agent and its preparation method. Background Technology

[0002] Fatigue, as a prevalent physiological and psychological state, has become a significant factor affecting health and quality of life in modern society. Its symptoms typically manifest as physical or mental exhaustion, difficulty concentrating, lack of motivation, and decreased interest in daily activities. Research has found that the mechanisms of fatigue are the result of multifactorial and multi-system interactions, involving neurotransmitter imbalances in the central nervous system, decreased energy metabolism efficiency, enhanced inflammatory responses in the immune system, and dysfunction of the endocrine system (such as the HPA axis). Although some progress has been made in fatigue research, its complex mechanisms are not yet fully understood, and further in-depth research is needed to better understand and address this global health problem.

[0003] Currently, there are various anti-fatigue drugs available, but most of them are often accompanied by significant side effects. In contrast, natural drugs, due to their higher safety profile, fewer side effects, and multi-target mechanisms of action, are gradually becoming a hot topic and key research area in this field. Summary of the Invention

[0004] In view of this, based on the functional properties of polysaccharides, the present invention provides a method for preparing the aforementioned immunomodulatory and anti-fatigue agent. The polysaccharide (LA) from black goji berries and the polysaccharide (HG) from *Fragaria pubescens* obtained by this method have high purity and better efficacy. A second objective of the present invention is to provide an immunomodulatory and anti-fatigue agent, which, through combined drug application, significantly enhances its immunomodulatory and anti-fatigue effects.

[0005] This invention provides an immunomodulatory and anti-fatigue agent composed of black goji berry polysaccharide and yellow-green mushroom polysaccharide, with a weight ratio of black goji berry polysaccharide to yellow-green mushroom polysaccharide of 1:2.

[0006] Black goji berry (Lycium ruthenicum Murr.) is a thorny shrub belonging to the genus Lycium in the Solanaceae family, possessing various medicinal values. Black goji berries are known for their kidney-tonifying and essence-boosting effects, liver-nourishing and vision-improving properties, blood-tonifying and calming effects, and thirst-quenching effects. Recent studies have discovered that black goji berries also exhibit various pharmacological activities, including antioxidant, anti-fatigue, anti-aging, and neuroprotective effects. Furthermore, research indicates that the polysaccharide components in black goji berries possess diverse biological activities, such as antioxidant, immunomodulatory, neuroprotective, and prebiotic activities.

[0007] *Floccularia luteovirens*, a large edible fungus widely distributed in the alpine meadows of the Qinghai-Tibet Plateau, possesses unique nutritional and medicinal value. In recent years, *Floccularia luteovirens* polysaccharides, as a major component of this fungus, have attracted widespread attention due to their significant biological activities. Studies have found that *Floccularia luteovirens* exhibits significant pharmacological effects, including antioxidant, immunomodulatory, and antitumor properties. Furthermore, *Floccularia luteovirens* polysaccharides demonstrate significant anti-fatigue effects through multiple mechanisms, such as regulating energy metabolism, reducing oxidative stress damage, and enhancing antioxidant capacity, providing a scientific basis for the development of novel anti-fatigue natural products.

[0008] This application demonstrates that the immunomodulatory and anti-fatigue activities of black wolfberry polysaccharide and yellow-green mushroom polysaccharide are significantly superior to those of single herbs, showing obvious compatibility advantages.

[0009] This invention also provides a method for preparing the aforementioned immunomodulatory and anti-fatigue agent, comprising the following steps:

[0010] 1) Black goji berries were harvested from Dongshagen, Dulan County, Haixi Mongolian and Tibetan Autonomous Prefecture, Qinghai Province, China. Yellow-green curly-haired mushroom fruiting bodies were harvested from Qilian, Haibei Prefecture, Qinghai Province. The black goji berry fruits and yellow-green curly-haired mushroom fruiting bodies were dried, ground, and sieved to obtain black goji berry fruit powder and yellow-green curly-haired mushroom fruiting body powder.

[0011] 2) The black goji berry powder was microwaved, then extracted with hot water, precipitated by ethanol fractionation, and vacuum dried to obtain black goji berry total polysaccharide (LRWP).

[0012] 3) The fruiting body powder of *Hygrophila globosum* was extracted with hot water, centrifuged, precipitated with ethanol, collected by centrifugation, and freeze-dried to obtain crude polysaccharide of *Hygrophila globosum* (HMGWP).

[0013] 4) Determine the purity of the black goji berry polysaccharide and yellow-green mushroom crude polysaccharide obtained in steps 2) and 3). The LRWP extracted by this method has the following contents: sugar content 54.7%, uronic acid content 0.9%, and protein content 1.3%; HMGWP has the following contents: sugar content 25.7%, uronic acid content 0.4%, and protein content 4.8%.

[0014] In the preparation method of the present invention, step 1) specifically includes the following operations:

[0015] The black goji berry fruit and the yellow-green curly hair mushroom fruit body were dried, ground into powder, and then passed through an 80-mesh sieve to obtain black goji berry fruit powder and yellow-green curly hair mushroom fruit body powder.

[0016] Drying not only facilitates grinding but also reduces microbial contamination, stabilizes polysaccharide components, and facilitates storage and transportation, while also reducing impurity content. Grinding further increases surface area, breaks down cell walls, and improves extraction efficiency and uniformity, facilitating subsequent processing. These two steps work together to significantly improve the extraction rate and purity of polysaccharides from black goji berries and *Fragaria leucocephala*.

[0017] In the preparation method of the present invention, in step 2), the microwave treatment, hot water extraction, ethanol fractionation precipitation, and chromatography treatment specifically refer to:

[0018] Black goji berry fruit powder was extracted using a microwave at 300-500W for 20-40 minutes. The extract was then soaked in an 80-95% ethanol solution at a ratio of (15-30):1 for 2-3 hours to obtain total polysaccharide LRWP from black goji berries. Microwave treatment has significant advantages in the extraction of polysaccharides from black goji berries. It not only significantly improves extraction efficiency and polysaccharide extraction rate but also maintains the bioactivity of polysaccharides, reduces the dissolution of impurities, and increases the purity of the extract.

[0019] In the preparation method of the present invention, in step 3), the hot water extraction and ethanol fractionation precipitation treatment specifically refers to:

[0020] The fruiting body powder of *Pleurotus eryngii* weighs approximately 500g and is crushed into 1-2cm pieces. 2 The fragments were added to 5L of pure water and soaked at room temperature for 4 hours. Then, they were boiled in water for 2 hours each time for a total of 3 extractions. After filtering with four layers of gauze, the filtrate was collected and concentrated to 4-5L at 80°C. 95% ethanol was added until the ethanol concentration in the mixture reached about 80%. The mixture was left to stand at room temperature overnight. The supernatant was discarded and the precipitate was collected by centrifugation. The precipitate was freeze-dried to obtain the crude polysaccharide HMGWP from Mushroom Yellow-green Hairy Mushroom.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] The combination of black goji berry polysaccharide and yellow-green mushroom polysaccharide showed significantly better immunomodulatory and anti-fatigue activities in mice than either herb alone, demonstrating a clear synergistic advantage. The combination significantly reduced the levels of pro-inflammatory cytokines and increased the levels of anti-inflammatory cytokines. Furthermore, in a mouse fatigue model, the combination of black goji berry polysaccharide and yellow-green mushroom polysaccharide significantly prolonged the time mice spent on a stick, reduced the time mice spent at rest in water, and decreased the levels of serum metabolites LA, CK, BUN, MDA, and ROS after fatigue exercise, while increasing the concentrations of SOD, CAT, and GSH-Px in mice after fatigue exercise.

[0023] The extraction methods for black goji berry polysaccharides and yellow-green mushroom polysaccharides are easy to operate and have high extraction efficiency. The LRWP obtained by this method has a sugar content of 54.7%, a uronic acid content of 0.9%, and a protein content of 1.3%; HMGWP has a sugar content of 25.7%, a uronic acid content of 0.4%, and a protein content of 4.8%.

[0024] This invention combines black goji berry polysaccharides and yellow-green mushroom polysaccharides to prepare an immunomodulatory and anti-fatigue agent. The immunomodulatory and anti-fatigue agent has been shown to have the following advantages:

[0025] (1) Significant immunomodulatory effects: The combined use of black wolfberry polysaccharide and yellow-green mushroom polysaccharide is significantly superior to that of single herbs, especially in LPS-induced RAW 264.7 macrophages, where it shows a clear synergistic advantage. By detecting inflammatory cytokines, it can significantly reduce pro-inflammatory cytokines and increase anti-inflammatory cytokines.

[0026] (2) Significant anti-fatigue effect: The combined use of black wolfberry polysaccharide and yellow-green mushroom polysaccharide is significantly superior to that of single herbs, especially in fatigue model mice, where it shows obvious compatibility advantages. Through fatigue rotarod test and forced swimming test, it can significantly prolong the time mice spend on the rotarod and reduce the time mice spend still in the water. This indicates its potential application value in anti-fatigue.

[0027] (3) The extraction process is simple and efficient: the extraction methods of black wolfberry polysaccharide and yellow-green mushroom polysaccharide are simple to operate and have high extraction efficiency, which ensures the purity and activity of the finished product and makes large-scale production possible.

[0028] The method for preparing an immunomodulatory and anti-fatigue agent provided by this invention, through a scientifically sound process, successfully achieves the effective combination of black wolfberry polysaccharide and yellow-green mushroom polysaccharide. This method not only significantly improves the clinical efficacy of traditional Chinese medicine, providing patients with a more efficient and safer treatment option, but also offers a novel approach and solution for fatigue relief, possessing broad market prospects and social value. Attached Figure Description

[0029] Figure 1 The effects of LA, HG, and LA+HG on the levels of inflammatory cytokines secreted by LPS-induced RAW 264.7 macrophages were compared. * P<0.05 and ** P<0.01 vs. the Control group. ## P<0.01 vs. the Model group.

[0030] Figure 2The effects of LA, HG, and LA+HG on the fatigue rotarod test and forced swimming test in fatigued mice were compared. * P<0.05 and ** P<0.01 vs. the Control group. ## P<0.01 vs. the Model group.

[0031] Figure 3 The effects of LA, HG, and LA+HG on oxidative stress-related biochemical indicators in fatigued mice were compared. * P<0.05 and ** P<0.01 vs. the Control group. ## P<0.01 vs. the Model group.

[0032] Figure 4 The effects of LA, HG, and LA+HG on fatigue-related metabolites in fatigued mice were compared. * P<0.05 and ** P<0.01 vs. the Control group. ## P<0.01 vs. the Model group. Detailed Implementation

[0033] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products. Example 1

[0034] Effects of the combination of black goji berry polysaccharide and yellow-green mushroom polysaccharide on LPS-induced RAW 264.7 macrophages

[0035] a. RAW 264.7 macrophages were divided into the 6 groups shown in Table 1:

[0036] Table 1 Group Experiments

[0037] Group LPS Drug dosage negative control group -- -- Model group 100 ng / mL -- Black goji berry polysaccharide group 100 ng / mL 150 μg / ml Yellow-green mushroom polysaccharide group 100 ng / mL 150 μg / ml Black goji berry polysaccharide + yellow-green mushroom polysaccharide (1:2) 100 ng / mL 50+100 μg / ml

[0038] b. Seed RAW 264.7 cells in 6-well plates and cultured until the cell density reached 70-80%. Add LPS (10 μg / mL) and incubate for 24 hours.

[0039] c. After 24 hours, remove the culture medium and wash the cells with PBS to remove residual LPS. Add culture medium containing different concentrations of compound traditional Chinese medicine, continue culturing the cells for 24 hours, and collect the culture supernatant.

[0040] d. Use an ELISA kit to detect the levels of TNF-α, IL-6, IL-1β and IL-10 respectively. Follow the instructions in the kit and measure the absorbance at 450 nm using a microplate reader.

[0041] Experimental results are as follows Figure 1 As shown, IL-1β, IL-6, and TNF-α, as pro-inflammatory cytokines, can rapidly initiate an immune response and promote the activation and proliferation of immune cells. IL-10, on the other hand, is an anti-inflammatory cytokine that reduces inflammation and maintains immune homeostasis by inhibiting the production of pro-inflammatory cytokines and the activation of immune cells. These cytokines interact in immune regulation, jointly maintaining the body's immune homeostasis. In the model group, the levels of IL-1β, IL-6, and TNF-α were significantly increased, while the level of IL-10 was significantly decreased. In the groups containing black goji berry polysaccharide, *Flammulina velutipes* polysaccharide, and a combination of both, the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α were significantly decreased, while the level of the anti-inflammatory cytokine IL-10 was significantly increased. This result indicates that both black goji berry polysaccharide and *Flammulina velutipes* polysaccharide have significant immunomodulatory effects, effectively inhibiting the excessive production of pro-inflammatory cytokines while promoting the secretion of anti-inflammatory cytokines, thereby restoring the body's immune homeostasis. Of particular note is that when black goji berry polysaccharide and yellow-green mushroom polysaccharide are used in combination, their immunomodulatory effect is significantly better than that of either polysaccharide used alone, and the difference is statistically significant.

[0042] Experiment Example 2

[0043] Effects of the combination of black goji berry polysaccharide and yellow-green mushroom polysaccharide on fatigue model mice

[0044] Experimental animals: C57BL / 6J male mice (20±2 g) were purchased from Changchun Yisi Experimental Animal Technology Co., Ltd. (Experimental animal license number: SYXK (Ji) 2019-0015).

[0045] a. The experimental animals were administered the drug by gavage for 14 consecutive days, with the dosages shown in Table 2:

[0046] Table 2 Dosage

[0047] Group Black goji berry polysaccharides Yellow-green mushroom polysaccharide physiological saline Blank control group -- -- 10 ml / kg Model group -- -- 10 ml / kg Black goji berry polysaccharide group 90 mg / kg -- -- Yellow-green mushroom polysaccharide group -- 90 mg / kg -- Black goji berry polysaccharide + yellow-green mushroom polysaccharide group 30 mg / kg 60 mg / kg --

[0048] b. Except for the control group, mice began swimming training 30 minutes after drug administration (for a total of 2 weeks, 6 days a week). The training was conducted in a circular plastic pool with a water depth of 20 cm, a water temperature of 28±2°C, and a diameter of 32 cm. The swimming time was 30 minutes on the first day, 45 minutes on the second day, and 60 minutes for the remaining days.

[0049] c. Mice underwent weighted free-range swimming adaptation in the first week. In the second week, a lead rope was wrapped around the mice's tails, with the weight of the lead rope being 2% of their body weight. In the third, fourth, fifth, and sixth weeks, the weight of the lead rope was increased to 4%, 6%, 8%, and 10%, respectively. The weighted swimming test was conducted on day 14.

[0050] d. On days 15-16, mice were placed on a rotating bar for training three times consecutively, each time for 5 minutes, with rotation speeds of 15 r / min, 18 r / min, and 18 r / min respectively. The rotation test was performed on day 17. On day 18, half an hour after drug administration, all mice were forced to swim without load for 15 minutes, and blood samples were then collected.

[0051] e. Place the collected blood sample in a plasma separator tube, let it stand for 30 min, then centrifuge at 3000 rpm for 15 min at 4°C and collect the supernatant. Use the blood supernatant to detect the levels of BUN, CK, LA, ROS, MDA, SOD, CAT, and GSH-Px according to the ELISA kit instructions. Measure the absorbance at 450 nm using a microplate reader and calculate the concentrations based on the standard curve.

[0052] Experimental results are as follows Figure 2 As shown. The experimental results are as follows. Figure 2 As shown in the figure. After sufficient training, we conducted weighted swimming and rotarod tests on mice on days 14 and 17, respectively, to assess their exercise endurance. In the rotarod test, compared with the control group, the average time spent on the rotarod was significantly reduced in the model group, indicating a significant decline in motor coordination under fatigue. However, the time spent on the rotarod was significantly prolonged in the black goji berry polysaccharide group, the yellow-green mushroom polysaccharide group, and the combination group of black goji berry polysaccharide and yellow-green mushroom polysaccharide. Among them, the time spent on the rotarod was the longest in the black goji berry polysaccharide and yellow-green mushroom polysaccharide group, indicating the most significant recovery of motor coordination.

[0053] In the forced swimming test, the exercise endurance of mice was assessed by observing the resting time of each group. The results showed that the resting time of the model group mice was significantly increased, indicating a significant decrease in exercise endurance under fatigue. Compared with the model group, the resting time of mice in the black goji berry polysaccharide group, the yellow-green mushroom polysaccharide group, and the combination group of black goji berry polysaccharide and yellow-green mushroom polysaccharide was significantly reduced, and the differences were statistically significant (P<0.01). In particular, the resting time of mice in the black goji berry polysaccharide and yellow-green mushroom polysaccharide combination group was even shorter, indicating the best effect on exercise endurance recovery. The above experimental results indicate that black goji berry polysaccharide, yellow-green mushroom polysaccharide, and the combination of black goji berry polysaccharide and yellow-green mushroom polysaccharide can all significantly improve the motor coordination and endurance level of mice, exhibiting good anti-fatigue effects, with the combination of black goji berry polysaccharide and yellow-green mushroom polysaccharide showing the most significant effect.

[0054] Experimental results are as follows Figure 3As shown, the accumulation of reactive oxygen species (ROS) in the body leads to lipid peroxidation, resulting in organ damage and pathological fatigue. Compared with the control group, the serum ROS level in the model group mice was significantly increased. However, the serum ROS concentrations in mice in the black goji berry polysaccharide group, the yellow-green mushroom polysaccharide group, and the combination group of black goji berry polysaccharide and yellow-green mushroom polysaccharide were all significantly reduced (P<0.05), and the reduction effect was most significant in the combination group of black goji berry polysaccharide and yellow-green mushroom polysaccharide.

[0055] Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) are important antioxidant enzymes in the human body. They can scavenge free radicals generated during metabolism, thereby reducing oxidative damage and muscle fatigue. Results showed that the serum levels of SOD, CAT, and GSH-Px in the model group mice were significantly lower than those in the control group. However, the serum levels of SOD, CAT, and GSH-Px in the black goji berry polysaccharide group, the yellow-green mushroom polysaccharide group, and the combination group of black goji berry polysaccharide and yellow-green mushroom polysaccharide were significantly higher than those in the model group, with the combination group showing the most significant increase. These results indicate that black goji berry polysaccharide, yellow-green mushroom polysaccharide, and the combination of black goji berry polysaccharide and yellow-green mushroom polysaccharide can effectively combat exercise-induced oxidative stress, not only reducing ROS production but also significantly increasing the levels of SOD, CAT, and GSH-Px, thus exerting antioxidant and anti-fatigue effects. Compared with using black goji berry polysaccharide or yellow-green mushroom polysaccharide alone, the regulatory effect of combining black goji berry polysaccharide and yellow-green mushroom polysaccharide is more significant.

[0056] Experimental results are as follows Figure 4 As shown in the figure, the main product of lipid peroxidation is malondialdehyde (MDA), and there is a close relationship between lipid peroxidation and fatigue. Compared with the model group, black goji berry polysaccharide, yellow-green mushroom polysaccharide, and the combination of black goji berry polysaccharide and yellow-green mushroom polysaccharide all significantly reduced the amount of MDA produced in mice, further demonstrating their antioxidant effects. It is worth noting that the combination of black goji berry polysaccharide and yellow-green mushroom polysaccharide had a more significant effect.

[0057] During strenuous exercise, lactate concentration and blood urea nitrogen (BUN) levels increase, which may lead to fatigue. Therefore, blood lactate concentration and BUN levels are also important indicators of fatigue. After forced swimming, lactate and BUN levels increased in mice, with the model group showing significantly higher serum lactate and BUN levels than the control group. The serum lactate and BUN levels in mice treated with black goji berry polysaccharide, *Amanita muscaria* polysaccharide, and a combination of both were significantly lower than in the model group, indicating that they effectively reduced lactate and BUN production in mice after exercise. The combination of black goji berry polysaccharide and *Amanita muscaria* polysaccharide showed the best improvement effect.

[0058] Creatine kinase (CK) levels are an important indicator for assessing the severity of exercise-induced injury. Serum CK levels in the model group mice were significantly higher than those in the control group. Black goji berry polysaccharide, *Flammulina velutipes* polysaccharide, and the combination of black goji berry polysaccharide and *Flammulina velutipes* polysaccharide all reduced CK levels, with the combination of black goji berry polysaccharide and *Flammulina velutipes* polysaccharide showing the most significant reducing effect. These results indicate that black goji berry polysaccharide, *Flammulina velutipes* polysaccharide, and the combination of black goji berry polysaccharide and *Flammulina velutipes* polysaccharide can effectively alleviate exercise-induced muscle damage, further confirming their anti-fatigue effects.

[0059] In conclusion, the experimental results of this study clearly demonstrate that the compound combination of black goji berry polysaccharide and yellow-green mushroom polysaccharide has a significant synergistic effect in immunomodulation and anti-fatigue, and its effect is significantly better than that of black goji berry polysaccharide or yellow-green mushroom polysaccharide alone. This discovery not only provides strong scientific evidence for the potential application of traditional Chinese medicine compound drugs in the fields of immunomodulation and anti-fatigue, but also provides valuable guidance for the development of related drugs, and has important theoretical and practical significance.

[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An immunomodulatory and anti-fatigue agent, characterized in that, It is composed of black goji berry polysaccharide and yellow-green mushroom polysaccharide, and the mass ratio of black goji berry polysaccharide to yellow-green mushroom polysaccharide is 1:(1-3). The method for preparing the black goji berry polysaccharide includes drying and pulverizing black goji berry fruit to obtain black goji berry fruit powder. The black goji berry powder was mixed with water and microwave-assisted extraction was performed. The extract was then fractionated and precipitated with ethanol and vacuum dried to obtain the total polysaccharides of black goji berries. The microwave power for microwave-assisted extraction is 300-500W, and the extraction time is 20-40 minutes.

2. The immunomodulatory and anti-fatigue agent according to claim 1, characterized in that, The ethanol is an aqueous solution with a volume percentage of 80-95%; the volume ratio of the aqueous ethanol solution to the extract is (15-30):1, and the soaking time is 2-3 hours.

3. The immunomodulatory and anti-fatigue agent according to claim 1, characterized in that, The preparation method of the polysaccharide of *Phyllostachys edulis* includes drying and pulverizing the fruiting bodies of *Phyllostachys edulis* to obtain *Phyllostachys edulis* fruiting body powder. The fruiting body powder of *Fragaria globosum* was extracted with hot water, centrifuged, and precipitated with ethanol. The precipitate was then collected by centrifugation and freeze-dried to obtain crude polysaccharide of *Fragaria globosum*.

4. The immunomodulatory and anti-fatigue agent according to claim 3, characterized in that, Before the hot water soaking, the process also includes soaking in pure water at room temperature for 4 hours; then boiling water extraction is performed, with each boiling extraction lasting 2 hours.

5. The immunomodulatory and anti-fatigue agent according to claim 4, characterized in that, The hot water extract was concentrated and then left to stand overnight with ethanol at a final concentration of 80%.

6. The method for preparing the immunomodulatory and anti-fatigue agent according to any one of claims 1-5, characterized in that, The immune modulator and anti-fatigue agent is obtained by mixing black goji berry polysaccharide and yellow-green mushroom polysaccharide according to the specified mass proportions.

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