Application of cistanche deserticola polysaccharide in preparation of product for protecting skin barrier
By preparing Cistanche polysaccharides A and B with specific molecular weight, the stability and effect problems of Cistanche polysaccharides in skin care products are solved, and the significant skin barrier protection effect is achieved, and it is used in cosmetics, oral products and pharmaceutical preparations.
Patent Information
- Application Number
- CN202510721529.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-30
- Publication Date
- 2025-08-15
AI Technical Summary
The application of Cistanche polysaccharide in skin care products in the prior art lacks clear quality control and stability, and cannot effectively exert its advantages in protecting skin barriers, which limits its application in skin care products.
By preparing Cistanche polysaccharide A and Cistanche polysaccharide B with a specific molecular weight, using monohydric alcohol soaking, water extraction, concentration, alcohol precipitation and membrane filtration, polysaccharide with a molecular weight of >800kDa was obtained, which was used to promote the proliferation of HaCaT cells, improve the expression of tight junction proteins between cells and prevent UVB damage.
Cistanche polysaccharides A and B significantly promote HaCaT cell proliferation at concentrations of 25μg/mL and 50μg/mL, improve the expression of tight junction protein, prevent UVB damage, and have a stronger skin barrier protection effect. It is used in cosmetics, oral products and pharmaceutical preparations.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of cosmetics or skin damage repair preparations, and particularly relates to the application of Cistanche deserticola polysaccharide in the preparation of skin barrier protection products. Background Art
[0002] Cistanche deserticola is the dried fleshy stem with scaly leaves of Cistanche deserticola YCMa or Cistanche tuhulosa (Schenk) Wight, both of the Orobanchaceae family. It has the functions of tonifying kidney yang, replenishing essence and blood, and moistening the intestines and relieving constipation. It is used to treat kidney yang deficiency, essence and blood deficiency, impotence and infertility, soreness of the waist and knees, weakness of the tendons and bones, and dry intestines and constipation.
[0003] In recent years, scholars have explored the skincare benefits of Cistanche deserticola. Literature and patents report that Cistanche deserticola water extracts, oligosaccharides, or facial masks and creams formulated with Cistanche deserticola extracts exhibit certain skincare benefits. However, the mechanisms and material basis for Cistanche deserticola's skincare benefits remain unclear, hindering accurate quality control and maintaining the stability of its efficacy. Existing technologies have also failed to identify polysaccharides in Cistanche deserticola that offer advantages in skincare. Consequently, the active ingredients in Cistanche deserticola have a very limited role in protecting the skin, limiting its application in skincare products.
[0004] Therefore, it is necessary to further explore the functions of Cistanche deserticola polysaccharides, reveal the specific effects of Cistanche deserticola polysaccharides on skin cells, and provide a guiding path for the refined development of functional skin care products and the research and development of drugs that protect the skin barrier. Summary of the Invention
[0005] After screening, the inventors discovered that polysaccharides of a specific molecular weight in Cistanche deserticola not only promote HaCaT cell proliferation and increase the expression of tight junction proteins between cells, but also prevent UVB damage to the skin barrier, improve cell migration rate, and promote wound healing. Their skin barrier protection activity is superior to that of Tremella fuciformis heteropolysaccharides and small molecule hyaluronic acid, and has high application value in the research and development of cosmetics, oral products, and pharmaceuticals related to skin barrier protection. The present invention specifically provides the following technical solutions.
[0006] The present invention provides an application of Cistanche deserticola polysaccharide in preparing a product for protecting skin barrier. Specifically, the Cistanche deserticola polysaccharide comprises Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B.
[0007] The preparation method of the Cistanche deserticola polysaccharide A comprises the following steps: pre-treating the Cistanche deserticola by soaking it in a monohydric alcohol, extracting it with water, concentrating it, and precipitating it with alcohol to obtain precipitated total polysaccharides and an alcohol precipitation supernatant; dissolving the total polysaccharide with 10 to 20 times its mass of water, filtering it with a membrane with a molecular weight cutoff of 800 kDa, collecting a portion with a molecular weight greater than 800 kDa, and obtaining the Cistanche deserticola polysaccharide A, wherein the polysaccharide content of the Cistanche deserticola polysaccharide A is greater than 50%.
[0008] The preparation method of Cistanche deserticola polysaccharide B is as follows: monohydric alcohol soaking pretreatment, water extraction, concentration, and alcohol precipitation are the same as those for the preparation of Cistanche deserticola polysaccharide A, except that: when the total polysaccharide is dissolved in water, the amount of water used is 2 to 10 times the mass of the total polysaccharide, 95% ethanol or anhydrous ethanol is added to make its concentration 10 to 40% for alcohol precipitation, and the precipitated polysaccharide is collected to obtain Cistanche deserticola polysaccharide B. The Cistanche deserticola polysaccharide B contains polysaccharides with a molecular weight greater than 800 kDa and a polysaccharide content greater than 50%.
[0009] Specifically, in the above application, the Cistanche deserticola polysaccharide A promotes HaCaT cell proliferation; promotes the expression of tight junction proteins between cells; improves cell migration rate, promotes wound healing; and prevents UVB damage to the skin barrier.
[0010] Specifically, in the above application, the Cistanche deserticola polysaccharide B promotes the proliferation of HaCaT cells; promotes the expression of tight junction proteins between cells; and prevents UVB damage to the skin barrier.
[0011] Furthermore, in the preparation of Cistanche deserticola polysaccharide A or Cistanche deserticola polysaccharide B, the monohydric alcohol is selected from: methanol, ethanol; the concentration of the monohydric alcohol is 30-68%, the amount is 4-10 times the mass of Cistanche deserticola, and the soaking time is 3-28 days.
[0012] Furthermore, in the preparation of Cistanche deserticola polysaccharide A or Cistanche deserticola polysaccharide B, during water extraction, the amount of water used is 4 to 12 times the mass of Cistanche deserticola, the water extraction temperature is 75 to 100° C., and the number of water extractions is 1 to 4 times, each time for 1 to 3 hours.
[0013] Furthermore, in the preparation of Cistanche deserticola polysaccharide A or Cistanche deserticola polysaccharide B, during concentration, the water extract is concentrated to 4-25% of its initial volume.
[0014] Furthermore, in the preparation of Cistanche deserticola polysaccharide A or Cistanche deserticola polysaccharide B, during the alcohol precipitation, 95% ethanol or anhydrous ethanol is added to the ethanol concentration of 60-90%.
[0015] Furthermore, in the above application, the skin barrier protection product includes: cosmetics, oral products and pharmaceutical preparations.
[0016] Furthermore, in the above application, the tight junction proteins include: Occludin protein and Claudin-1 protein.
[0017] Compared with the prior art, the present invention "Application of Cistanche deserticola polysaccharide in the preparation of skin barrier protection products" has the following beneficial effects: the present invention pre-treats Cistanche deserticola with a low-concentration monohydric alcohol, and then uses water extraction, concentration, alcohol precipitation, membrane filtration and other steps to prepare Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B. Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B have many advantages, including the following aspects:
[0018] 1. The present invention has been experimentally confirmed that at concentrations of 25 μg / mL and 50 μg / mL, the survival rate of HaCaT cells treated with Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B is higher, indicating that Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B promote the proliferation of HaCaT cells ( Figure 1 ).
[0019] 2. The present invention has been confirmed by experiments that at concentrations of 25 μg / mL and 50 μg / mL, both Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B can promote the expression of tight junction proteins (Claudin-1 and Occludin) between cells ( Figure 2 、 Figure 3 ).
[0020] 3. The present invention has been confirmed by experiments that Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B have a good protective effect on preventing the decrease in expression of intercellular tight junction proteins (Claudin-1 and Occludin) caused by UVB, and can be used to prevent skin barrier damage caused by UVB ( Figure 4 、 Figure 5 ).
[0021] Fourth, the present invention also confirmed that Cistanche deserticola polysaccharide A can promote HaCaT cell migration, further indicating that Cistanche deserticola polysaccharide A can promote wound healing and is expected to be used for the repair of skin damage, etc. ( Figure 6 、 Figure 7 ).
[0022] 5. At a concentration of 50 μg / mL, the effect of the Cistanche deserticola polysaccharide A on promoting HaCaT cell proliferation and Claudin-1 expression was stronger than that of Tremella heteropolysaccharide and small molecule hyaluronic acid ( Figure 11 、 Figure 12 At concentrations of 25 μg / mL and 50 μg / mL, the Cistanche deserticola polysaccharide A has a stronger promoting effect on the expression of Occludin than Tremella heteropolysaccharide and small molecule hyaluronic acid ( Figure 13 ).
[0023] Based on the above-mentioned properties of Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B, they are used to prepare products related to protecting the skin barrier and preventing UVB damage to the skin, and are expected to play a role in the preparation of cosmetics, oral products, and pharmaceutical preparations. The preparation methods of Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B are simple, easy to produce and transform, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 The survival rate of HaCaT cells after intervention with Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B at 25 μg / mL (low concentration) and 50 μg / mL (high concentration), respectively.
[0025] Figure 2 This is the effect of Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B on Claudin-1 expression after intervention at 25μg / mL (low concentration) and 50μg / mL (high concentration), respectively.
[0026] Figure 3 This is the effect of Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B on Occludin expression after intervention at 25μg / mL (low concentration) and 50μg / mL (high concentration), respectively.
[0027] Figure 4 This is the protective effect of Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B in preventing the decrease of Claudin-1 expression caused by UVB.
[0028] Figure 5 This is the protective effect of Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B in preventing the decrease of Occludin expression caused by UVB.
[0029] Figure 6 The migration rate of HaCaT cells after intervention with Cistanche deserticola polysaccharide A at 50 μg / mL (low concentration) and 100 μg / mL (high concentration) (24h).
[0030] Figure 7 The migration rate of HaCaT cells after intervention with Cistanche deserticola polysaccharide A at 50 μg / mL (low concentration) and 100 μg / mL (high concentration) (48 h).
[0031] Figure 8 This is the effect of Cistanche deserticola polysaccharide A on the survival rate of HaCaT cells under 50μg / mL (low concentration) and 100μg / mL (high concentration) conditions.
[0032] Figure 9 This is the effect of Cistanche deserticola polysaccharide A on the expression of tight junction protein Claudin-1 under 50μg / mL (low concentration) and 100μg / mL (high concentration) conditions.
[0033] Figure 10This is the effect of Cistanche deserticola polysaccharide A on the expression of tight junction protein Occludin under 50μg / mL (low concentration) and 100μg / mL (high concentration) conditions.
[0034] Figure 11 The survival rate of HaCaT cells after intervention with Cistanche deserticola polysaccharide A, Tremella fuciformis heteropolysaccharide, and hyaluronic acid at 50 μg / mL (low concentration) and 100 μg / mL (high concentration), respectively.
[0035] Figure 12 This is the effect of Cistanche deserticola polysaccharide A, Tremella fuciformis heteropolysaccharide, and hyaluronic acid on Claudin-1 expression after intervention at 50 μg / mL (low concentration) and 100 μg / mL (high concentration), respectively.
[0036] Figure 13 This is the effect of Cistanche deserticola polysaccharide A, Tremella fuciformis heteropolysaccharide, and hyaluronic acid on Occludin expression after intervention at 25μg / mL (low concentration) and 50μg / mL (high concentration), respectively. DETAILED DESCRIPTION
[0037] The technical solutions of the present invention are described clearly and completely below with reference to the embodiments. It is obvious that the embodiments described are only a portion of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are also within the scope of protection of the present invention.
[0038] In the following examples, the preparation method of the Cistanche deserticola polysaccharide A is as follows:
[0039] Alcohol soaking: Clean the Cistanche deserticola and soak it in a 30-68% monohydric alcohol (methanol, ethanol, etc.) at room temperature for 3-28 days. The amount of monohydric alcohol used should be 4-10 times the mass of the Cistanche deserticola.
[0040] Water extraction: After soaking, remove the Cistanche deserticola and extract it with water at 75-100℃ for 1-4 times, each time for 1-3 hours. The amount of water used should be 4-12 times the weight of the Cistanche deserticola.
[0041] Concentration: Concentrating the aqueous extract to 4-25% of its initial volume to obtain a concentrated aqueous extract;
[0042] Alcohol precipitation: adding 95% ethanol or anhydrous ethanol to the concentrated solution to adjust the concentration of ethanol in the concentrated solution to 60-90%, thereby obtaining alcohol-precipitated total polysaccharides and a supernatant;
[0043] The total polysaccharide is dissolved in 10-20 times the mass of water, filtered with a membrane with a molecular weight cut-off of 800 kDa, and the portion with a molecular weight greater than 800 kDa is collected to obtain Cistanche deserticola polysaccharide A, wherein the polysaccharide content of the Cistanche deserticola polysaccharide A is greater than 50%.
[0044] The preparation method of the Cistanche deserticola polysaccharide B is as follows:
[0045] The method of monohydric alcohol soaking pretreatment, water extraction, concentration and alcohol precipitation is the same as that of preparing Cistanche deserticola polysaccharide A, except that: when the above-mentioned total polysaccharide is dissolved in water, the amount of water used is 2 to 10 times the mass of the total polysaccharide, and then 95% ethanol or anhydrous ethanol is added to make its concentration 10 to 40% for alcohol precipitation, and the precipitated polysaccharide is collected to obtain Cistanche deserticola polysaccharide B. The Cistanche deserticola polysaccharide B contains polysaccharides with a molecular weight greater than 800 kDa and a polysaccharide content greater than 50%.
[0046] Example 1
[0047] This example describes the preferred preparation method of Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B.
[0048] 1. Preparation of Cistanche deserticola polysaccharide A
[0049] Alcohol soaking: 500 g of Cistanche deserticola was cleaned and soaked in 45% ethanol at room temperature for 20 days. The amount of ethanol used was 10 times the weight of Cistanche deserticola (5 kg).
[0050] Water extraction: After soaking, remove the Cistanche deserticola and extract it in water at 100°C for 3 times, each time for 2 hours. The amount of water used is 4 times the weight of Cistanche deserticola (2.0 kg).
[0051] Concentration: Concentrate the aqueous extract to 20% (400 mL) of its initial volume to obtain a concentrate of the aqueous extract;
[0052] Alcohol precipitation: adding anhydrous ethanol to the concentrated solution to adjust the ethanol concentration to 80% to obtain alcohol-precipitated total polysaccharides and a supernatant;
[0053] The total polysaccharide was dissolved in water at an amount of 20 times the mass of the total polysaccharide, and then filtered through a membrane with a molecular weight cutoff of 800 kDa, and the fraction with a molecular weight greater than 800 kDa was collected and vacuum-dried at -50°C to obtain Cistanche deserticola polysaccharide A. The polysaccharide content of the Cistanche deserticola polysaccharide A was greater than 50%.
[0054] 2. Preparation of Cistanche deserticola polysaccharide B
[0055] In the preparation of Cistanche deserticola polysaccharide B, the methods of alcohol soaking, water extraction, concentration, and alcohol precipitation are the same as those for the preparation of Cistanche deserticola polysaccharide A, except that when the total polysaccharide is dissolved in water, the amount of water used is 6 times the total polysaccharide, and then alcohol precipitation is performed with ethanol at a final concentration of 25%. The precipitated polysaccharide is collected and vacuum dried at -50°C to obtain Cistanche deserticola polysaccharide B. The Cistanche deserticola polysaccharide B contains polysaccharides with a molecular weight greater than 800 kDa and a polysaccharide content greater than 50%.
[0056] Example 2
[0057] This example describes the effect of Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B on promoting HaCaT cell proliferation. Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B were prepared by the method in Example 1.
[0058] HaCaT cells were prepared with complete culture medium to a volume of 5 × 10 4 Cells / mL of single cell suspension were inoculated into 96-well plates (inoculation volume 100 μL / well), cultured at 37 ° C, 5% CO2 for 24 hours, and after adherence, the culture medium was discarded. Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B were used for intervention, respectively, and blank group HaCaT cells (n=5) were prepared at the same time. After 48 hours of intervention, the culture medium was discarded, and 100 μL of 10% CCK-8 solution was added to each well. The cells were continued to be incubated in the incubator. After 1 hour, the OD value at 450 nm was measured with a microplate reader to calculate the HaCaT cell survival rate (%). The experimental results are shown in Figure 1 .
[0059] Depend on Figure 1 It can be seen that at a concentration of 25 μg / mL (low concentration), both Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B can promote the proliferation of HaCaT cells. The promoting effects of Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B increased with increasing concentration, and significantly promoted the proliferation of HaCaT cells at 50 μg / mL (high concentration) (P < 0.05), indicating that the two have comparable effects.
[0060] Example 3
[0061] This example describes how Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B promote the expression of tight junction proteins Claudin-1 and Occludin. Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B are prepared by the method in Example 1.
[0062] HaCaT cells were prepared with complete culture medium to a volume of 5 × 10 4 Cells were suspended at a concentration of 10 cells / mL and inoculated into culture flasks. After 24 hours of culture at 37°C and 5% CO2, the culture medium was discarded and cells were treated with 25 μg / mL and 50 μg / mL of Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B, respectively. A blank control group (n=3) was also prepared. After 48 hours of treatment, cell lysates were collected and the levels of tight junction proteins Claudin-1 and Occludin were measured using ELISA kits.
[0063] Elisa kit detection method: set up standard wells and test sample wells, add 100μL of standard or test sample to each well, mix well, cover with plate sticker, and incubate at 37℃ for 2h. Discard the liquid, spin dry, add 100μL of biotin-labeled antibody working solution to each well, cover with a new plate sticker, and incubate at 37℃ for 1h. Discard the liquid, spin dry, wash the plate 3 times, and spin dry. Add 100μL of horseradish peroxidase-labeled avidin working solution to each well, cover with a new plate sticker, and incubate at 37℃ for 1h. Discard the liquid, spin dry, wash the plate 5 times, and spin dry. Add 90μL of substrate solution to each well in sequence, and color at 37℃ in the dark for 15-30min. Add 50μL of stop solution to each well to terminate the reaction. Within 5min after the reaction is terminated, use an enzyme reader to measure the OD value of each well at 450nm in sequence, and obtain the concentration of Claudin-1 and Occludin according to the standard curve. The test results are shown in Figure 2 、 Figure 3 .
[0064] Depend on Figure 2 It can be seen that the effects of Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B in promoting Claudin-1 expression are comparable, and both increase with increasing concentrations, and have significant effects at 25 μg / mL (low concentration) and 50 μg / mL (high concentration) (P < 0.05).
[0065] Depend on Figure 3 It can be seen that the effects of Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B in promoting occludin expression are comparable, and both increase with increasing concentrations, and have significant effects at 25 μg / mL (low concentration) and 50 μg / mL (high concentration) (P < 0.05).
[0066] Example 4
[0067] This example describes the effects of Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B on preventing UVB-induced barrier damage.
[0068] HaCaT cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 units / mL penicillin, and 100 mg / mL streptomycin at 37°C and 5% CO2. 5 The irradiation test was conducted in a culture dish with 10 cells / well. The experiment set up blank group, UVB group, polysaccharide A group, and polysaccharide B group. The treatment method of each group was as follows:
[0069] 1) Blank group: After HaCaT cells adhered to the wall, the culture medium was discarded and replaced with fresh DMEM culture medium.
[0070] 2) UVB group: After HaCaT cells adhered to the wall, they were exposed to 60 mJ·cm -2 After irradiation, the cells were cultured for 48 h.
[0071] 3) Polysaccharide A group: After HaCaT cells adhered to the wall, 50 μg / mL of Cistanche deserticola polysaccharide A was added and then 60 mJ·cm -2 After irradiation, the cells were cultured for 48 h.
[0072] 4) Polysaccharide B group: After HaCaT cells adhered to the wall, 50 μg / mL of Cistanche deserticola polysaccharide B was added and then 60 mJ·cm -2 After irradiation, the cells were cultured for 48 h.
[0073] Cells were collected and the expression of tight junction proteins Claudin-1 and Occludin in HaCaT cells of each group was detected by ELISA. The experimental results are shown in Figure 4 、 5 .
[0074] Depend on Figure 4 、 Figure 5 It can be seen that compared with the blank group, the expression levels of Claudin-1 and Occludin were significantly reduced after UVB irradiation (P < 0.05), indicating that UVB irradiation has a damaging effect on HaCaT cells. Compared with the UVB group, polysaccharide A and polysaccharide B significantly promoted the expression of Claudin-1 and Occludin (P < 0.05), indicating that Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B can prevent UVB-induced barrier damage and protect the skin.
[0075] Example 5
[0076] This example describes the effect of Cistanche deserticola polysaccharide A on cell migration rate.
[0077] Based on the above effects of Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B, one of the polysaccharides was selected for cell migration test. In this example, Cistanche deserticola polysaccharide A was used as an example.
[0078] HaCaT cells were prepared with complete culture medium to a density of 5 × 10 5 A single cell suspension of 10 cells / mL was inoculated into a 24-well plate at a volume of 500 μL / well. After the cells adhered, the intervention test was performed. The experiment was divided into a blank group, a positive drug group, and a Cistanche deserticola polysaccharide A group. The treatment methods for each group were as follows:
[0079] 1) Blank group: After HaCaT cells adhered to the wall, the complete medium was discarded and replaced with new complete medium.
[0080] 2) Positive drug group: After HaCaT cells adhered to the wall, the complete culture medium was discarded and the cells were treated with 10% fetal bovine serum.
[0081] 3) Cistanche deserticola polysaccharide A group: After HaCaT cells adhered to the wall, the complete culture medium was discarded and the cells were intervened with 50 μg / mL and 100 μg / mL of Cistanche deserticola polysaccharide A, respectively.
[0082] The intervention time for each group was 48 h. After the intervention, a straight scratch area was gently made with a gun tip. Except for the positive drug group, the other groups were replaced with serum-free culture medium, and the scratch area was photographed at 0 h, 24 h, and 48 h, respectively. The scratch area and HaCaT cell migration rate were calculated using ImageJ.
[0083] Figure 6 The migration rate of HaCaT cells in each treatment group at 24 hours under 50 μg / mL (low concentration) and 100 μg / mL (high concentration) conditions. Figure 7 The migration rate of HaCaT cells in each treatment group at 48h under the conditions of 50μg / mL (low concentration) and 100μg / mL (high concentration) is shown. Figure 6 、 Figure 7 It can be seen that at a concentration of 50 μg / mL for 24 hours and 48 hours, Cistanche deserticola polysaccharide A significantly promoted the migration of HaCaT cells (P < 0.05), further proving that Cistanche deserticola polysaccharide A can promote wound healing and is expected to be used for the repair of skin injuries.
[0084] Example 6
[0085] This example describes the effects of Cistanche deserticola polysaccharide A at other concentrations on HaCaT cell proliferation.
[0086] HaCaT cells were prepared with complete culture medium to a volume of 5 × 10 4 A single-cell suspension of 100 μL cells / mL was inoculated into a 96-well plate (inoculation volume 100 μL / well) and cultured at 37°C, 5% CO2 for 24 hours. After adherence, the culture medium was discarded and the cells were treated with 50 μg / mL and 100 μg / mL of Cistanche deserticola polysaccharide A, respectively. A blank control group of HaCaT cells (n=5) was also prepared. After 48 hours of intervention, the culture medium was discarded and 100 μL of 10% CCK-8 solution was added to each well. The cells were incubated in the incubator for another hour. The OD value at 450 nm was measured using a microplate reader after 1 hour, and the HaCaT cell survival rate (%) was calculated.
[0087] Figure 8 The following are the effects of Cistanche deserticola polysaccharide A on the survival rate of HaCaT cells at 50 μg / mL (low concentration) and 100 μg / mL (high concentration). Figure 8 It can be seen that at both concentrations, the survival rate of HaCaT cells treated with Cistanche deserticola polysaccharide A was higher than that of the blank group, indicating that Cistanche deserticola polysaccharide A can also promote HaCaT cell proliferation at other concentrations.
[0088] Example 7
[0089] This example describes the effects of Cistanche deserticola polysaccharide A at other concentrations on the expression of tight junction proteins Claudin-1 and Occludin between HaCaT cells.
[0090] HaCaT cells were prepared with complete culture medium to a volume of 5 × 10 4 Cells were inoculated into culture flasks with a single cell suspension of 100 μg / mL cells / mL and cultured at 37°C, 5% CO2 for 24 hours. After adherence, the culture medium was discarded and cells were treated with 50 μg / mL (low concentration) and 100 μg / mL (high concentration) of Cistanche deserticola polysaccharide A, respectively. At the same time, blank control cells (n=3) were prepared. After 48 hours of intervention, cell lysates were collected and the levels of tight junction proteins Claudin-1 and Occludin were detected by Elisa kit. The Elisa kit detection method was the same as in Example 3. The experimental results are shown in Figure 9 、 Figure 10 .
[0091] Figure 9 The effect of Cistanche deserticola polysaccharide A on the expression of tight junction protein Claudin-1 at 50μg / mL and 100μg / mL respectively. Figure 9 It can be seen that at both concentrations, after intervention with Cistanche deserticola polysaccharide A, the expression level of Claudin-1 was significantly increased compared with the blank group (P < 0.05), indicating that Cistanche deserticola polysaccharide A can also promote the expression of Claudin-1 at other concentrations.
[0092] Figure 10 The effect of Cistanche deserticola polysaccharide A on the expression of tight junction protein Occludin at 50μg / mL and 100μg / mL respectively. Figure 10 It can be seen that at both concentrations, the expression level of occludin was higher after intervention with Cistanche deserticola polysaccharide A than in the blank group, indicating that Cistanche deserticola polysaccharide A can also promote the expression of occludin at other concentrations.
[0093] Example 8
[0094] The examples describe the comparative effects of Cistanche deserticola polysaccharide A, Tremella fuciformis heteropolysaccharide (≥1000 kDa), and small molecule hyaluronic acid (200-400 kDa).
[0095] 1) Comparison of effects on HaCaT cell proliferation
[0096] According to the test method in Example 2, the effects of Cistanche deserticola polysaccharide A, Tremella fuciformis heteropolysaccharide, and small molecule hyaluronic acid on HaCaT cell proliferation were compared, and the intervention concentrations were set at 50 μg / mL (low concentration) and 100 μg / mL (high concentration). Figure 11 .Depend on Figure 11It can be seen that at both concentrations, Cistanche deserticola polysaccharide A can promote the proliferation of HaCaT cells, and its effect is better than that of Tremella heteropolysaccharide and small molecule hyaluronic acid, and 50 μg / mL is the best.
[0097] 2) Comparison of the effects on the expression of tight junction proteins Claudin-1 and Occludin
[0098] According to the experimental method in Example 3, the effects of Cistanche deserticola polysaccharide A, Tremella fuciformis heteropolysaccharide, and small molecule hyaluronic acid on the expression of tight junction proteins Claudin-1 and Occludin were compared. For Claudin-1, the intervention concentration was set to 50 μg / mL (low concentration) and 100 μg / mL (high concentration). For Occludin, the intervention concentration was set to 25 μg / mL (low concentration) and 50 μg / mL (high concentration). The experimental results are shown in Figure 12 、 Figure 13 .
[0099] Depend on Figure 12 It can be seen that compared with the blank group, under the conditions of 50μg / mL and 100μg / mL, Cistanche polysaccharide A, Tremella heteropolysaccharide, and small molecule hyaluronic acid can significantly promote the expression of Claudin-1 (P<0.05), and under the condition of 50μg / mL, the effect of Cistanche polysaccharide A in promoting the expression of Claudin-1 is better than Tremella heteropolysaccharide and small molecule hyaluronic acid.
[0100] Depend on Figure 13 It can be seen that compared with the blank group, Cistanche deserticola polysaccharide A significantly promoted the expression of Occludin at 25 μg / mL and 50 μg / mL (P < 0.05), and its promoting effect was better than that of Tremella heteropolysaccharide and small molecule hyaluronic acid.
[0101] The embodiments described above are only some of the embodiments of the present invention, not all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but merely represents selected embodiments of the present invention. All other embodiments obtained without creative effort and through deduction and substitution by a person of ordinary skill in the art based on the concept of the present invention are within the scope of protection of the present invention.
Claims
1. The application of Cistanche deserticola polysaccharide in the preparation of a product for protecting the skin barrier is characterized by: Cistanche deserticola polysaccharides include Cistanche deserticola polysaccharide A and Cistanche deserticola polysaccharide B; The preparation method of the Cistanche deserticola polysaccharide A comprises the following steps: pre-treating the Cistanche deserticola by soaking it in monohydric alcohol, extracting it with water, concentrating it, and precipitating it with alcohol to obtain the precipitated total polysaccharide and the alcohol precipitation supernatant; The total polysaccharide is dissolved in 10 to 20 times the mass of water, and filtered through a membrane with a molecular weight cutoff of 800 kDa to collect a portion with a molecular weight greater than 800 kDa to obtain Cistanche deserticola polysaccharide A, wherein the polysaccharide content of the Cistanche deserticola polysaccharide A is greater than 50%; The preparation method of Cistanche deserticola polysaccharide B is as follows: monohydric alcohol soaking pretreatment, water extraction, concentration, and alcohol precipitation are the same as those for the preparation of Cistanche deserticola polysaccharide A, except that: when the total polysaccharide is dissolved in water, the amount of water used is 2 to 10 times the mass of the total polysaccharide, 95% ethanol or anhydrous ethanol is added to make its concentration 10 to 40% for alcohol precipitation, and the precipitated polysaccharide is collected to obtain Cistanche deserticola polysaccharide B. The Cistanche deserticola polysaccharide B contains polysaccharides with a molecular weight greater than 800 kDa and a polysaccharide content greater than 50%.
2. The use according to claim 1, characterized in that The Cistanche deserticola polysaccharide A promotes the proliferation of HaCaT cells; Promote the expression of tight junction proteins between cells; Prevent UVB from damaging the skin barrier; Improve cell migration rate and promote wound healing.
3. The use according to claim 1, characterized in that The Cistanche deserticola polysaccharide B promotes the proliferation of HaCaT cells; Promote the expression of tight junction proteins between cells; Prevent UVB from damaging the skin barrier.
4. The use according to claim 1, characterized in that In the preparation of Cistanche deserticola polysaccharide A or Cistanche deserticola polysaccharide B, the monohydric alcohol is selected from: methanol, ethanol; the concentration of the monohydric alcohol is 30-68%, the amount is 4-10 times the mass of Cistanche deserticola, and the soaking time is 3-28 days.
5. The use according to claim 1, characterized in that In the preparation of Cistanche deserticola polysaccharide A or Cistanche deserticola polysaccharide B, during water extraction, the amount of water used is 4 to 12 times the mass of Cistanche deserticola, the water extraction temperature is 75 to 100° C., the water extraction times are 1 to 4 times, each time for 1 to 3 hours.
6. The use according to claim 1, characterized in that In the preparation of Cistanche deserticola polysaccharide A or Cistanche deserticola polysaccharide B, during concentration, the water extract is concentrated to 4-25% of its initial volume.
7. The use according to claim 1, characterized in that In the preparation of Cistanche deserticola polysaccharide A or Cistanche deserticola polysaccharide B, during the alcohol precipitation, 95% ethanol or anhydrous ethanol is added to the ethanol concentration of 60-90%.
8. The use according to claim 1, characterized in that The skin barrier protection products include cosmetics, oral products and pharmaceutical preparations.
9. The use according to claim 1, characterized in that The tight junction proteins include: Occludin protein and Claudin-1 protein.