A polysaccharide of rhizoma dioscoreae nigri, a preparation method and application thereof, and an ultraviolet light damage repair agent and application thereof

Abstract

The present application provides a polysaccharide of rhizoma dioscoreae nigri, a preparation method and application thereof, and an ultraviolet light damage repair agent and application thereof, and relates to the technical field of polysaccharides. The polysaccharide of rhizoma dioscoreae nigri provided by the present application comprises one or more of the following: a rhizoma dioscoreae nigri involucre crude polysaccharide (polysaccharide 37-42 wt%, uronic acid 36-40 wt%, protein 146-151 mg / g), a rhizoma dioscoreae nigri fruit crude polysaccharide (polysaccharide 52-56 wt%, uronic acid 16-18 wt%, protein 41-47 mg / g) and a rhizoma dioscoreae nigri cap crude polysaccharide (polysaccharide 57-61 wt%, uronic acid 33-35 wt%, protein 84-94 mg / g). The polysaccharide of rhizoma dioscoreae nigri provided by the present application can improve the HSF cell survival rate and antioxidant enzyme activity after UVB light damage, reduce the MDA content and inhibit the increase of ROS level, has the performance of resisting photoaging, can be used as an antioxidant to cope with oxidative stress, and has a wide application prospect in food, cosmetics, medicine and the like.

Description

Technical Field

[0001] This invention relates to the field of polysaccharide technology, specifically to a bamboo fungus polysaccharide and its preparation method and application, and an ultraviolet light damage repair agent and its application. Background Technology

[0002] Mid-wave ultraviolet radiation (UVB, 280~320nm), also known as mid-wave erythema-inducing ultraviolet radiation, causes erythema in the human body. It promotes mineral metabolism and vitamin D formation, but long-term or excessive exposure can cause skin damage, leading to erythema, abnormal pigmentation, wrinkles, and skin cancer in severe cases. UVB stimulation of the skin causes a large accumulation of reactive oxygen species (ROS) in the body, activating cellular oxidative stress, leading to loosening of collagen fiber structure and degradation of elastin, causing tissue damage and accelerating skin aging. Furthermore, ROS can cause lipid peroxidation damage, generating malondialdehyde (MDA), impairing membrane function and inducing apoptosis. With the increasing demand for anti-photoaging products, the selection of raw materials for these products has become an important issue.

[0003] Bamboo fungus ( Dictyophora indusiata Bamboo fungus (Dictyophora indica) is a rare saprophytic fungus that grows in forests or broad-leaved areas. Due to its beautiful appearance, unique taste, and rich nutritional value, it is known as the "Queen of Fungi" and the "King of Mountain Delicacies." A mature bamboo fungus can be divided into three parts: the cap, the volva, and the fruiting body. Polysaccharides are important bioactive substances in bamboo fungus, possessing antioxidant, immunomodulatory, and anti-inflammatory properties. However, bamboo fungus polysaccharides that repair UVB photodamage to human skin fibroblasts (HSF) have not yet been reported. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a bamboo fungus polysaccharide, its preparation method and application, and a UV damage repair agent and its application. The bamboo fungus polysaccharide provided by this invention has excellent repair ability on UVB photodamaged human skin fibroblasts.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a bamboo fungus polysaccharide, comprising one or more of the following: bamboo fungus volvulus crude polysaccharide, bamboo fungus fruiting body crude polysaccharide, and bamboo fungus cap crude polysaccharide;

[0007] The composition of the crude polysaccharide from the bamboo fungus includes: 37-42 wt% polysaccharide, 36-40 wt% uronic acid, and 146-151 mg / g protein.

[0008] The composition of the crude polysaccharide from the fruiting body of the bamboo fungus includes: 52-56 wt% polysaccharide, 16-18 wt% uronic acid, and 41-47 mg / g protein.

[0009] The composition of the crude polysaccharide from the bamboo fungus cap includes: 57-61 wt% polysaccharide, 33-35 wt% uronic acid, and 84-94 mg / g protein.

[0010] Preferably, the monosaccharides comprising the crude polysaccharide of *Dictyophora indica* include fucose, galactose, glucose, mannose, and glucuronic acid, and the molar ratio of fucose, galactose, glucose, mannose, and glucuronic acid is 1.3:4.6:16.9:2.4:22.6.

[0011] Preferably, the monosaccharides comprising the crude polysaccharide of the bamboo fungus fruiting body include fucose, galactose, glucose, mannose and glucuronic acid, and the molar ratio of fucose, galactose, glucose, mannose and glucuronic acid is 1.1:4.4:35.0:3.9:2.1.

[0012] Preferably, the monosaccharides comprising the crude polysaccharide of bamboo fungus include fucose, galactose, glucose, mannose and glucuronic acid, and the molar ratio of fucose, galactose, glucose, mannose and glucuronic acid is 6.7:3.1:50.9:23.0:2.4.

[0013] This invention provides a method for preparing the bamboo fungus polysaccharide described in the above technical solution.

[0014] The preparation method of the crude polysaccharide from bamboo fungus includes the following steps: collecting the inner layer gel of bamboo fungus, pulping the inner layer gel and then centrifuging it for the first time, and ultrafiltration the obtained gel supernatant to obtain the crude polysaccharide from bamboo fungus.

[0015] The preparation method of the crude polysaccharide of bamboo fungus fruiting body includes the following steps: first water extraction of bamboo fungus fruiting body powder, first alcohol precipitation of the obtained first water extract to obtain crude polysaccharide of bamboo fungus fruiting body;

[0016] The preparation method of the crude polysaccharide from bamboo fungus cap includes the following steps: performing a second water extraction on bamboo fungus cap powder, and performing a second alcohol precipitation on the obtained second water extract to obtain crude polysaccharide from bamboo fungus cap.

[0017] Preferably, the pulping time is 3-5 minutes;

[0018] The ultrafiltration has a molecular weight cutoff of 10 kDa.

[0019] Preferably, the solid-liquid ratio of the bamboo fungus fruiting body powder to the first water extraction water is 1g:8~12mL;

[0020] The temperature of the first water extraction is 90~100℃, and the time is 1.5~2.5h;

[0021] The first water extraction process further includes a second centrifugal separation of the obtained first water extraction system, and the supernatant obtained is concentrated to obtain the first water extract.

[0022] The volume ratio of the first aqueous extract to the alcohol used for the first alcohol precipitation is 1:0.6~1.2;

[0023] The alcohol used for the first alcohol precipitation includes ethanol;

[0024] The first alcohol precipitation was carried out at a temperature of 3-5°C for 10-14 hours.

[0025] Preferably, the solid-liquid ratio of the bamboo fungus cap powder to the second water extraction water is 1g:8~12mL;

[0026] The temperature of the second water extraction is 90~100℃, and the time is 1.5~2.5h;

[0027] The second water extraction process further includes a third centrifugal separation of the obtained second water extraction system, and the supernatant obtained is concentrated to obtain the second water extract.

[0028] The volume ratio of the second aqueous extract to the alcohol used for the second alcohol precipitation is 1:0.6-1.2;

[0029] The second alcohol used for precipitation includes ethanol.

[0030] The second alcohol precipitation is carried out at a temperature of 3-5°C for 10-14 hours.

[0031] This invention provides an ultraviolet light damage repair agent, the effective ingredient of which includes bamboo fungus polysaccharide as described in the above technical solution or bamboo fungus polysaccharide prepared by the above technical solution.

[0032] This invention provides the application of bamboo fungus polysaccharide described in the above-described technical solution, bamboo fungus polysaccharide prepared by the above-described technical solution, or ultraviolet light damage repair agent described in the above-described technical solution in cosmetics or pharmaceutical preparations.

[0033] This invention provides a bamboo fungus polysaccharide, comprising one or more of the following: Dictyophora indusiatavolva gel polysaccharide (DGP), Dictyophora indusiata polysaccharide (DIP), and Dictyophora indusiata pileus polysaccharide (DPP). The DGP composition includes: 37-42 wt% polysaccharide, 36-40 wt% uronic acid, and 146-151 mg / g protein. The DIP composition includes: 52-56 wt% polysaccharide, 16-18 wt% uronic acid, and 41-47 mg / g protein. The DPP composition includes: 57-61 wt% polysaccharide, 33-35 wt% uronic acid, and 84-94 mg / g protein. The bamboo fungus polysaccharide provided by this invention can improve the survival rate and antioxidant enzyme activity of HSF cells after UVB photodamage, and inhibit the increase of MDA content and ROS level in HSF cells after UVB photodamage. This indicates that the bamboo fungus polysaccharide provided by this invention has anti-photoaging and antioxidant capabilities, and can be used as an antioxidant to cope with oxidative stress. It provides a reference for applications in cosmetics, medicine, etc., and broadens the application scope of bamboo fungus.

[0034] This invention provides a method for preparing bamboo fungus polysaccharides as described in the above technical solution. The preparation method provided by this invention utilizes widely available raw materials, has low cost, simple processes, and is easy to operate, making it suitable for industrial production. Attached Figure Description

[0035] Figure 1 FT-IR spectra of DIP, DPP, and DGP;

[0036] Figure 2 SEM images of DIP, DPP, and DGP;

[0037] Figure 3 Figure showing the effects of DIP, DPP, and DGP on HSF cell viability;

[0038] Figure 4 Figure showing the effects of DIP, DPP, and DGP on HSF cell viability after UVB photodamage;

[0039] Figure 5 The graph shows the effect of DIP, DPP and DGP on ROS levels in HSF (percentage of ROS-positive cells in flow cytometry).

[0040] Figure 6 Bar chart showing the effects of DIP, DPP, and DGP on ROS levels in HSF;

[0041] Figure 7 The graph shows the effects of DIP, DPP, and DGP on the level of SOD under oxidative stress.

[0042] Figure 8 The graph shows the effects of DIP, DPP, and DGP on the oxidative stress level MDA.

[0043] Figure 9 The effect of DIP, DPP and DGP on oxidative stress level CAT is shown in the figure.

[0044] Figure 10 The graph shows the effects of DIP, DPP, and DGP on the oxidative stress level GSH-Px. Detailed Implementation

[0045] This invention provides a bamboo fungus polysaccharide, comprising one or more of the following: bamboo fungus volvulus crude polysaccharide, bamboo fungus fruiting body crude polysaccharide, and bamboo fungus cap crude polysaccharide.

[0046] In this invention, the crude polysaccharide from *Dictyophora indica* comprises: 37-42 wt% polysaccharide, preferably 39.47 ± 1.59 wt%; 36-40 wt% uronic acid, preferably 38.64 ± 0.64 wt%; and 146-151 mg / g protein, preferably 148.23 ± 2.15 mg / g. In this invention, the monosaccharides comprising the crude polysaccharide from *Dictyophora indica* preferably include fucose, galactose, glucose, mannose, and glucuronic acid, and the molar ratio of fucose, galactose, glucose, mannose, and glucuronic acid is preferably 1.3:4.6:16.9:2.4:22.6. In this invention, the weight-average molar mass (M...) of the crude polysaccharide from *Dictyophora indica* is... w The preferred value is (50~52)×10 3 g / mol, more preferably 51.29 × 10 g / mol. 3 g / mol; the number-average molar mass (M) of the crude polysaccharide from the bamboo fungus stalk. n The preferred value is (37~39)×10 3 g / mol, more preferably 37.96 × 10 g / mol. 3 g / mol; the molar mass distribution (M) of the crude polysaccharide from the bamboo fungus stalk. w / M n The ratio is preferably 1 to 2, and more preferably 1.351.

[0047] In this invention, the crude polysaccharide from the fruiting body of *Dictyophora indica* comprises: 52-56 wt% polysaccharide, preferably 54.27 ± 1.60 wt%; 16-18 wt% uronic acid, preferably 16.97 ± 0.96 wt%; and 41-47 mg / g protein, preferably 44.43 ± 2.45 mg / g. In this invention, the monosaccharides comprising the crude polysaccharide from the fruiting body of *Dictyophora indica* preferably include fucose, galactose, glucose, mannose, and glucuronic acid, and the molar ratio of fucose, galactose, glucose, mannose, and glucuronic acid is preferably 1.1:4.4:35.0:3.9:2.1. In this invention, the weight-average molar mass (Mi) of the crude polysaccharide from the fruiting body of *Dictyophora indica* is... w The preferred value is (2200~2300)×10 3 g / mol, more preferably 2110×10 g / mol 3 g / mol; the number-average molar mass (M) of the crude polysaccharide from the fruiting body of the bamboo fungus. n The preferred value is (1600~1800)×10 3 g / mol, more preferably 1678 × 10 g / mol 3 g / mol; the molar mass distribution of the crude polysaccharide from the fruiting body of the bamboo fungus (M w / M n The value is preferably 1 to 2, and more preferably 1.257.

[0048] In this invention, the composition of the crude polysaccharide from *Fungiidae* cap includes: 57-61 wt% polysaccharide, preferably 58.64 ± 1.44 wt%; 33-35 wt% uronic acid, preferably 34.16 ± 0.32 wt%; and 84-94 mg / g protein, preferably 89.04 ± 4.15 mg / g. In this invention, the monosaccharides constituting the crude polysaccharide from *Fungiidae* cap preferably include fucose, galactose, glucose, mannose, and glucuronic acid, and the molar ratio of fucose, galactose, glucose, mannose, and glucuronic acid is preferably 6.7:3.1:50.9:23.0:2.4. In this invention, the weight-average molar mass (Mi) of the crude polysaccharide from *Fungiidae* cap is... w The preferred value is (2100~2300)×10 3 g / mol, more preferably 2185×10 g / mol, 3 g / mol; the number-average molar mass (M) of the crude polysaccharide from the bamboo fungus cap. n The preferred value is (1000~2000)×10 3 g / mol, more preferably 1384 × 10 g / mol 3 g / mol; the molar mass distribution of the crude polysaccharide from the bamboo fungus cap (M w / M n The preferred value is 1 to 2, and more preferably 1.579.

[0049] The present invention provides a method for preparing crude polysaccharide from bamboo fungus volvulus as described above, comprising the following steps: collecting the inner layer gel of bamboo fungus volvulus, pulping the inner layer gel and then centrifuging it for the first time, and ultrafiltration of the obtained gel supernatant to obtain crude polysaccharide from bamboo fungus volvulus.

[0050] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0051] In this invention, the outer and inner skins of the bamboo fungus volva are removed first, and then the inner gel is collected.

[0052] In this invention, the pulping time is preferably 3-5 minutes, more preferably 5-5 minutes; the pulping temperature is preferably room temperature.

[0053] In this invention, the rotation speed of the first centrifugation is preferably 8000~10000 rpm, more preferably 9000~10000 rpm; the time of the first centrifugation is preferably 15~20 min, more preferably 15~18 min.

[0054] In this invention, the molecular weight cutoff of the ultrafiltration is preferably 10 kDa (retaining materials with a molecular weight greater than 10 kDa); the temperature of the ultrafiltration is preferably room temperature; the pressure difference (ΔP) of the ultrafiltration is preferably 0.4~0.8 bar, more preferably 0.6 bar; and the rotation speed of the ultrafiltration is preferably 200~300 r / min, more preferably 250 r / min.

[0055] Following ultrafiltration, the present invention preferably further includes concentrating and drying the retained portion to obtain crude polysaccharide from *Dictyophora indica*. In this invention, the concentration temperature is preferably 50-60°C, more preferably 55°C. The present invention does not specifically limit the concentration method; any concentration method well-known to those skilled in the art can be used, such as vacuum concentration. In this invention, the drying preferably includes freeze-drying. The present invention does not specifically limit the temperature and time of the freeze-drying, as long as the solvent is completely removed.

[0056] The present invention provides a method for preparing crude polysaccharide from bamboo fungus fruiting bodies as described in the above technical solution, comprising the following steps: subjecting bamboo fungus fruiting body powder to a first water extraction, subjecting the obtained first water extract to a first alcohol precipitation, thereby obtaining crude polysaccharide from bamboo fungus fruiting bodies.

[0057] In this invention, the solid-liquid ratio of the bamboo fungus fruiting body powder to the water used for the first water extraction is preferably 1g:10mL. In this invention, the temperature of the first water extraction is preferably 90~100℃, more preferably 95~100℃; the extraction time is preferably 1.5~2.5h, more preferably 2h.

[0058] The present invention preferably further includes a second centrifugal separation of the obtained first water extraction system after the first water extraction, and the supernatant obtained is concentrated to obtain the first water extract. In the present invention, the rotation speed of the second centrifugation is preferably 8000~10000 rpm, more preferably 9000~10000 rpm; the second centrifugation time is preferably 15~20 min, more preferably 15~18 min. In the present invention, the concentration temperature is preferably 50~60℃, more preferably 55℃; the volume of the first water extract is preferably 30~70% of the volume of the supernatant, more preferably 40~60%, and even more preferably 50%; the present invention does not have a special limitation on the concentration method, and a concentration method well known to those skilled in the art can be used, such as vacuum concentration.

[0059] In this invention, the volume ratio of the first aqueous extract to the alcohol used for the first alcohol precipitation is preferably 1:0.6~1.2, more preferably 1:1. In this invention, the alcohol used for the first alcohol precipitation includes ethanol; the alcohol is preferably anhydrous. In this invention, the temperature of the first alcohol precipitation is preferably 3~5℃, more preferably 4℃, and the time of the first alcohol precipitation is preferably 10~14h, more preferably 12h.

[0060] Following the first alcohol precipitation, the present invention preferably further includes subjecting the first alcohol precipitation system obtained from the first alcohol precipitation to a second centrifugation separation, washing the resulting solid component with alcohol, and then drying it to obtain crude polysaccharide from the fruiting body of *Dictyophora indica*. In the present invention, the rotation speed of the second centrifugation separation is preferably 8000-10000 rpm, more preferably 9000-10000 rpm; the time of the second centrifugation separation is preferably 15-20 min, more preferably 15-18 min. In the present invention, the alcohol used for washing preferably includes ethanol, and the alcohol is preferably anhydrous alcohol; the number of alcohol washings is preferably 3-4 times. In the present invention, the drying preferably includes freeze-drying. The present invention does not have special limitations on the temperature and time of the freeze-drying, as long as the solvent is completely removed.

[0061] The present invention provides a method for preparing the crude polysaccharide of bamboo fungus cap as described in the above technical solution, comprising the following steps: subjecting bamboo fungus cap powder to a second water extraction, subjecting the obtained second water extract to a second alcohol precipitation, and obtaining the crude polysaccharide of bamboo fungus cap.

[0062] In this invention, the solid-liquid ratio of the bamboo fungus cap powder to the water used for the second water extraction is preferably 1g:10mL. In this invention, the temperature of the second water extraction is preferably 90~100℃, more preferably 95~100℃; the extraction time is preferably 1.5~2.5h, more preferably 2h.

[0063] Following the second water extraction, the present invention further includes subjecting the obtained second water extraction system to a third centrifugal separation, and concentrating the obtained supernatant to obtain the second water extract. In this invention, the rotation speed of the third centrifugation is preferably 8000-10000 rpm, more preferably 9000-10000 rpm; the third centrifugation time is preferably 15-20 min, more preferably 15-18 min. In this invention, the concentration temperature is preferably 50-60℃, more preferably 55℃; the volume of the second water extract is preferably 30-70% of the volume of the supernatant, more preferably 40-60%, and even more preferably 50%; the present invention does not specifically limit the concentration method, and any concentration method well known to those skilled in the art can be used, such as vacuum concentration.

[0064] In this invention, the volume ratio of the second aqueous extract to the alcohol used for second alcohol precipitation is preferably 1:0.6~1.2, more preferably 1:1. In this invention, the alcohol used for second alcohol precipitation includes ethanol; the alcohol is preferably anhydrous. In this invention, the temperature for second alcohol precipitation is preferably 3~5℃, more preferably 4℃, and the precipitation time is preferably 10~14h, more preferably 12h.

[0065] Following the second alcohol precipitation, the present invention preferably further includes a third centrifugal separation of the second alcohol precipitation system, followed by alcohol washing and drying of the resulting solid component to obtain crude polysaccharide from the fruiting body of *Dictyophora indica*. In the present invention, the centrifugal speed for the third centrifugation is preferably 8000-10000 rpm, more preferably 9000-10000 rpm; the centrifugation time for the third centrifugation is preferably 15-20 min, more preferably 15-18 min. In the present invention, the alcohol used for the alcohol washing preferably includes ethanol, and the alcohol is preferably anhydrous alcohol; the number of alcohol washings is preferably 3-4 times. In the present invention, the drying preferably includes freeze-drying. The present invention does not have specific limitations on the temperature and time of the freeze-drying, as long as the solvent is completely removed.

[0066] This invention provides an ultraviolet light damage repair agent, the effective ingredient of which includes bamboo fungus polysaccharide as described in the above-described technical solution or bamboo fungus polysaccharide prepared by the above-described technical solution. In this invention, the ultraviolet light includes mid-wavelength ultraviolet light.

[0067] This invention provides the application of bamboo fungus polysaccharide, bamboo fungus polysaccharide prepared by the method described above, or the ultraviolet damage repair agent described above in cosmetics or pharmaceutical preparations. In this invention, the preferred application method includes the use of bamboo fungus polysaccharide in cosmetics or pharmaceuticals, preferably as an ultraviolet damage repair agent.

[0068] To further illustrate the present invention, the following detailed descriptions of bamboo fungus polysaccharide and its preparation method and application, as well as ultraviolet light damage repair agent and its application, are provided in conjunction with the accompanying drawings and embodiments. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0069] Example 1

[0070] The waste material from the post-harvest volva of *Dictyophora indica* was collected. The purple volva and white inner skin were removed, and the inner gel was collected. The gel was pulped for 5 minutes using a pulper, centrifuged at 10,000 rpm for 15 minutes, and the supernatant was collected for ultrafiltration. The retained fraction was concentrated at 55°C and then freeze-dried to obtain crude polysaccharide from the volva of *Dictyophora indica* (denoted as DGP). The ultrafiltration conditions were: molecular weight cutoff of 10 kDa, room temperature, ΔP = 0.6 bar, and rotation speed of 250 rpm.

[0071] Example 2

[0072] The dried fruiting bodies of *Dictyophora indica* were pulverized, and the resulting powder was mixed with distilled water. The mixture was heated to 100°C and extracted with water for 2 hours under stirring. The extract was then centrifuged at 10,000 rpm for 15 minutes, and the supernatant was collected. The supernatant was concentrated to 50% of its volume at 55°C. Anhydrous ethanol was added to the aqueous extract, and the mixture was precipitated at 4°C for 12 hours. The extract was then centrifuged at 10,000 rpm for 15 minutes, and the precipitate was washed three times with anhydrous ethanol and freeze-dried to obtain crude polysaccharide from the fruiting bodies of *Dictyophora indica* (denoted as DIP). The solid-liquid ratio of the fruiting body powder (dry weight) to distilled water was 1 g:10 mL; the volume ratio of the aqueous extract to the anhydrous ethanol used for precipitation was 1:1.

[0073] Example 3

[0074] After drying, the bamboo fungus cap was pulverized. The resulting bamboo fungus cap powder was mixed with distilled water and heated to 100℃. Water extraction was performed for 2 hours with stirring, followed by centrifugation at 10,000 rpm for 15 minutes. The supernatant was collected and concentrated to 50% of its volume at 55℃. Anhydrous ethanol was added to the resulting water extract, and alcohol precipitation was performed at 4℃ for 12 hours. Centrifugation was then performed at 10,000 rpm for 15 minutes. The precipitate was washed three times with anhydrous ethanol and then freeze-dried to obtain crude bamboo fungus cap polysaccharide (denoted as DPP). The solid-liquid ratio of bamboo fungus cap powder (dry weight) to distilled water was 1 g:10 mL; the volume ratio of the water extract to the anhydrous ethanol used for alcohol precipitation was 1:1.

[0075] Test Example 1

[0076] 1. Compositional analysis of the crude polysaccharides prepared in Examples 1-3

[0077] 2 mg of crude polysaccharide was placed in a round-bottom flask, and 3 mL of 2 mol / L trifluoroacetic acid (TFA) was added. Hydrolysis was carried out at 110 °C for 4 h. After hydrolysis, the product was dried using a nitrogen evaporator, and 3 mL of methanol was added repeatedly until no sour smell remained. The hydrolysis product was dissolved in ultrapure water, centrifuged, diluted, and filtered through a 0.22 μm filter membrane. Analysis was performed using a Dionex ICS-2500 ion chromatography system equipped with a CarboPac™ PA20 detection column (3 mm × 150 mm). A pulsed ampoule detector was used for detection at 30 °C. The mobile phase consisted of deionized water, 0.25 mol / L NaOH, and 1 mol / L NaAc. Fucose (Fuc), arabinose (Ala), rhamnose (Rha), galactose (Gal), glucose (Glc), xylose (Xyl), mannose (Man), galacturonic acid (GalA), and glucuronic acid (GlcA) were used as standards. The compositional analysis results of the crude polysaccharides prepared in Examples 1-3 are shown in Tables 1-2.

[0078] Table 1. Compositional analysis results of the crude polysaccharides prepared in Examples 1-3

[0079]

[0080] Note: All values ​​are expressed as mean ± standard deviation (n=3). Different lowercase letters in the same row indicate statistically significant differences (p<0.05).

[0081] As shown in Table 1, the polysaccharide content of DPP is higher than that of DIP and DGP, at 58.64%; the polysaccharide content of DGP is 39.47%; in terms of protein content, DGP has the highest protein content compared to other parts of bamboo fungus, at 148.23 mg / g; in terms of uronic acid content, DGP has a uronic acid content of 38.46%, which is higher than that of DIP and DPP, indicating that bamboo fungus polysaccharide is an acidic polysaccharide.

[0082] Table 2. Monosaccharide (molar ratio) of the crude polysaccharides prepared in Examples 1-3

[0083]

[0084] As shown in Table 2, DIP, DPP, and DGP are all composed of fucose, galactose, glucose, mannose, and glucuronic acid. The molar ratio of DIP is 1.1:4.4:35.0:3.9:2.1; the molar ratio of DPP is 6.7:3.1:50.9:23.0:2.4; and the molar ratio of DGP is 1.3:4.6:16.9:2.4:22.6. It can be seen that glucose and glucuronic acid are the main monosaccharides constituting DGP, which is an acidic polysaccharide.

[0085] 2. Molecular weight analysis of the crude polysaccharides prepared in Examples 1-3

[0086] Molecular weight was determined using high-performance gel permeation chromatography (HPSEC). Samples were prepared into 5 mg mL solutions. -1 The solution was completely dissolved, and the supernatant was collected, filtered through a 0.22 μm membrane, and then analyzed by HPSEC-MALLS-RI. The mobile phase was 0.05 mol / L. -1 NaH2PO4 and 0.02% (w / v) sodium azide. The results are shown in Table 3.

[0087] Table 3. Molecular weight analysis of the crude polysaccharides prepared in Examples 1-3

[0088]

[0089] Table 3 shows that the molecular weight of DIP is 2110 kDa, DPP is 2185 kDa, and DGP is 51.29 kDa. It can be seen that the molecular weight of DGP is much smaller than that of polysaccharides from other parts of bamboo fungus, classifying it as a small-molecule polysaccharide. The molecular weight distribution of polysaccharides is commonly measured using M... w / M n To measure. M w / M n The higher the value, the wider the molar mass distribution. DGP has an Mw / Mn ratio of 1.351, indicating that DGP has a narrow molecular weight distribution.

[0090] 3. Fourier transform infrared spectroscopy analysis of the crude polysaccharides prepared in Examples 1-3

[0091] A 2.0 mg lyophilized sample was placed at a designated position on a Nicolet iS50 infrared spectrometer for scanning, with the spectral acquisition range being 500–4000 cm⁻¹. -1 The resolution was set to 4 cm. -1 .

[0092] The infrared spectral structures of DIP, DPP and DGP are as follows: Figure 1 As shown, by Figure 1 It can be known that 3303cm -1 The absorption peaks at the left and right represent the stretching vibration of -OH. In fact, at 3400 cm⁻¹... -1 The nearby broad wavenumbers correlate with the OH band of carboxylic acids, thus indicating the presence of acidic monosaccharides in DGPs, corresponding to the monosaccharide composition results. 2912 cm⁻¹ -1 The nearby weak absorption peaks correspond to the antisymmetric and symmetric stretching vibrations of the -CH2 and -CH3 groups. (1732 and 1605 cm⁻¹) -1 The absorption bands on the left and right are attributed to the stretching vibration of the esterified carboxyl group (-COOR) and the asymmetric stretching of the carboxylate anion group (-COO-), respectively. 1409 cm⁻¹-1 The absorption peak may be attributed to the in-plane bending vibration of the aldehyde group -CHO. (1029 cm⁻¹) -1 The absorption peak at that position is due to the asymmetric stretching vibration of the D-pyranose ring, the absorption of lactones and hydroxyl groups in the pyranose ring, and is caused by the stretching vibration of CO in the COC ring and the angular vibration of OH in COH. This is a typical infrared spectral signal of dextran.

[0093] 4. Scanning electron microscopy of the crude polysaccharides prepared in Examples 1-3

[0094] Take 1 mg of each of the different parts of bamboo fungus polysaccharide after crushing, spray with gold for 15 min, and use an S48000 SEM at a vacuum environment with an accelerating voltage of 15 kV to observe the microstructure of the polysaccharide samples at magnifications of 50, 100, and 1000 times.

[0095] Figure 2 SEM images of DIP, DPP, and DGP at 50x, 100x, and 1000x magnification are shown. The SEM results at 50x and 100x magnification reveal that the polysaccharides from different parts of the bamboo fungus are distributed in a sheet-like pattern, with the polysaccharide fragments of DIP and DPP being larger than those of DGP. At 1000x magnification, the surface of DGP polysaccharide appears loose, porous, and rough, while the surfaces of DIP and DPP polysaccharides are smoother and denser, with DGP exhibiting a more compact surface, which may explain their higher apparent viscosity.

[0096] 5. Test on the UVB photodamage repair performance of the crude polysaccharides prepared in Examples 1-3

[0097] 5.1 Effects on the viability of human skin fibroblasts (HSF)

[0098] Human skin fibroblasts (HSFs) were preserved in DMEM complete medium containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin. They were cultured in a thermo- and humidified incubator at 37°C and 5% CO2. HSFs in logarithmic growth phase were seeded at 200 μL into 96-well plates at a cell concentration of 1 × 10⁶ cells / well. 4 Cells were cultured in 37°C (5% CO2) incubators for 24 hours, then washed twice with PBS. Before washing, the supernatant was discarded, and different concentrations (5, 10, 25, 50, 100, 200, 400, 800 μg / mL) of DIP, DPP, and DGP were added accordingly. PBS was added to the control group. Cell viability was assessed by MTT assay after 24 hours of culture. Results are shown below. Figure 3 As shown. By Figure 3It was found that DIP, DPP, and DGP at concentrations of 25, 50, 100, 200, and 400 μg / mL had no toxic effect on HSF. The survival rate of HSF in the 400 μg / mL DGP treatment group was 86.16%, and the survival rate in the 800 μg / mL DGP treatment group was 74.40%. Therefore, concentrations of 100, 200, and 400 μg / mL were selected as the concentrations for subsequent experiments.

[0099] 5.2 Protective effect on the viability of HSF cells damaged by UVB light

[0100] HSF in the logarithmic growth phase was seeded into 96-well plates at a density of 1 × 10⁻⁶. 4 Cells were cultured at 37°C in a 5% CO2 incubator for 24 hours until cell adhesion was achieved. The supernatant was discarded, and the cells were washed twice with PBS. Different concentrations (100, 200, 400 μg / mL) of DIP, DPP, and DGP were added to the treatment groups, while PBS was added to the blank and model groups. Cells were cultured again for 24 hours. After 24 hours, the culture medium was discarded, and the cells were incubated with 40 mJ / cm² water. 2 Cells were irradiated with UVB, then the PBS was discarded, and complete culture medium was added again. Cells were cultured for 24 hours. After 24 hours, the absorbance was measured using the MTT assay, and cell viability was calculated. Results are as follows: Figure 4 As shown.

[0101] Depend on Figure 4 As can be seen, compared with the HSF model group damaged by UVB, the three concentrations of DIP, DPP, and DGP (100, 200, and 400 μg / mL) all protected the survival of HSF cells damaged by UVB in a dose-dependent manner. Furthermore, compared with DIP and DPP, DGP showed a more significant reduction in HSF cell death caused by UVB damage, with the HSF survival rate in the 400 μg / mL DGP treatment group reaching 65.71%.

[0102] 5.3 Effects on ROS levels in HSF cells damaged by UVB light

[0103] The human body possesses an enzymatic antioxidant system, such as GSH-Px, CAT, and SOD, to maintain a dynamic redox balance between free radicals and ROS within cells. These antioxidants can prevent oxidative damage to cells and tissues, neutralize free radicals and ROS, and limit their harmful effects on cells. Intracellular ROS was measured using the fluorescent probe DCFH-DA to investigate whether 100, 200, and 400 μg / mL of DIP, DPP, and DGP could prevent UVB-induced cellular oxidative stress. The specific steps were as follows: HSF in logarithmic growth phase was seeded into 24-well plates, 1 × 10⁶ cells per well. 5After 24 hours of culture, the supernatant was discarded. Different concentrations (100, 200, 400 μg / mL) of DIP, DPP, and DGP were added to the treatment groups, while PBS was added to the blank and model groups. Cells were cultured again for 24 hours, with three replicate wells for each group. After culture, the original culture medium was replaced with DMEM containing 10 M DCFH-DA. Twenty minutes later, the cells were washed three times with PBS. Fluorescence intensity was quantitatively detected using flow cytometry, and images were taken using a fluorescence microscope.

[0104] Figure 5 The graph shows the effects of DIP, DPP, and DGP on ROS levels in HSF (the percentage of ROS-positive cells in flow cytometry). Figure 6 for Figure 5 The corresponding bar chart. (From...) Figure 5 and Figure 6 It was found that the intracellular ROS levels after treatment with crude polysaccharides from different parts of bamboo fungus were significantly lower than those in the UVB model group, but still higher than those in the control group without UVB treatment, demonstrating the antioxidant capacity of bamboo fungus polysaccharides. Furthermore, comparing the effects of DIP, DPP, and DGP on intracellular ROS levels in HSF cells, all three polysaccharides showed a dose-dependent reduction in intracellular ROS levels, with DGP exhibiting the strongest ability to reduce intracellular ROS levels.

[0105] The activities of intracellular antioxidant enzymes, including SOD, MDA, CAT, and GSH-Px, were measured, and the results are as follows: Figures 7-10 As shown. By Figures 7-10 As can be seen, UVB damage significantly decreased SOD, CAT, and GSH-Px levels in HSF cells, while treatment with different parts of bamboo fungus increased the activity of antioxidant enzymes in HSF cells and inhibited the increase in MDA content. Specifically, from Figure 7 It was found that, compared with the UVB control group, the protective effect of DGP on SOD activity in HSF was dose-dependent in the DGP treatment groups, with the 400 μg / mL DGP treatment group showing the best effect. Furthermore, comparing the DIP, DPP, and DGP treatment groups revealed that the SOD activity in HSF of the 400 μg / mL DGP treatment group was significantly higher than that of the other dosage groups. Figure 8 As can be seen, the lipid peroxidation experiment showed that the content of lipid peroxides (MDA) in HSF significantly increased after UVB damage. Compared with the UVB model group, the MDA content in the DGP treatment group decreased in a dose-dependent manner. Furthermore, comparing the MDA content in HSF after DIP, DPP, and DGP treatments revealed that 400 μg / mL DGP treatment had a good inhibitory effect on the increase in MDA content in HSF caused by UVB damage. Figure 9It was found that in the study on the effect of CAT activity, the protective effect of the DGP treatment group against the decrease in CAT activity of HSF after UVB damage was dose-dependent. Comparison of CAT activity in HSF treated with DIP, DPP, and DGP revealed that the 100 μg / mL DIP and 400 μg / mL DGP treatment groups had more significant protective effects against the decrease in CAT activity of HSF caused by UVB damage. Figure 10 Studies on the effects of DGP on GSH-Px activity revealed that the protective effect of DGP treatment against the decrease in GSH-Px in HSF caused by UVB damage was dose-dependent. Comparing the effects of DIP, DPP, and DGP treatments, it was found that 200 μg / mL DIP and 400 μg / mL DGP treatments had a more significant protective effect against GSH-Px activity in HSF.

[0106] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on the present invention without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of bamboo fungus polysaccharide in the preparation of ultraviolet light damage repair agent, wherein the bamboo fungus polysaccharide includes one or more of the following: bamboo fungus volvulus crude polysaccharide, bamboo fungus fruiting body crude polysaccharide, and bamboo fungus cap crude polysaccharide; The crude polysaccharide from the bamboo fungus includes: Polysaccharides 37-42 wt%, uronic acid 36-40 wt%, protein 146-151 mg / g; The composition of the crude polysaccharide from the fruiting body of the bamboo fungus includes: 52-56 wt% polysaccharide, 16-18 wt% uronic acid, and 41-47 mg / g protein. The composition of the crude polysaccharide from the bamboo fungus cap includes: 57-61 wt% polysaccharide, 33-35 wt% uronic acid, and 84-94 mg / g protein; The crude polysaccharide from the bamboo fungus includes monosaccharides such as fucose, galactose, glucose, mannose, and glucuronic acid, with a molar ratio of 1.3:4.6:16.9:2.4:22.

6. The crude polysaccharide from the fruiting body of the bamboo fungus comprises monosaccharides including fucose, galactose, glucose, mannose, and glucuronic acid, wherein the molar ratio of fucose, galactose, glucose, mannose, and glucuronic acid is 1.1:4.4:35.0:3.9:2.

1. The crude polysaccharide from bamboo fungus cap comprises monosaccharides including fucose, galactose, glucose, mannose, and glucuronic acid, with a molar ratio of fucose, galactose, glucose, mannose, and glucuronic acid of 6.7:3.1:50.9:23.0:2.

4.

2. The application according to claim 1, characterized in that, The preparation method of the crude polysaccharide from bamboo fungus includes the following steps: collecting the inner layer gel of bamboo fungus, pulping the inner layer gel and then centrifuging it for the first time, and ultrafiltration the obtained gel supernatant to obtain the crude polysaccharide from bamboo fungus. The preparation method of the crude polysaccharide of bamboo fungus fruiting body includes the following steps: first water extraction of bamboo fungus fruiting body powder, first alcohol precipitation of the obtained first water extract to obtain crude polysaccharide of bamboo fungus fruiting body; The preparation method of the crude polysaccharide from bamboo fungus cap includes the following steps: performing a second water extraction on bamboo fungus cap powder, and performing a second alcohol precipitation on the obtained second water extract to obtain crude polysaccharide from bamboo fungus cap.

3. The application according to claim 2, characterized in that, The pulping time is 3-5 minutes; The ultrafiltration has a molecular weight cutoff of 10 kDa.

4. The application according to claim 2, characterized in that, The solid-liquid ratio of the bamboo fungus fruiting body powder to the first water extraction water is 1g:8~12mL; The temperature of the first water extraction is 90~100℃, and the time is 1.5~2.5h; The first water extraction process further includes a second centrifugal separation of the obtained first water extraction system, and the supernatant obtained is concentrated to obtain the first water extract. The volume ratio of the first aqueous extract to the alcohol used for the first alcohol precipitation is 1:0.6~1.2; The alcohol used for the first alcohol precipitation includes ethanol; The first alcohol precipitation was carried out at a temperature of 3-5°C for 10-14 hours.

5. The application according to claim 2, characterized in that, The solid-liquid ratio of the bamboo fungus cap powder to the second water extraction water is 1g:8~12mL; The temperature of the second water extraction is 90~100℃, and the time is 1.5~2.5h; The second water extraction process further includes a third centrifugal separation of the obtained second water extraction system, and the supernatant obtained is concentrated to obtain the second water extract. The volume ratio of the second aqueous extract to the alcohol used for the second alcohol precipitation is 1:0.6-1.2; The second alcohol used for precipitation includes ethanol. The second alcohol precipitation is carried out at a temperature of 3-5°C for 10-14 hours.

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