Anti-aging uses of Deer Rutaceae polysaccharide

Abstract

This invention provides the anti-aging applications of deer antler polysaccharide (CRWP-S), belonging to the field of traditional Chinese medicine technology. Specifically, this invention provides the use of CRWP-S in alleviating skin aging. CRWP-S exhibits good anti-photoaging effects in mice with photoaging skin. The prepared compound drug shows significant alleviating effects on UVB-induced skin aging in mice: wrinkles on the mouse skin surface are reduced, water content is increased, epidermal thickness is thinned, and dryness, redness, and peeling are significantly alleviated, while the collagen content in the epidermis is increased. The yield of CRWP-S extracted by this method is 2.81% (based on the dry weight of the raw material).

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology and relates to the anti-aging uses of deer antler polysaccharide. Background Technology

[0002] Photoaging of the skin is mainly caused by oxidative stress induced by ultraviolet (UV) radiation, characterized by collagen degradation, elastic fiber breakage, and cellular DNA damage. UV radiation can activate reactive oxygen species (ROS) pathways, leading to overexpression of matrix metalloproteinases (MMPs) and accelerating skin matrix degradation. Currently, while synthetic antioxidants (such as vitamin C derivatives) are effective, they suffer from poor stability and low transdermal absorption rates.

[0003] Reindeer lichen (Cladonia rangiferina), also known as caribou lichen, is a light-colored, shrub-like lichen belonging to the Cladoniaceae family. It grows in well-drained, open environments, primarily found in alpine tundra, and is an extremely cold-hardy organism. In Traditional Chinese Medicine, the branched form of Cladonia rangiferina is used in medicinal cuisine for over 500 years. It is often blanched and eaten as a salad to treat restlessness accompanied by a feeling of suffocation, dry throat, phlegm, blurred vision, and jaundice. However, research on the effects of other active substances in Cladonia rangiferina is limited. Summary of the Invention

[0004] In view of this, the present invention provides the anti-aging use of deer antler polysaccharide, which has strong free radical scavenging ability (DPPH, ABTS⁺) and reducing power, and has no metal chelating effect, and can significantly alleviate cell senescence induced by UVB.

[0005] This invention provides the application of deer antler polysaccharide in the preparation of anti-aging products. The preparation method of the deer antler polysaccharide includes the following steps: drying and pulverizing the deer antler dendrites, defatting and extracting with hot water, collecting the supernatant by centrifugation, concentrating and adding ethanol to a final concentration of 80%, collecting the precipitate after standing to obtain a crude product, dissolving the crude product and performing freeze-thaw cycles until no precipitate is obtained, taking the supernatant and freeze-drying to obtain the deer antler polysaccharide (abbreviated as CRWP-S).

[0006] Preferably, the hot water extraction includes extraction in water at 90°C for 3-8 hours each time, for a total of 2-5 extractions.

[0007] This invention also provides a skin conditioning agent to alleviate skin aging, the main component of which is deer antler polysaccharide. The preparation method of the deer antler polysaccharide includes the following steps: drying and pulverizing the deer antler dendrites, defatting and extracting with hot water, collecting the supernatant by centrifugation, concentrating and adding ethanol to a final concentration of 80%, allowing it to stand and collecting the precipitate to obtain a crude product, dissolving the crude product and performing freeze-thaw cycles until no precipitate is found, taking the supernatant and freeze-drying it to obtain the CRWP-S.

[0008] This invention also provides the application of the above-mentioned skin conditioning agent in the preparation of products that alleviate skin photoaging.

[0009] The present invention also provides a product for alleviating photoaging of the skin, comprising the above-mentioned skin conditioning agent.

[0010] Compared with existing technologies, the skin conditioning agent for alleviating skin aging of the present invention has the following beneficial effects:

[0011] 1. Significantly reduces the activity of β-galactosidase, a marker of aging.

[0012] In the HaCaT cell model irradiated by UVB, the activity of β-galactosidase in the treated group cells decreased with increasing CRWP-S concentration, indicating a decrease in the proportion of senescent cells. This suggests that CRWP-S can significantly alleviate UVB-induced cell senescence.

[0013] 2. Reverses UV-induced cell cycle arrest and restarts the skin cell proliferation cycle.

[0014] The expression level of p53 and its active form (serine phosphorylation) can serve as a G2 checkpoint marker; a high p-p53 / p53 ratio may indicate cell cycle arrest in the G2 / M phase. In the treatment group, the ratio of phosphorylated p53 to p53 increased after UVB irradiation, indicating cell cycle arrest. In the treatment group, this ratio decreased significantly, reducing the proportion of senescent cells.

[0015] 3. Activates the endogenous antioxidant defense system and inhibits photoaging inflammatory responses.

[0016] UVB irradiation causes intracellular ROS accumulation, disrupts the antioxidant defense system, and triggers intracellular inflammatory responses. Compared with the experimental group, the treatment group showed significantly increased expression of HO-1 and IL-6.

[0017] 4. Low toxicity and cell-protective effects

[0018] CCK-8 assays showed that CRWP-S (≤400 μg / mL) could promote the growth of HaCaT cells and protect them from apoptosis induced by UVB irradiation. Attached Figure Description

[0019] Figure 1 To demonstrate the protective effect of CRWP-S on HaCaT cells, NT in the figure represents the blank control group, ns P > 0.05 vs. the UVB group, ***P < 0.05 vs. the NT group;

[0020] Figure 2 The effect of CRWP-S on β-galactosidase in HaCaT cells;

[0021] Figure 3 The effect of CRWP-S on HaCaT cell cycle proteins;

[0022] Figure 4 The effect of CRWP-S on IL-6 expression in HaCaT cells. Detailed Implementation

[0023] This invention provides a skin conditioning agent to alleviate skin aging, the main components of which include CRWP-S.

[0024] This invention targets and inhibits the UV-induced ROS-MMP pathway through CRWP-S, and its anti-photoaging effect is significantly better than that of traditional single photoprotective agents (such as vitamin C or physical sunscreens), demonstrating multi-dimensional synergistic repair advantages.

[0025] The preparation method of the skin conditioning agent for relieving skin aging according to the present invention includes the following steps:

[0026] The deer stamens were dried and pulverized, then defatted by ethanol reflux, extracted with hot water, and subjected to ethanol fractionation and precipitation followed by protein removal. The CRWP-S was obtained by freeze-thaw fractionation and purification.

[0027] The dendritic portion of *Deer antler filament* was dried at 50°C for 3 days and then ground into powder. After removing hydrophobic compounds with 95% ethanol under reflux for 12 hours, the ground material was extracted three times (6 hours each time) with hot water (90°C, 1:20 w / v). The extract was collected by centrifugation at 3000×g for 20 min, concentrated to 1 / 10 of the initial volume, and then mixed with ethanol at 4°C until up to 80% overnight. The solution was centrifuged at 3000×g for 20 min, the precipitate was collected, and vacuum dried. The dried precipitate was then dissolved in water (5% w / v) and deproteinized using the Sevage method to obtain crude polysaccharide (CRWP, crude polysaccharide yield 3.69%). Subsequently, CRWP was separated into two fractions by a freeze-thaw process (freezing at -86°C, thawing at 4°C, and repeating the process until no precipitate was formed). The supernatant was CRWP-S, accounting for 76.28% of the crude polysaccharide.

[0028] Based on the dry weight of the raw material, the yield of CRWP-S was determined to be 2.81%. After hot water extraction and ethanol precipitation, crude polysaccharide CRWP was obtained with a yield of 3.69% (based on the dry weight of the raw material). CRWP was then separated by freeze-thaw fractionation, with CRWP-S (the supernatant fraction) accounting for 76.28% of the crude polysaccharide CRWP.

[0029] Total carbohydrate content was determined using glucose as a standard. Galacturonic acid content was determined using the m-hydroxydiphenyl colorimetric method, using galacturonic acid as a standard; the results showed no galacturonic acid content. Protein content was determined using the Coomassie brilliant blue method, using bovine serum albumin as a standard; the protein content was 6.13%. Starch content was determined using the potassium iodide method, using soluble starch as a standard; the results indicated no starch content. Endotoxin levels were analyzed using a horseshoe crab (LAL) reagent kit; the concentration was <0.05 EU / mL.

[0030] The homogeneity and molecular weight of the polysaccharide components were identified using high-performance gel permeation chromatography (HPGPC). The monosaccharide composition of the polysaccharide components was analyzed by pre-column derivatization HPLC using 1-phenyl-3-methyl-5-pyrazolone (PMP). The deer antler stamen described in this invention is a traditional medicinal organism, and its fractional component CRWP-S (comprising 76.28% of the crude polysaccharide) exhibits strong antioxidant activity. CRWP-S is a heteropolysaccharide (molecular weight 15-200 kDa), composed of glucose, mannose, and galactose (molar ratio 11.23:1.27:1.00), and contains both α / β-glycosidic bonds (FT-IR characteristic peak 845.6 cm⁻¹). -1 and 931.9 cm -1 Its ability to scavenge DPPH free radicals (EC) 50 Its concentration of 760.69 mg TE / g and its cytoprotective effects suggest that it has the potential to become a natural photosensitizer.

[0031] The CRWP-S described in this invention possesses moderate cellular oxidative damage protection capabilities, primarily achieved by inhibiting ROS accumulation, mitigating lipid peroxidation, and enhancing the activity of endogenous antioxidant enzymes (SOD, CAT). It exhibits strong free radical scavenging capabilities (DPPH, ABTS⁺) and reducing power, and has no metal chelating effect. Therefore, the antioxidant function of CRWP-S is effective in cell models. The CRWP-S described in this invention can inhibit UVB-induced senescence of immortalized human keratinocytes (HaCaT) by reducing ROS and decreasing β-galactosidase activity, thus possessing moderate cellular oxidative damage protection capabilities, primarily achieved by inhibiting ROS accumulation, mitigating lipid peroxidation, and enhancing the activity of endogenous antioxidant enzymes (SOD, CAT). Therefore, the antioxidant function of CRWP-S is effective in cell models.

[0032] This discovery provides irreplaceable scientific evidence for CRWP-S as a novel photoaging repair agent. Its "ROS-MMP pathway targeted blockade and barrier synergistic regeneration" mechanism opens up new pathways for photodamage protection, especially in the development of combined photodynamic therapy or functional cosmetics, which has significant industrialization value. Simultaneously, this research lays a theoretical foundation for the sustainable utilization and ecological-economic integration of lichen resources in high-altitude and cold regions, possessing both environmental friendliness and commercial potential.

[0033] The core anti-aging mechanism of CRWP-S described in this invention lies in the regulation of the Nrf2 / NF-κB dual pathway and collagen metabolism remodeling, thereby achieving protection against photoaging. This invention can utilize CRWP-S to prepare various dosage forms, including common dosage forms such as oral preparations, injections, or others. Innovative dosage forms, such as microneedle patches or liposome sprays, are also possible. Furthermore, products using CRWP-S as the active ingredient can be used in combination with LED phototherapy and retinoids. In addition to its anti-aging applications, the products of this invention can also be used for extended indications, such as radiation dermatitis and hormone-dependent skin atrophy.

[0034] Unless otherwise specified in the following examples, the conditions were performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products. Example 1

[0035] CRWP-S exhibits cell-protective and cell-growth-promoting effects in a UVB-induced HaCaT cell senescence model.

[0036] a. Establishing a UVB-induced HaCaT cell senescence model

[0037] HaCaT cells were cultured in vitro for 2 to 3 generations. When the cells were in the logarithmic growth phase, the cells were collected and divided into groups of 5 × 10⁻⁶ cells / year. 3 The cells were seeded at a density of / wells into 96-well cell culture plates. After the cells adhered overnight, they were irradiated with UVB lamps for 10 minutes.

[0038] b. Drug toxicity testing

[0039] HaCaT cells were cultured in vitro for 2 to 3 generations. When the cells were in the logarithmic growth phase, the cells were collected and divided into groups of 5 × 10⁻⁶ cells / year. 3 The cells were seeded at a density of / wells into 96-well cell culture plates and allowed to adhere overnight.

[0040] c. Add different doses of the test substance (50, 100, 200, 400 μg / ml) dissolved in cell culture medium. Simultaneously, set up a blank group containing only culture medium and a negative control group containing only cells, with 5 parallel wells in each group. Furthermore, fill the edge wells with sterile PBS buffer to avoid interference from edge effects on the experimental results. Incubate in a cell culture incubator for 24 h.

[0041] d. Carefully aspirate the supernatant from each well and add fresh cell culture medium containing 10 μL of CCK-8 solution. After incubating in a cell culture incubator for 2 hours, aspirate the cell culture medium and measure the absorbance of each well in the 96-well plate at 490 nm using a microplate reader. Cell proliferation inhibition rate (%) = (OD negative control group - OD experimental group) / (OD negative control group - OD blank group) × 100.

[0042] cck8 results are as follows Figure 1 As shown, CRWP-S can promote the growth of HaCaT cells and has a significant protective effect against UVB-induced cell damage.

[0043] Example 2

[0044] CRWP-S inhibits β-galactosidase activity

[0045] a. HaCaT cells in the logarithmic growth phase were divided into the following six groups, and after UVB irradiation, the corresponding drugs were added to each group:

[0046] Table 1 HaCaT cell grouping

[0047] Group Drug concentration Control group -- UVB group -- UVB+VC group 100μg / ml UVB+CRWP-S 50μg / ml group 50μg / ml UVB+CRWP-S 100μg / ml group 100μg / ml UVB+CRWP-S 200μg / ml group 200μg / ml UVB+CRWP-S 400μg / ml group 400μg / ml

[0048] b. HaCaT cells were cultured overnight in 6-well plates, and then various concentrations of the drug were added. The supernatant was carefully removed, and the cells were gently washed once with PBS buffer. The cells were then fixed in the fixative solution provided in the kit at room temperature for 10 min, followed by two gentle washes with PBS buffer. SA-β-Gal staining working solution was added, and the cells were incubated overnight at 37°C in a CO2-free incubator. During incubation, the plates were sealed with sealing film to prevent reagent evaporation from interfering with the experimental results. After incubation, the cells were observed under an optical microscope to observe changes in SA-β-Gal activity.

[0049] The experimental results are as follows Figure 2 As shown in the figure. The results showed that the β-galactosidase activity in the experimental group was significantly increased, indicating a significant increase in the proportion of senescent cells. In the drug group, the β-galactosidase activity decreased significantly with increasing drug concentration.

[0050] Example 3

[0051] CRWP-S reduces the proportion of cells in growth cycle arrest.

[0052] a. HaCaT cells in the logarithmic growth phase were divided into the following six groups, and after UVB irradiation, the corresponding drugs were added to each group.

[0053] Table 2 HaCaT cell grouping

[0054] Group Drug concentration Control group -- UVB group -- UVB+VC group 100μg / ml UVB+CRWP-S 50μg / ml group 50μg / ml UVB+CRWP-S 100μg / ml group 100μg / ml UVB+CRWP-S 200μg / ml group 200μg / ml UVB+CRWP-S 400μg / ml group 400μg / ml

[0055] b. The expression levels of p53 and p-p53 proteins in HaCaT cells were detected using Western blot. The experimental results are as follows: Figure 3 As shown, UVB irradiation increased the ratio of phosphorylated p53 to p53 in cells, while the treatment group showed a decrease in the ratio of phosphorylated p53 to p53.

[0056] Example 4

[0057] CRWP-S inhibits the expression of pro-inflammatory factors

[0058] Cell supernatant from Example 3 was collected, and the expression of the pro-inflammatory factor IL-6 was detected by ELISA. The experimental results are as follows: Figure 4 As shown, IL-6 expression increased significantly after UVB irradiation, while IL-6 expression decreased in the treatment group.

[0059] 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. The application of deer antler polysaccharide in the preparation of anti-skin aging drugs, characterized in that, The preparation method of the deer stamen polysaccharide includes the following steps: drying and pulverizing the deer stamen dendrites, defatting and extracting with hot water, centrifuging to collect the supernatant, concentrating and adding ethanol to a final concentration of 80%, allowing it to stand and collecting the precipitate to obtain the crude product, dissolving the crude product and performing freeze-thaw cycles until no precipitate is found, taking the supernatant and freeze-drying it to obtain the deer stamen polysaccharide.

2. The application according to claim 1, characterized in that, The hot water extraction involves extraction in water at 90°C for 3-8 hours each time, for a total of 2-5 extractions.

3. The application of a skin conditioning agent in the preparation of a drug to alleviate photoaging of the skin, characterized in that, The main component of the skin conditioning agent is deer antler polysaccharide. The preparation method of the deer antler polysaccharide includes the following steps: drying and pulverizing the deer antler dendrites, defatting and extracting with hot water, collecting the supernatant by centrifugation, concentrating and adding ethanol to a final concentration of 80%, collecting the precipitate after standing to obtain the crude product, dissolving the crude product and performing freeze-thaw cycles until there is no precipitate, taking the supernatant and freeze-drying to obtain the deer antler polysaccharide.

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