Application of ultramicro-grade antler powder in anti-aging

Abstract

The invention discloses an application of ultramicro-grade antler powder in anti-aging, and belongs to the technical field of health-care products. The size of the superfine antler powder is smaller than 1 micron, the superfine antler powder reaches the superfine level, and the solubility, dispersity and chemical reactivity of the superfine antler powder can be remarkably improved, so that the superfine antler powder can be more easily absorbed by a human body, and the curative effect of the medicine is enhanced. The ultramicro-grade antler powder prepared by the invention can improve the immune level of thymus and spleen organs, reduce the levels of IL-2 and TNF-alpha in serum, improve the reduction of autonomous movement ability and memory impairment caused by aging, and inhibit kidney and colon tissue damage caused by aging.

Description

Technical Field

[0001] The invention relates to the technical field of health care products, and in particular to application of ultra-fine red deer antler powder in anti-aging. Background Art

[0002] Red deer antlers: branched, usually divided into 4-6 branches, with a total length of 50-120cm. The main branch is curved, 3-6cm in diameter, disc-shaped at the base, with irregular tumor-like protrusions on it, commonly known as "pearl discs", and often with sparse and small holes around it. The side branches mostly extend to one side, the first branch is close to the pearl disc, extending at almost right angles or obtuse angles to the main trunk, the second branch extends close to the first branch, commonly known as "sit-on-the-ground branches"; the second branch is far away from the third branch. The surface is gray-brown or gray-yellow, shiny, the tip of the horn is smooth, the middle and lower parts often have wart-like protrusions, commonly known as "bone nails", and have intermittent longitudinal spindles of varying lengths, commonly known as "bitter melon ridges". Red deer horns are hard, the outer circle of the cross section is boney, gray-white or slightly light brown, the middle part is mostly gray-brown or blue-gray, with honeycomb holes; odorless, slightly salty taste.

[0003] Since Chinese medicine powder can be used to prepare other preparations, or can be taken directly with boiled water or medicinal soup or decocted with other medicines, it is widely used for its characteristics of improving effective utilization, saving medicinal resources, and flexible compatibility. Red deer antler powder is also included in it. However, most of the red deer antler products on the market are roughly processed and the product quality is uneven. The time of antler collection is also unclear. Its processing technology, quality and animal source will have a serious impact on its efficacy. Therefore, it is urgent to develop a more efficient red deer antler powder preparation process to ensure the safety and effectiveness of red deer antler powder as a health product. Research on the anti-aging effect of red deer antler will promote the emergence of new red deer antler drugs through further scientific preparation technology, which is of great significance for enriching the drug treatment effect of red deer antler, preventing the threat to the health of deer populations caused by illegal velvet antler mining, and promoting the healthy development of red deer breeding industry. Summary of the invention

[0004] In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide an application of ultra-fine red deer antler powder in anti-aging, so as to improve the safety and effectiveness of red deer antler powder as a health product and enrich the medicinal therapeutic effects of red deer antler.

[0005] The technical solution of the present invention to solve the above technical problem is as follows: provide an application of ultra-fine red deer antler powder in anti-aging, wherein the particle size of the ultra-fine red deer antler powder is less than 1 μm.

[0006] Furthermore, the anti-aging effect of ultra-fine red deer antler powder can improve the immune level of the thymus and spleen organs.

[0007] Furthermore, anti-aging properties of ultra-fine red deer antler powder can reduce serum IL-2 and TNF-α levels.

[0008] Furthermore, anti-aging ultra-fine red deer antler powder can improve the reduction of autonomous movement ability and memory impairment caused by aging.

[0009] Furthermore, anti-aging properties as ultra-fine grade red deer antler powder can inhibit kidney tissue damage caused by aging.

[0010] Furthermore, anti-aging properties include ultra-fine red deer antler powder which can inhibit colon tissue damage caused by aging.

[0011] Furthermore, anti-aging ultra-fine red deer antler powder can significantly increase the richness and diversity of intestinal flora in aged mice.

[0012] Furthermore, the richness and diversity of intestinal flora in aging mice can be significantly increased, and ultra-fine red deer antler powder can increase the enrichment of beneficial bacteria in the intestines of aging mice.

[0013] The invention provides a health-care product capable of resisting aging, and the health-care product comprises ultra-fine red deer antler powder.

[0014] The present invention has the following beneficial effects:

[0015] (1) The ultrafine red deer antler powder of the present invention has a size of less than 1 μm, reaching the ultrafine level, which can significantly improve the solubility, dispersibility, and chemical reactivity of the red deer antler powder, thereby making the red deer antler powder more easily absorbed by the human body, thereby enhancing the efficacy of the drug;

[0016] (2) The dissolution rate of a drug is generally proportional to the specific surface area of ​​the drug particles, while the specific surface area is inversely proportional to the particle size. Ultrafine red deer antler powder has a small particle size and a large specific surface area, which increases the contact area with the gastrointestinal mucosa and enhances adhesion, allowing it to stay in the digestive tract for a longer time and allowing the drug to be more fully absorbed, thereby improving the bioavailability of red deer antler powder;

[0017] (3) Ultrafine red deer antler powder can improve the immune level of the thymus and spleen organs, reduce the levels of IL-2 and TNF-α in serum, improve the reduction of autonomous motor ability and memory damage caused by aging, and inhibit the kidney and colon tissue damage caused by aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of mouse grouping;

[0019] Figure 2 is the organ index of mouse spleen and thymus;

[0020] Figure 3 is the plasma IL-2 and TNF-α levels in mice;

[0021] Figure 4 This is the open field movement path for mice;

[0022] Figure 5 This is the movement path of the Y-shaped maze of mice;

[0023] Figure 6 HE staining of mouse kidney;

[0024] Figure 7 HE staining of mouse colon;

[0025] Figure 8 is the gene expression level of Claudin-1, Occludin, Claudin-3 and Zo-1 in mice;

[0026] Fig. 9 The effect of ultra-fine red deer antler powder on the abundance of intestinal flora in mice;

[0027] Fig.10 The effect of ultra-fine red deer antler powder on the intestinal flora structure of mice;

[0028] Fig.11 The evolutionary branch diagram and feature diagram for LEfSe analysis;

[0029] Fig.12 This is a bar graph showing the distribution of LDA values ​​for LEfSe analysis. DETAILED DESCRIPTION

[0030] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0031] Example 1 Preparation of ultra-fine red deer antler powder

[0032] The antlers of red deer were collected, impurities were removed, sliced ​​and dried, and ground by a grinder to obtain red deer antler powder. The red deer antler powder was dissolved in 20% ethanol solution, ultrasonically treated for 15 minutes, filtered, and freeze-dried in a freeze dryer for 2 days to obtain ultra-fine red deer antler powder.

[0033] Example 2 Anti-aging effect of ultra-fine red deer antler powder

[0034] 40 male C57BL / 6 mice were evenly divided into four groups. The control group was fed normally and injected with normal saline subcutaneously at the back of the neck every morning. The other three groups were continuously administered with D-galactose, with 100 mg / kg of D-galactose injected subcutaneously at the back of the neck every morning, and the injection volume was 10 mL / kg / d, for 8 consecutive weeks to construct a mouse aging model; ultrafine red deer antler powder was gavaged into the mice (4 g / kg) every day as the ultrafine red deer antler powder group; metformin treatment (oral, 300 mg / kg / d) was used as the positive group (the prior art "Metformin decelerates aging clock in male monkeys, Yuanhan Yang, Cell, 2024" revealed that metformin can effectively delay multi-organ aging in primates); the mice were treated for 8 weeks, and then the mice were euthanized to obtain blood and brain, kidney, colon and other tissues (see Figure 1 ).

[0035] (1) The regulatory effect of ultra-fine deer antler powder on immune level

[0036] ① Thymus and spleen organ index: Aseptically separate the thymus and spleen of mice, weigh them, and calculate the organ index of thymus / spleen: organ index = organ weight / mouse body weight. Organ immune function is evaluated by organ index, and an increase in organ index indicates improved immune function.

[0037] Depend on Figure 2 It can be seen that compared with the control group, the thymus and spleen organ indexes of aged mice were significantly reduced. On the contrary, the thymus and spleen organ indexes of the metformin-treated group and the ultra-fine red deer antler powder group were significantly increased compared with those of the aged mice, and the indexes returned to the level of the control group. There was no significant difference between the group with ultra-fine red deer antler powder and the control group and the positive group. The above results show that the supplementation of ultra-fine red deer antler powder can improve the immune level of the thymus and spleen organs of aged mice.

[0038] ② IL-2 and TNF-α levels in plasma: After aseptic blood collection, the IL-2 and TNF-α levels in mouse plasma were detected by ELISA kits. The changes in IL-2 and TNF-α levels can reflect the immune status and degree of inflammatory response of mice.

[0039] Depend on Figure 3 It can be seen that compared with the control group, the levels of IL-2 and TNF-α in the plasma of aging mice increased significantly. However, compared with the aging mice, the positive group and the ultra-fine red deer antler powder group had significantly reduced levels of IL-2 and TNF-α in the plasma, and the index returned to the level of the control group. There was no significant difference between the group supplemented with ultra-fine red deer antler powder and the control group and the positive mice. The above results show that the supplementation of ultra-fine red deer antler powder can reduce inflammatory factors in the serum of aging mice.

[0040] (2) The regulatory effect of ultra-fine deer antler powder on the brain

[0041] The open field test and Y-maze test were used to evaluate the regulatory effect of ultrafine deer antler powder on the aging brain.

[0042] ① Open field test: It is generally used to evaluate autonomous behavior and exploratory behavior in a novel environment, and it has been observed in the open field test that motor activity decreases with age. The open field consists of a 50×50 cm square bottom surface, which is divided into 16 small squares of 12.5×12.5 cm (4×4) by white stripes. The square bottom surface is subdivided into peripheral and central areas, where the central area includes 4 central squares (2×2), and the peripheral area includes 12 square grids (40 cm high) close to the surrounding walls. The walking trajectory is recorded for 5 minutes with a camera throughout the process. Between each test, the open field was wiped clean with 75% alcohol to remove the mouse odor marks to avoid affecting the subsequent behavioral activities of the mice and interfering with the test results.

[0043] Depend on Figure 4 It can be seen that compared with the control group, the central distance of the aging group mice was significantly reduced; compared with the aging group mice, the central distance explored by the positive group and the ultra-fine red deer antler powder group mice was significantly increased, and this indicator returned to the level of the control group. There was no significant difference between the ultra-fine red deer antler powder group and the control group and the positive group mice. The above results show that the supplementation of ultra-fine red deer antler powder can improve the reduction of autonomous movement ability of mice caused by aging.

[0044] ②Y-maze experiment: short-term working memory was tested. Compared with young mice, the working and spatial memory of old mice was reduced. The Y-maze device (16 cm high, 32 cm long, 10 cm wide at the bottom) was used for behavioral analysis. The walking trajectory was recorded for 5 minutes with a camera. Between each test, the Y-maze was cleaned with 75% alcohol to remove the mouse odor marks to avoid affecting the subsequent behavioral activities of the mice and interfering with the test results.

[0045] Depend on Figure 5 It can be seen that compared with the control group, the exploration distance of the new arm of the aging group mice was significantly reduced; compared with the aging group mice, the exploration distance of the new arm of the positive group and the ultra-fine red deer antler powder group was significantly increased, and this index returned to the level of the control group. There was no significant difference between the ultra-fine red deer antler powder group and the control group and the positive group mice. The above results show that the supplementation of ultra-fine red deer antler powder can improve the damage of short-term working memory of mice caused by aging.

[0046] (3) The regulatory effect of ultra-fine deer antler powder on the kidneys

[0047] Observe the morphology of glomeruli and tubules by microscopy: Aseptically collect kidney tissue, perform paraffin sections and HE (hematoxylin-eosin) staining, observe the kidney structure under a microscope, and evaluate kidney aging. The kidney fixed in formaldehyde is trimmed into small square pieces with a length and width of 0.5 cm, placed in an embedding box for step-by-step dehydration, and then transparentized with xylene and embedded in paraffin. The embedded paraffin tissue is sliced ​​with a thickness of 5 μm, and then placed in a slicer. After the slices are completely unfolded, they are sliced ​​and placed on a slice dryer for baking, and then transferred to a 37°C constant temperature box for baking. HE staining of kidney paraffin sections is performed using a HE staining kit.

[0048] Depend on Figure 6 It can be seen that the results of HE staining showed that the renal tissue structure of the mice in the control group was normal, the glomerular structure was clear, the glomerular diameter was normal, the renal capsule cavity was obvious, the renal tubules were tightly arranged, and the structure was complete. The glomeruli of the mice in the aging group were obviously atrophied, the diameter became smaller, the renal capsule cavity became larger, the renal tubules around the renal body were loosely arranged, the tubular lumen was obvious, and the boundaries of the epithelial cells on the tubular wall were unclear and blurred. The mice in the positive group and the ultra-fine red deer antler powder group could significantly improve the structural damage of the kidneys, the glomeruli were normal in morphology, the surrounding renal tubules were tightly arranged, and the structure was obviously restored. The results show that supplementing with ultra-fine red deer antler powder can inhibit the damage of renal tissue caused by aging and play a protective role on the kidneys.

[0049] (4) The regulatory effect of ultra-fine deer antler powder on the colon

[0050] ① Observe the colon morphology under a microscope: The colon tissue was collected aseptically, and paraffin sections and HE (hematoxylin-eosin) staining were performed. The colon and its villus structure were observed under a microscope to evaluate intestinal aging.

[0051] Depend on Figure 7 It can be seen that the colon tissue structure of the mice in the control group was intact, the villi were uniform and slender, arranged in finger-like shapes, the epithelial cells did not fall off, and the intestinal glands were arranged tightly; the colon villi of the mice in the aging group were shortened, swollen and hypertrophic, the tissue mucosal layer was damaged, the local mucosal epithelial cells fell off, the intestinal glands in the lamina propria were arranged loosely, the gaps were widened, and the intestinal mucosa became thinner; the colon villi length of the mice in the ultra-fine red deer antler powder group was significantly longer than that of the mice in the aging group, there was no hypertrophy, the mucosal layer and epithelial cells did not fall off, and the intestinal glands in the lamina propria were arranged tightly. The results show that supplementing with ultra-fine red deer antler powder can inhibit the damage of colon tissue caused by aging and play a protective role on the intestine.

[0052] ②The effect of ultra-fine red deer antler powder on tight junctions in the intestine of mice: Claudin-1 and Claudin-3 both belong to the Claudin family and are transmembrane proteins related to the formation of tight junctions between cells. Tight junctions play an important role in maintaining epithelial integrity, and the loss of tight junction structure may lead to changes in intercellular permeability, thereby affecting tissue physiology and metabolism. Occludin and zonula occludens 1 (Zo-1) are also representative proteins among tight junction proteins, which are mainly responsible for sealing intercellular junctions, maintaining cell permeability, and participating in maintaining the integrity of vascular endothelium.

[0053] Depend on Figure 8 As shown in the AD graphs, compared with the control group, the expression of Claudin-1, Claudin-3, Occludin and Zo-1 genes in the colon of aged mice was significantly reduced, while compared with aged mice, the expression of Claudin-1 and Occludin genes in the intestine of the positive group and the ultra-fine red deer antler powder group was significantly increased, and this index returned to the level of the control group. There was no significant difference between the group with ultra-fine red deer antler powder and the control group and the positive mice, indicating that the addition of deer antler powder can alleviate the damage of tight junctions in the intestine of aged mice.

[0054] ③ The effect of ultra-fine red deer antler powder on the intestinal flora structure of mice: The representative bacteria with differences between different groups were identified, and a total of 20 mouse intestinal microbial samples (n=5) were obtained and sequenced to generate V1-V9 16S rRNA gene maps, among which, Fig. 9 Figure A is the OTU quantity graph, Figure B is the rarefaction curve, and Figure C is the Shannon curve; Fig.10 Figure A shows the rank abundance curve, Figure B shows the principal component analysis (PCA), and Figure C shows the relative abundance at the phylum level of the microbial community; Fig.11 Figure A in the middle is the evolutionary branch diagram and characteristic diagram of LEfSe analysis. Fig.12 This is a bar graph showing the distribution of LDA values ​​for LEfSe analysis.

[0055] like Fig. 9 As shown in Figure A, OTU is a taxonomic operational unit, which is a symbol set for a taxonomic unit (strain, species, genus, group, etc.) in phylogenetic studies or population genetics studies for the convenience of analysis. All sequences can be divided into OTUs according to different similarity levels, and each OTU corresponds to a representative sequence. Through the OTU number distribution analysis, it was found that the number of OTUs in the intestinal flora of aging mice decreased, while the number of OTUs in the intestinal flora of mice supplemented with ultra-micro-grade red deer antler powder increased to a certain extent.

[0056] α diversity reflects the richness and diversity of microbial community diversity. Among them, Shannon curve and rarefaction curve are used to measure species diversity, which is affected by species richness and species evenness in the sample community. Fig. 9 As shown in Figures B and C, the dilution curve and Shannon curve of aged mice increased more slowly, indicating that the richness of intestinal flora in aged mice was lower than that in other groups.

[0057] In addition, the graded abundance curve is a curve graph that sorts the abundance of each sample by size and draws a graph based on its relative abundance. It is mainly used to simultaneously explain the richness and uniformity of species contained in the sample. The richness of the species is reflected by the length of the curve on the horizontal axis. The wider the curve, the richer the species composition. Fig.10 As shown in Figure A, among the four groups, the curve of aging mice on the horizontal axis is the narrowest, while the grade abundance curve of ultra-fine red deer antler powder-fed mice is the widest, indicating that ultra-fine red deer antler powder has a positive regulatory effect on the abundance of intestinal flora in aging mice.

[0058] Bray-Curtis principal coordinate analysis (PCA) was used to further evaluate the beta diversity of the intestinal flora to measure the degree of similarity of the intestinal flora. Fig.10 As shown in Figure B, the intestinal flora of mice in the aging group was significantly different from that in other groups.

[0059] The present invention also compared the abundance of 10 bacteria at the phylum level. Fig.10 As shown in Figure C, the Firmicutes / Bacteroidota in the intestinal flora of aged mice decreased significantly, while the Verrucomicrobiota in the ultra-micro deer antler powder group increased significantly. Studies have shown that the ratio of Firmicutes to Bacteroidetes is of great significance in the health and disease state of the body. Changes in this ratio are closely related to a variety of physiological and pathological states, especially in intestinal health and inflammatory bowel disease. With age, especially from middle age to old age, the ratio of Firmicutes to Bacteroidetes increases significantly, which may be related to age-dependent changes in intestinal flora, which in turn affects intestinal health. Therefore, our results show that the intestinal health of aged mice declines, and the supplementation of ultra-micro deer antler powder can restore intestinal health.

[0060] In order to further identify the differential bacteria between the groups, LEfSe was used to determine the core flora of each group. Fig.11 and Fig.12As shown, LEfSe analysis identified that the conditional pathogens Pantoea_rodasii and Xanthomonadaceae were significantly enriched in aged mice, and beneficial bacteria such as g_Candidatus Arthromitus, Lachnospiraceae_NK4A136_group and Muribaculaceae were enriched in the intestinal flora of mice supplemented with ultra-micro-grade red deer antler powder.

[0061] The above results collectively indicate that, compared with the control group, the richness and diversity of intestinal flora in aged mice were significantly reduced, the intestinal health declined, the beneficial bacteria decreased, and the abundance of conditional pathogens increased; on the contrary, ultra-fine red deer antler powder can significantly increase the richness and diversity of intestinal flora in aged mice, improve the intestinal health of aged mice, promote the enrichment of beneficial bacteria in the intestines of aged mice and inhibit the abundance of harmful bacteria.

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The application of ultra-fine red deer antler powder in anti-aging, characterized in that: The particle size of the ultra-fine red deer antler powder is less than 1 μm.

2. The use of the ultra-fine red deer antler powder in anti-aging according to claim 1, characterized in that: The anti-aging effect is that the ultra-fine red deer antler powder can improve the immune level of the thymus and spleen organs.

3. The use of the ultra-fine red deer antler powder in anti-aging according to claim 1, characterized in that: The anti-aging effect is that the ultra-fine red deer antler powder can reduce the levels of IL-2 and TNF-α in serum.

4. The use of the ultra-fine red deer antler powder in anti-aging according to claim 1, characterized in that: The anti-aging effect is that the ultra-fine red deer antler powder can improve the reduction of autonomous movement ability and memory damage caused by aging.

5. The use of the ultra-fine red deer antler powder in anti-aging according to claim 1, characterized in that: The anti-aging effect is that the ultra-fine red deer antler powder can inhibit kidney tissue damage caused by aging.

6. The use of the ultra-fine red deer antler powder in anti-aging according to claim 1, characterized in that: The anti-aging effect is that the ultra-fine red deer antler powder can inhibit colon tissue damage caused by aging.

7. The use of the ultra-fine red deer antler powder in anti-aging according to claim 1, characterized in that: The anti-aging effect is that the ultra-fine red deer antler powder can significantly increase the richness and diversity of the intestinal flora of aging mice.

8. The use of the ultra-fine red deer antler powder in anti-aging according to claim 7, characterized in that: The invention can significantly increase the richness and diversity of the intestinal flora of aging mice, and the ultra-fine red deer antler powder can increase the enrichment of beneficial bacteria in the intestines of aging mice.

9. A health product capable of resisting aging, characterized in that: The health care product includes ultra-fine red deer antler powder.

Images