Preparation method of panax notoginseng polysaccharide, product and application thereof

Abstract

This invention relates to a method for preparing Panax notoginseng polysaccharides, as well as the products and applications thereof. The preparation method includes: extracting Panax notoginseng by mixing it with an ethanol-water solution; further extracting the residue; mixing the extract with a flocculant; filtering; collecting the filtrate; mixing the filtrate with ethanol and water; centrifuging; collecting the precipitate; subjecting the precipitate to enzymatic depolymerization treatment; inactivating the enzyme; centrifuging; and collecting the supernatant to obtain the final product. The polysaccharide obtained by the method of this invention has a high content and excellent solubility. Furthermore, this invention reveals that Panax notoginseng polysaccharides can regulate key genes of the skin barrier. Specifically, the Panax notoginseng polysaccharides obtained using the method provided by this invention can regulate multiple skin barrier genes, synergistically enhancing skin barrier function from multiple aspects, and have great potential for application in cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for preparing Panax notoginseng polysaccharide, its products, and applications. Background Technology

[0002] The skin is the largest organ in the human body, maintaining the body's integrity and functional homeostasis. It is the first line of defense against pathogens, ultraviolet radiation, and physical and chemical substances. The skin's physiological barrier function is mainly accomplished by the epidermis, which is the outermost layer of the skin. The epidermis has a biological barrier to prevent pathogen invasion, a physical barrier to prevent mechanical damage, and a biochemical barrier with antibacterial activity. The stratum corneum plays a crucial role in maintaining the integrity of the skin barrier function. Formed and continuously renewed by keratinocytes, the stratum corneum undergoes constant renewal during keratinization. During this process, keratinocytes form a protein- and lipid-rich outer membrane, composed of several proteins. Filamentin (FLG) and its metabolites are key components for maintaining normal skin barrier function. FLG and lobelin (LOR) are linked to the stratum corneum via keratin, forming a stable mechanical structure. During keratinocyte migration, FLG is enzymatically degraded and further dissociated into pyrrolidine carboxylic acid (PCA). PCA is a major component of natural moisturizing factor (NMF), thus FLG plays a vital role in moisturizing and barrier integrity. Aquaporin-3 (AQP3) not only increases skin hydration but also plays a crucial role in skin damage and wound repair. In addition to the physical barrier, keratinocytes synthesize and secrete various antimicrobial peptides (AMPs) during differentiation. AMPs interact and bind to the lipid membrane, rapidly eliminating microorganisms upon contact with the epidermis. In addition, keratinocytes can secrete related AMPs, such as β-defensins (HBD-1, HBD-2, HBD-3) and antimicrobial peptide LL-37. When the skin barrier is damaged or pathogens invade, they can quickly trigger an immune response to protect the skin from harm.

[0003] Panax notoginseng (Burk.) FH Chen, a plant belonging to the genus Panax in the family Araliaceae, is known for its dried roots and rhizomes. It has a long history of medicinal use, including dispersing blood stasis, stopping bleeding, reducing swelling, and relieving pain. Current research on Panax notoginseng mainly focuses on its saponin components, which have effects such as promoting blood circulation, improving myocardial ischemia, antiarrhythmic effects, anti-shock effects, sedation, anti-aging, and anti-tumor effects.

[0004] Currently, most applications of Panax notoginseng polysaccharides are concentrated in anti-tumor, immunomodulatory, and liver-protective applications. For example, CN109354629A provides an application of neutral Panax notoginseng polysaccharide in the treatment of bone marrow suppression, low immunity, and in combination therapy for tumors, as well as in health foods. CN102585029B discovered a Panax notoginseng polysaccharide with immunomodulatory, liver-protective, bone defect repair-promoting, tumor-inhibiting, blood sugar-lowering, and antiviral effects. The above research on active Panax notoginseng polysaccharides focuses on applications in pharmaceuticals and health products, with limited research on cosmetic applications. CN115141289B provides an acidic Panax notoginseng polysaccharide, its preparation method, and its applications. This polysaccharide has whitening and antioxidant effects; however, this target polysaccharide is only an acidic Panax notoginseng polysaccharide, resulting in low yield. Furthermore, the preparation process requires prolonged dialysis for desalination, leading to low production efficiency and hindering industrialization.

[0005] In summary, the skin barrier is a crucial mechanism for maintaining skin health; therefore, protecting the skin barrier is an important means of solving skin problems. We have an obligation to find a low-toxicity and highly effective active substance to provide solutions to skin problems. Panax notoginseng polysaccharides, as one of the active ingredients in Panax notoginseng, are characterized by low toxicity and significant effects, showing great application potential in the cosmetics field. However, large-molecule polysaccharides are characterized by poor solubility, poor bioavailability, and difficulty in application, which are problems that urgently need to be solved in their development and utilization. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a method for preparing Panax notoginseng polysaccharide, as well as its products and applications.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a method for preparing Panax notoginseng polysaccharide, the method comprising:

[0009] (1) The residue of Panax notoginseng obtained by mixing and extracting with ethanol aqueous solution was extracted by heating and reflux with water to obtain the extract;

[0010] (2) Filter the extract under reduced pressure, then mix it with the flocculant, filter again, and collect the filtrate;

[0011] (3) Mix the filtrate with ethanol, centrifuge, and collect the precipitate;

[0012] (4) Mix the precipitate with the enzyme for enzymatic hydrolysis, centrifuge, and the product is obtained.

[0013] The preparation of Panax notoginseng polysaccharides using the method described in this invention yields products with high Panax notoginseng polysaccharide content. Furthermore, the enzymatic hydrolysis process not only further improves the solubility and bioavailability of Panax notoginseng polysaccharides but also enhances their protective effect on the skin barrier.

[0014] Preferably, the mass percentage of ethanol in the ethanol aqueous solution in step (1) is 80-90%, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0015] This invention uses 80-90% ethanol to extract small molecule medicinal components to the maximum extent, and then uses water to heat and reflux extract the residue for further development and utilization. This allows the medicinal value of Panax notoginseng to be fully utilized and achieves sustainable use. In addition, ethanol at this concentration can remove inactive substances such as monosaccharides and sucrose to a large extent.

[0016] Preferably, the mass ratio of Panax notoginseng residue to water is 1:(10-20), where the specific point values ​​in (10-20) can be selected from 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0017] Preferably, the extraction time is independently 1-2 h, such as 1 h, 1.1 h, 1.2 h, 1.3 h, 1.4 h, 1.5 h, 1.6 h, 1.7 h, 1.8 h, 1.9 h, 2 h, etc. Other specific point values ​​within the above range can be selected, and will not be elaborated here.

[0018] Preferably, the extraction is performed 1-3 times, such as 1 time, 2 times, 3 times, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0019] Preferably, the mass of the flocculant is 0.5-1% of the mass of the extract, such as 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0020] Preferably, the flocculant comprises any one or a combination of at least two of chitosan, citric acid, carrageenan, or gelatin.

[0021] Preferably, the flocculant is a combination of chitosan and citric acid.

[0022] Preferably, the mass ratio of chitosan to citric acid in the flocculant is 1:(1-2), wherein the specific point values ​​in (1-2) can be selected from 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0023] Preferably, the final concentration of ethanol in step (3) is 40-60%, such as 40%, 42%, 45%, 48%, 50%, 52%, 55%, 58%, 60%, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0024] Preferably, the enzyme in step (4) includes any one or a combination of at least two of β-glucosidase, cellulase, and pectinase.

[0025] Preferably, the enzyme in step (4) is a combination of β-glucosidase and cellulase.

[0026] Preferably, the mass ratio of β-glucosidase to cellulase is 1:(0.1-1), wherein the specific values ​​in (0.1-1) can be selected from 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0027] Preferably, the amount of enzyme added in step (4) is 0.5%-2%, such as 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, etc. Other specific values ​​within the above range can be selected, and will not be elaborated further.

[0028] Preferably, the enzymatic hydrolysis time in step (4) is 1-2 hours and the temperature is 40-50℃.

[0029] The enzymatic hydrolysis time can be selected from 1 h, 1.1 h, 1.2 h, 1.3 h, 1.4 h, 1.5 h, 1.6 h, 1.7 h, 1.8 h, 1.9 h, 2 h, etc., and the temperature can be selected from 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, etc. Other specific values ​​within the above range can be selected, and will not be elaborated further.

[0030] Preferably, the pH value of the enzymatic hydrolysis in step (4) is 5-6, such as 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, etc. Other specific values ​​within the above range can be selected, and will not be described in detail again.

[0031] Preferably, step (4) further includes enzyme inactivation after enzymatic hydrolysis.

[0032] In a second aspect, the present invention provides a Panax notoginseng polysaccharide prepared according to the preparation method of Panax notoginseng polysaccharide described in the first aspect.

[0033] Thirdly, the present invention provides an application of the Panax notoginseng polysaccharide according to the second aspect in the preparation of cosmetics with skin barrier protection function.

[0034] This invention is the first to discover that Panax notoginseng polysaccharides can upregulate the expression of skin barrier-related genes. Furthermore, the Panax notoginseng polysaccharides obtained by precipitation with 40% ethanol and water, after enzymatic hydrolysis and polymerization, can simultaneously regulate the expression of key skin barrier genes FLG, LOR, AQP3, HBD-1, HBD-3, and LL-37, thus synergistically enhancing the protective function of the skin barrier and showing strong application potential in cosmetics.

[0035] Preferably, the amount of Panax notoginseng polysaccharide added to the cosmetic with skin barrier protection function is 1-10%, such as 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0036] Preferably, the cosmetic includes lotion, cream, spray, or mask.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] Firstly, this invention employs 80-90% ethanol-water extraction, which maximizes the extraction of small-molecule medicinal components and ensures the full utilization of Panax notoginseng's medicinal components. Furthermore, 80-90% ethanol-water extraction removes inactive substances such as monosaccharides and sucrose from Panax notoginseng, ensuring the efficacy of the polysaccharide extract in the residue and ultimately achieving sustainable utilization.

[0039] Secondly, the present invention provides a method for preparing Panax notoginseng polysaccharide with skin barrier protection function. The preparation method adopts enzymatic depolymerization treatment to depolymerize the extracted Panax notoginseng polysaccharide polymer into smaller polymer fragments, thereby improving its solubility and increasing the bioavailability of polysaccharide. This technology can solve the product application problem caused by the difficulty in dissolving large molecular polysaccharides.

[0040] Thirdly, this invention provides an application of the Panax notoginseng polysaccharide described in the second aspect in the preparation of cosmetics with skin barrier protection function. This invention is the first to discover that Panax notoginseng polysaccharide can upregulate the expression of skin barrier-related genes, and that the Panax notoginseng polysaccharide obtained by precipitation with 40% ethanol and water, after enzymatic hydrolysis and polymerization, yields a more active polysaccharide polymer that can simultaneously regulate the expression of key skin barrier genes FLG, LOR, AQP3, HBD-1, HBD-3, and LL-37, synergistically protecting the skin barrier and possessing strong application potential in cosmetics. Attached Figure Description

[0041] Figure 1 This is an HPLC chromatogram for monosaccharide composition analysis.

[0042] Figure 2 This is the result of the expression of the filaggrin (FLG) gene.

[0043] Figure 3 This is the result of the expression of the lobe protein (LOR) gene.

[0044] Figure 4 This is the result of the expression of the aquaporin 3 (AQP3) gene.

[0045] Figure 5 This is the result of the expression of the human β-defensin 1 (HBD-1) gene.

[0046] Figure 6 This is the result of the expression of the human β-defensin 2 (HBD-3) gene.

[0047] Figure 7 This is the result of the expression of the antimicrobial peptide LL-37 gene. Detailed Implementation

[0048] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0049] The sources of the active ingredients in the products involved in the following examples and comparative examples are as follows (only the active ingredients are shown; other necessary excipients contained in commercially available raw materials are not described):

[0050] The medicinal material Panax notoginseng originates from Wenshan, Yunnan;

[0051] The keratinocytes (HaCaT) were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences;

[0052] Chitosan was sourced from Shanghai Yuanye Biotechnology Co., Ltd.

[0053] Citric acid is sourced from Sinopharm Chemical Reagent Co., Ltd.

[0054] β-glucosidase is sourced from Shandong Longket Enzyme Preparation Co., Ltd.

[0055] The cellulase was sourced from Shandong Longket Enzyme Preparation Co., Ltd.

[0056] Papain is sourced from Shandong Longkete Enzyme Preparation Co., Ltd.

[0057] Example 1

[0058] This embodiment provides a method for preparing Panax notoginseng polysaccharide, the preparation method comprising:

[0059] (1) Crush the dried Panax notoginseng roots and pass them through a No. 2 sieve to obtain Panax notoginseng root powder;

[0060] (2) Extract the Panax notoginseng root powder by heating and refluxing with 85% ethanol and water for 2 h, collect the extract, and dry the residue for later use.

[0061] (3) The residue of Panax notoginseng was extracted by heating and reflux with water. The liquid-to-solid ratio was 1:10. Each extraction lasted 2 hours and was repeated twice. The extract was filtered under reduced pressure while hot.

[0062] (4) Add flocculant for clarification. The amount of flocculant is 0.8%. The flocculant includes chitosan and citric acid in a mass ratio of 1:1.5. Stir for 30 minutes, filter under reduced pressure, and collect the filtrate.

[0063] (5) Add anhydrous ethanol to the clear liquid for alcohol precipitation until the final ethanol concentration is 40%. Place it in a refrigerator at 4°C overnight to allow it to fully precipitate. Centrifuge and collect the precipitate.

[0064] (6) Redissolve the precipitate in water, add β-glucosidase and cellulase to the solution in a ratio of 1:0.5 and a dosage of 1%, adjust the pH to 5.0, keep warm at 40℃ for 2 hours, and inactivate the enzyme at 85℃ for 10 minutes.

[0065] (7) Centrifuge at 5000 r / min for 15 min, collect the precipitate, freeze dry, and obtain Panax notoginseng polysaccharide.

[0066] Example 2

[0067] This embodiment provides a method for preparing Panax notoginseng polysaccharide, the preparation method is as follows:

[0068] (1) Crush the dried Panax notoginseng roots and pass them through a No. 2 sieve to obtain Panax notoginseng root powder;

[0069] (2) Extract the Panax notoginseng root powder by heating and refluxing with 80% ethanol and water for 1.5 h, collect the extract, and dry the residue for later use.

[0070] (3) The residue of Panax notoginseng was extracted by heating and reflux with water. The liquid-to-solid ratio was 1:15. Each extraction lasted 1.5 h and was performed once. The extract was filtered under reduced pressure while hot.

[0071] (4) Add flocculant for clarification. The amount of flocculant is 1%. The flocculant includes chitosan and citric acid in a mass ratio of 1:2. Stir for 30 minutes, filter under reduced pressure, and collect the filtrate.

[0072] (5) Add anhydrous ethanol to the clear liquid for alcohol precipitation until the final ethanol concentration is 40%. Place it in a refrigerator at 4°C overnight to allow it to fully precipitate. Centrifuge and collect the precipitate.

[0073] (6) Redissolve the precipitate in water, add β-glucosidase and cellulase to the solution in a ratio of 1:0.1, and use 2% of the enzyme. Adjust the pH to 5.5, keep warm at 45°C for 1.5 h, and inactivate the enzyme at 85°C for 10 min.

[0074] (7) Centrifuge at 5000 r / min for 15 min, collect the precipitate, freeze dry, and obtain Panax notoginseng polysaccharide.

[0075] Example 3

[0076] This embodiment provides a method for preparing Panax notoginseng polysaccharide, the preparation method is as follows:

[0077] (1) Crush the dried Panax notoginseng roots and pass them through a No. 2 sieve to obtain Panax notoginseng root powder;

[0078] (2) Extract the Panax notoginseng root powder by heating and refluxing with 90% ethanol and water for 1 h, collect the extract, and dry the residue for later use.

[0079] (3) The residue of Panax notoginseng was extracted by heating and reflux with water. The liquid-to-solid ratio was 1:20. Each extraction lasted 1 hour and was repeated 3 times. The extract was filtered under reduced pressure while hot.

[0080] (4) Add flocculant for clarification. The amount of flocculant is 0.5%. The flocculant includes chitosan and citric acid in a mass ratio of 1:1. Stir for 30 minutes, filter under reduced pressure, and collect the filtrate.

[0081] (5) Add anhydrous ethanol to the clear liquid for alcohol precipitation until the final ethanol concentration is 40%. Place it in a refrigerator at 4°C overnight to allow it to fully precipitate. Centrifuge and collect the precipitate.

[0082] (6) Redissolve the precipitate in water, add β-glucosidase and cellulase to the solution in a ratio of 1:0.1, use 0.5% of enzyme, adjust the pH to 6, keep warm at 50℃ for 1 h, and inactivate the enzyme at 85℃ for 10 min.

[0083] (7) Centrifuge at 5000 r / min for 15 min, collect the precipitate, freeze dry, and obtain Panax notoginseng polysaccharide.

[0084] Example 4

[0085] This embodiment provides a method for preparing Panax notoginseng polysaccharide. The only difference between this method and Example 1 is that step (6) is to "redissolve the precipitate in water, add β-glucosidase to the solution at a dosage of 1%, adjust the pH to 5.0, keep warm at 40°C for 2 hours, and inactivate the enzyme at 85°C for 10 minutes". Other operations remain unchanged.

[0086] Example 5

[0087] This embodiment provides a method for preparing Panax notoginseng polysaccharide. The only difference between this method and Example 1 is that step (6) is to "redissolve the precipitate in water, add cellulase to the solution at a dosage of 1%, adjust the pH to 5.0, keep warm at 40°C for 2 hours, and inactivate the enzyme at 85°C for 10 minutes". Other operations remain unchanged.

[0088] Example 6

[0089] This embodiment provides a method for preparing Panax notoginseng polysaccharide. The only difference between this method and Example 1 is that step (5) is to "add anhydrous ethanol to the clarified liquid for alcohol precipitation until the final ethanol concentration is 40%, place it in a refrigerator at 4°C overnight, wait for it to fully precipitate, centrifuge, collect the precipitate, discard the 40% ethanol precipitate, continue to add ethanol to the clarified liquid until the final concentration is 60%, place it in a refrigerator at 4°C overnight, wait for it to fully precipitate, centrifuge, and collect the precipitate". Other operations remain unchanged.

[0090] Comparative Example 1

[0091] This embodiment provides a method for preparing Panax notoginseng polysaccharide, which differs from Example 1 only in that step (6) is omitted, while other operations remain unchanged.

[0092] Comparative Example 2

[0093] This embodiment provides a method for preparing Panax notoginseng polysaccharide, which differs from Example 6 only in that step (6) is omitted, while other operations remain unchanged.

[0094] Comparative Example 3

[0095] This embodiment provides a method for preparing Panax notoginseng polysaccharide. The only difference between this method and Example 1 is that step (6) is to "redissolve the precipitate with water, add papain to the solution at a dosage of 1%, adjust the pH to 5.0, keep warm at 40°C for 2 hours, and inactivate the enzyme at 85°C for 10 minutes". Other operations remain unchanged.

[0096] Test Example 1

[0097] Determination of polysaccharide content, monosaccharide composition and molecular weight

[0098] (1) Determination of polysaccharide content

[0099] The polysaccharide content in samples 1-6 and Comparative Examples 1-3 was determined using the sulfuric acid-phenol method. The test method is as follows:

[0100] ① Preparation of standard solution: Prepare a 0.1 mg / mL glucose standard solution by successively pipetting 0, 0.1, 0.2, 0.4, 0.8, 1.6, and 2 mL into test tubes, adding ddH2O to each test tube to make up to 2 mL, and set aside for use.

[0101] ② Sample solution preparation: Accurately weigh the sample powder and add ddH2O to prepare a 0.1 mg / mL sample solution for testing. 5% phenol solution preparation: Accurately weigh 2.5 g of phenol solid powder, add 47.5 mL of ddH2O and dissolve at 75℃. Store in the dark.

[0102] ③ Reaction of standard solution and sample solution: 1 mL of the prepared standard solution and sample solution were sequentially added to test tubes. 0.5 mL of 5% phenol solution and 2.5 mL of concentrated sulfuric acid solution were added to initiate the reaction. The mixture was gently shaken. ddH2O was used as a blank control. The absorbance was measured at 490 nm using an ELISA reader. A standard curve was plotted with concentration on the x-axis and absorbance on the y-axis. The absorbance of the sample solution was substituted into the standard curve for calculation. The polysaccharide content determination results are shown in Table 1.

[0103] Table 1

[0104]

[0105] As shown in Table 1, the extract obtained by the preparation method described in this invention has a high polysaccharide content. The increase in polysaccharide content in the test results may be related to its improved solubility, and the improved solubility can enhance the bioavailability of polysaccharides.

[0106] (2) Determination of monosaccharide composition

[0107] This invention investigated the monosaccharide composition of Panax notoginseng polysaccharides obtained by fractional alcohol precipitation (Examples 1 and 6). The experimental methods are as follows:

[0108] ① Hydrolysis reaction: Trifluoroacetic acid (TFA) was used to hydrolyze the Panax notoginseng polysaccharides obtained in Examples 1 and 6. 5 mg of Panax notoginseng polysaccharide sample was accurately weighed, 2 mL of 2 M TFA was added, and hydrolysis was carried out at 110 °C for 2 h. After complete hydrolysis, the sample was dried by a nitrogen blower to completely remove TFA.

[0109] ② Derivatization reaction: Derivatization was performed using 1-phenyl-3-methyl-5-pyrazolone (PMP) reagent. Quantitatively weighed D-glucose, D-galactose, D-mannose, D-xylose, L-rhamnose, L-arabinose, D-fructose, D-galacturonic acid, D-glucuronic acid standards, and two polysaccharide hydrolysate samples (Examples 1 and 6) were added to 0.5 mL of 0.5 M PMP reagent and 0.5 mL of 0.3 M NaOH solution and reacted at 70 °C for 30 min. After cooling, 0.5 mL of 0.3 M HCl and 0.5 mL of ddH2O were added for neutralization, and an equal volume of chloroform solution was added for extraction by shaking. The supernatant was collected and filtered through a 0.22 μM filter membrane for testing.

[0110] ③ High-performance liquid chromatography (HPLC): Qualitative and quantitative analysis of the derivatized samples was performed using HPLC. The chromatographic conditions were as follows: Phase A was phosphate buffer (pH=7.2), and Phase B was acetonitrile; gradient elution was performed: 0-3 min: 15% B, 3-4 min: 15-18% B, 4-10 min: 18% B, 10-25 min: 18-40% B; column type: Agilent ZORBAX SB-C18, 4.6×150 mm; flow rate: 0.8 mL / min; column temperature: 30℃; DAD: 250 nm; injection volume: 5 μL.

[0111] The polysaccharide composition is shown in Table 2.

[0112] Table 2

[0113]

[0114] As shown in Table 2, the monosaccharide composition of Examples 1 and 6 is the same, but the proportion of monosaccharides is different. In Example 1, the proportion of galactose is higher, while in Example 6, the proportion of glucose is higher.

[0115] Test Example 2

[0116] Physical barrier genes were tested in Examples 1, 4-6, and Comparative Examples 1-2.

[0117] Test method:

[0118] Keratinocytes (HaCaT) were seeded in 12-well plates and incubated overnight in an incubator (37℃, 5% CO2). When the cell layer coverage reached 60%, cells were divided into groups and treated with 1 mL of the drug per well. Except for the blank control group, the Panax notoginseng polysaccharide treatment groups were treated with a dose of 120 μg / mL. Each group had three replicates. Cells were incubated for 24 h in an incubator (37℃, 5% CO2). After 24 h of culture, the culture medium was discarded, and the cells were washed twice with PBS. 1 mL of RNAiso Plus was added to each well, and the cells were lysed by pipetting. Total RNA was extracted using an RNA extraction kit. After reverse transcription to cDNA, quantitative real-time PCR was performed for detection. -ΔΔct Perform the result calculation.

[0119] The primer information for quantitative real-time mRNA detection is as follows:

[0120] Table 3

[0121]

[0122] GraphPad Prism was used for plotting, and the results are expressed as Mean ± SD. t-tests were used for comparisons between groups. "*" indicates P < 0.05 compared to the normal control group, "**" indicates P < 0.01 compared to the normal control group, and "***" indicates P < 0.001 compared to the normal control group. FLG gene expression is shown in Table 4, LOR gene expression in Table 5, and AQP3 gene expression in Table 6.

[0123] Table 4

[0124]

[0125] Table 5

[0126]

[0127] Table 6

[0128]

[0129] The upward adjustment rate is shown in Table 7.

[0130]

[0131] Note: Based on the statistical analysis results in Table 4-6, groups with no statistically significant differences were not analyzed for upregulation rates.

[0132] Table 7

[0133]

[0134] Experimental results show that Panax notoginseng polysaccharides have the effect of upregulating skin physical barrier-related proteins FLG, LOR, and AQP3, and have great potential for maintaining the integrity of skin barrier function. The Panax notoginseng polysaccharides prepared in Examples 1, 4, 5, and 6 upregulated the expression of FLG, LOR, and AQP3 genes, exhibiting strong skin barrier repair function, with Example 1 showing superior regulatory effect. The Panax notoginseng polysaccharides prepared in Comparative Examples 1 and 2 only significantly upregulated AQP3, showing some protective effect on skin barrier function. Therefore, Panax notoginseng polysaccharides obtained under different treatments all have the potential to protect skin barrier function, while the Panax notoginseng polysaccharides prepared by the method provided in this invention simultaneously upregulated multiple physical barrier genes, exhibiting superior skin barrier protection effect.

[0135] Test Example 3

[0136] Examples 1, 4-6, and Comparative Examples 1-2 were tested for biochemical barrier genes.

[0137] Test method:

[0138] Keratinocytes (HaCaT) were seeded in 6-well plates and incubated overnight in an incubator (37℃, 5% CO2). When the cell layer coverage reached 60%, cells were divided into groups and treated with 2 mL of the drug per well. Except for the blank control group, the Panax notoginseng polysaccharide treatment groups were given a dose of 120 μg / mL for incubation. Each group had three replicates. Cells were incubated for 24 h in an incubator (37℃, 5% CO2). After 24 h of culture, the culture medium was discarded, and the cells were washed twice with PBS. 1 mL of RNAisoPlus was added to each well, and the cells were lysed by pipetting. Total RNA was extracted using an RNA extraction kit. After reverse transcription to cDNA, quantitative real-time PCR was performed for detection. -ΔΔct Perform the result calculation.

[0139] The primer information for quantitative real-time mRNA detection is shown in Table 8:

[0140] Table 8

[0141]

[0142] GraphPad Prism was used for plotting, and the results are expressed as Mean ± SD. The t-test was used for comparison between groups. "*" indicates P < 0.05 compared with the normal control group, "**" indicates P < 0.01 compared with the normal control group, and "***" indicates P < 0.001 compared with the normal control group.

[0143] The expression of HBD-1 gene is shown in Table 9, the expression of HBD-3 gene is shown in Table 10, and the expression of LL-37 gene is shown in Table 11.

[0144] Table 9

[0145]

[0146] Table 10

[0147]

[0148] Table 11

[0149]

[0150] The upward adjustment rate is shown in Table 12.

[0151]

[0152] Note: Based on the statistical analysis results in Table 9-11, groups with no statistically significant differences were not analyzed for upregulation rates.

[0153] Table 12

[0154]

[0155] The main antimicrobial peptides present in the skin are human β-defensins (HBDs) and LL-37. HBDs are expressed in human epithelial cells, with HBD-1 being mainly expressed in keratinocytes and having antibacterial effects. HBD-3 is expressed in the epidermis and has antibacterial and immunomodulatory effects. Studies have shown that HBD-3 plays an important role in maintaining skin homeostasis. The antimicrobial peptide LL-37 is expressed in human skin keratinocytes, neutrophils, and sebaceous gland cells. LL-37 is of great significance in resisting microbial invasion and enhancing skin barrier function. The above experimental results show that the Panax notoginseng polysaccharides prepared in Examples 1, 4, and 5 significantly increased the expression of HBD-1, HBD-3, and LL-37 genes, exhibiting superior effects in enhancing skin barrier function, with Example 1 showing the most significant regulatory effect. The Panax notoginseng polysaccharide prepared in Example 6 significantly increased the expression of HBD-1 and LL-37 genes, demonstrating some skin barrier function enhancement. The Panax notoginseng polysaccharide prepared in Comparative Example 1 only upregulated the expression of the antimicrobial peptide LL-37 gene, showing some potential for skin barrier repair. The Panax notoginseng polysaccharide prepared in Comparative Example 2 significantly upregulated both HBD-1 and LL-37, demonstrating some effect in enhancing skin barrier function. Therefore, the Panax notoginseng polysaccharides obtained under different treatments have good effects in maintaining skin barrier function. In particular, the Panax notoginseng polysaccharides prepared by the method provided in this invention can simultaneously regulate multiple skin biochemical barrier genes, synergistically exerting skin barrier protection function from multiple aspects, and possessing superior application potential.

[0156] The above experimental results demonstrate that the method for preparing Panax notoginseng polysaccharides provided by this invention possesses the ability to regulate multiple key genes of the skin's physical and biochemical barriers, exhibiting significant potential for application in cosmetics. This invention utilizes ethanol precipitation at different concentrations to obtain polysaccharide components with different structures, followed by enzymatic depolymerization at specific sites of the polysaccharides to obtain Panax notoginseng polysaccharides with enhanced skin barrier gene regulatory functions and superior regulatory effects. Furthermore, this technique overcomes the application difficulties caused by the inability to dissolve large-molecule polysaccharides in Panax notoginseng, improving industrial feasibility and demonstrating immense application potential.

[0157] The applicant declares that this invention illustrates a method for preparing Panax notoginseng polysaccharide, its products, and applications through the above embodiments. However, this invention is not limited to the above embodiments, meaning that it does not necessarily depend on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

[0158] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0159] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A method for preparing Panax notoginseng polysaccharide, characterized in that, The preparation method includes: (1) The residue of Panax notoginseng obtained by mixing and extracting with ethanol aqueous solution was extracted by heating and reflux with water to obtain the extract; (2) Filter the extract under reduced pressure, then mix it with the flocculant, filter again, and collect the filtrate; (3) Mix the filtrate with ethanol, centrifuge, and collect the precipitate; (4) Mix the precipitate with the enzyme for enzymatic hydrolysis, centrifuge, and the product is obtained; In step (3), the final concentration of ethanol is 40%-60%; The enzymes described in step (4) include β-glucosidase and / or cellulase; The amount of enzyme added in step (4) is 0.5%-2%; The enzymatic hydrolysis in step (4) takes 1-2 hours, at a temperature of 40-50°C, and at a pH of 5-6.

2. The method for preparing Panax notoginseng polysaccharide according to claim 1, characterized in that, Step (1) The ethanol aqueous solution contains 80-90% ethanol by mass.

3. The method for preparing Panax notoginseng polysaccharide according to claim 1, characterized in that, The mass ratio of Panax notoginseng residue to water is 1:(10-20).

4. The method for preparing Panax notoginseng polysaccharide according to claim 1, characterized in that, The extraction time is independently 1-2 hours.

5. The method of claim 1, wherein the method is characterized by, The extraction is performed 1-3 times.

6. The method of claim 1, wherein the method is characterized by, The mass of the flocculant is 0.5-1% of the mass of the extract.

7. The method of claim 1, wherein the method is characterized by, The flocculant includes any one or a combination of at least two of chitosan, citric acid, carrageenan, or gelatin.

8. The method of claim 7, wherein the preparation of the panax polysaccharide is characterized by, The flocculant is a combination of chitosan and citric acid.

9. The method for preparing Panax notoginseng polysaccharide according to claim 8, characterized in that, The mass ratio of chitosan to citric acid in the flocculant is 1:(1-2).

10. The method for preparing Panax notoginseng polysaccharide according to claim 1, characterized in that, The enzyme in step (4) is a combination of β-glucosidase and cellulase.

11. The method of claim 10, wherein the preparation of the panax polysaccharide is characterized by, The mass ratio of β-glucosidase to cellulase is 1:(0.1-1).

12. The method of claim 1, wherein the preparation of the panax polysaccharide is characterized by, Step (4) involves enzyme inactivation after enzymatic hydrolysis.

13. The Panax notoginseng polysaccharide prepared by the method according to any one of claims 1-12.

14. The application of the Panax notoginseng polysaccharide according to claim 13 in the preparation of cosmetics with skin barrier protection function.

15. Use according to claim 14, characterized in that, The amount of Panax notoginseng polysaccharide added to the cosmetic with skin barrier protection function is 1-10%.

16. The use according to claim 14, characterized in that, The cosmetics include lotions, creams, sprays, or masks.

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