Composition and application thereof in preparation of product with effect of improving activity of lymphatic endothelial cells

Abstract

The invention particularly discloses a composition and application thereof in preparation of a product with an effect of improving the activity of lymphatic endothelial cells. The composition is prepared from a schisandra chinensis fruit extract and aescin, the weight ratio of the schisandra chinensis fruit extract to the aescin is (1-3): (0.1-0.3). Furthermore, the composition also comprises a passion fruit extract. The weight ratio of the schisandra chinensis fruit extract to the passion fruit extract to the aescin is (1-3): (1-3): (0.1-0.3). Researches show that the composition can improve the activity of lymphatic endothelial cells by enhancing the mitochondrial function and promoting cell autophagy. The composition disclosed by the invention has the effect of improving the activity of lymphatic endothelial cells; therefore, the compound has important application value when being used as an active ingredient for preparing medicines or cosmetics with the effect of improving the activity of lymphatic endothelial cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a composition and application thereof in the preparation of a product with the function of improving the activity of lymphatic endothelial cells. BACKGROUND

[0002] With the deepening of consumers' understanding of skin health, modern skin care concepts have gradually shifted from traditional surface moisturizing, whitening and anti-wrinkling to comprehensive conditioning of skin microecological environment, barrier function and internal circulation system. In recent years, the skin lymphatic system, as a key network for maintaining tissue homeostasis, removing metabolic waste and regulating immune response, has attracted increasing attention in skin health and anti-aging. Among them, lymphatic endothelial cells, as the main constituent cells of the lymphatic vessel wall, their structural integrity and physiological activity directly determine the efficiency of skin lymphatic drainage function.

[0003] Studies have shown that the initial lymphatic vessels in the skin are blind-end structures, widely distributed in the superficial dermis, with high permeability, which can effectively absorb macromolecular substances, inflammatory factors and immune cells in the interstitial space. This process is not only crucial for maintaining skin hydration balance and preventing edema, but also plays a "cleaner" role in removing free radicals, aged cell fragments and pro-inflammatory mediators. However, with age, ultraviolet radiation (photoaging), environmental pollution and chronic inflammatory stimuli, the density of skin lymphatic vessels gradually decreases, and the proliferation, migration and lumen formation ability of lymphatic endothelial cells decrease, leading to blocked lymphatic return. Its clinical manifestations include: facial contour relaxation, periorbital and cheek edema, dull skin color, increased skin sensitivity, and delayed wound repair, which seriously affects the overall appearance and health status of the skin.

[0004] Therefore, it is of great application value to provide a product with the function of improving the activity of lymphatic endothelial cells. SUMMARY

[0005] In order to overcome at least one of the technical problems existing in the prior art, the present application provides a composition and application thereof in the preparation of a product with the function of improving the activity of lymphatic endothelial cells.

[0006] The technical scheme of the present application is as follows: The present application first provides a composition comprising Schisandra chinensis fruit extract and aescin.

[0007] The present application provides a brand new composition, studies have shown that the composition composed of Schisandra chinensis fruit extract and aescin has the function of improving the activity of lymphatic endothelial cells.

[0008] Preferably, the weight ratio of the Schisandra chinensis fruit extract and aescin is 1-3:0.1-0.3.

[0009] Most preferably, the weight ratio of the Schisandra chinensis fruit extract and the aescin is 1:0.2.

[0010] Preferably, the composition further comprises a Passiflora edulis extract.

[0011] The inventors have found in further studies that the addition of a Passiflora edulis extract to a composition consisting of a Schisandra chinensis fruit extract and an aescin further improves the effect of increasing the viability of lymphatic endothelial cells.

[0012] Preferably, the composition comprises a Schisandra chinensis fruit extract, a Passiflora edulis extract and an aescin.

[0013] Preferably, the weight ratio of the Schisandra chinensis fruit extract, the Passiflora edulis extract and the aescin is 1-3:1-3:0.1-0.3.

[0014] Most preferably, the weight ratio of the Schisandra chinensis fruit extract, the Passiflora edulis extract and the aescin is 1:1:0.2.

[0015] The Schisandra chinensis fruit extract can be obtained commercially or prepared by the inventors.

[0016] The Passiflora edulis extract can be obtained commercially or prepared by the inventors. Preferably, the Schisandra chinensis fruit extract is prepared by the following method: The dried Schisandra chinensis fruit is extracted with water. After the extraction is completed, the extract is separated. The extract is concentrated and dried to obtain the Schisandra chinensis fruit extract.

[0017] Preferably, the ratio of the dried Schisandra chinensis fruit to water is 1 kg:5-10 L.

[0018] Most preferably, the ratio of the dried Schisandra chinensis fruit to water is 1 kg:8 L.

[0019] Preferably, the extraction is a heated reflux extraction, and the extraction temperature is 80-100°C, and the extraction time is 1-2 h.

[0020] Most preferably, the extraction is a heated reflux extraction, and the extraction temperature is 100°C, and the extraction time is 1.5 h.

[0021] Preferably, the Passiflora edulis extract is prepared by the following method: The dried Passiflora edulis is ground and passed through a 80-200 mesh sieve. The Passiflora edulis is then extracted with water. After the extraction is completed, the extract is separated. The extract is concentrated and dried to obtain the Passiflora edulis water extract. The Passiflora edulis extract is obtained by taking the Passiflora edulis water extract.

[0022] Preferably, the ratio of the dried Passiflora edulis to water is 1 kg:5-10 L.

[0023] Most preferably, the ratio of the dry Physalis pruinosa to water is 1kg:8L.

[0024] Preferably, the extraction refers to heating reflux extraction, the extraction temperature is 80-100℃, and the extraction time is 1-2h.

[0025] Most preferably, the extraction refers to heating reflux extraction, the extraction temperature is 100℃, and the extraction time is 1.5h.

[0026] Further preferably, the Physalis pruinosa water extract is subjected to a macroporous resin column; first, 3-6 times the column volume of 31-33% ethanol aqueous solution is used to elute and remove impurities; then, 3-6 times the column volume of 46-48% ethanol aqueous solution is used to elute, and the eluate eluted by the 46-48% ethanol aqueous solution is collected, concentrated and dried to obtain the Physalis pruinosa extract.

[0027] Most preferably, the Physalis pruinosa water extract is subjected to a macroporous resin column; first, 5 times the column volume of 32% ethanol aqueous solution is used to elute and remove impurities; then, 5 times the column volume of 47% ethanol aqueous solution is used to elute, and the eluate eluted by the 47% ethanol aqueous solution is collected, concentrated and dried to obtain the Physalis pruinosa extract.

[0028] Preferably, the macroporous resin column is a D101 type macroporous resin column.

[0029] The inventors have further found in research that, in the composition consisting of the Schisandra chinensis fruit extract and the aescin, further adding the Physalis pruinosa extract obtained by elution of the macroporous resin column according to the above method can further greatly improve the effect of improving the lymphatic endothelial cell activity compared with adding the Physalis pruinosa extract obtained by water extraction only.

[0030] In addition, the inventors have also found in research that the elution condition of the macroporous resin column is very critical; in the composition consisting of the Schisandra chinensis fruit extract and the aescin, it is necessary to further add the Physalis pruinosa extract obtained by elution of the macroporous resin column according to the elution condition of the macroporous resin column of the present application, and the effect of improving the lymphatic endothelial cell activity can be further greatly improved compared with adding the Physalis pruinosa extract obtained by water extraction only; however, the effect of improving the lymphatic endothelial cell activity cannot be further greatly improved by adding the Physalis pruinosa extract obtained by elution of other macroporous resin columns.

[0031] The present application also provides a use of the above-mentioned composition in the preparation of a product with enhanced mitochondrial function.

[0032] The application further provides application of the composition in preparation of a product with the function of promoting autophagy of cells.

[0033] The application further provides application of the composition in preparation of a product with the function of improving activity of lymphatic endothelial cells.

[0034] The inventors have further found in the mechanism research that the composition can also enhance mitochondrial function and promote autophagy of cells, and thus it can be inferred that the composition improves activity of lymphatic endothelial cells by enhancing mitochondrial function and promoting autophagy of cells.

[0035] Preferably, the product is a medicine or a cosmetic.

[0036] Beneficial effects: the application provides a brand-new composition; researches show that the composition has the function of improving activity of lymphatic endothelial cells; further researches show that the composition can also enhance mitochondrial function and promote autophagy of cells; and thus it can be inferred that the composition improves activity of lymphatic endothelial cells by enhancing mitochondrial function and promoting autophagy of cells.

[0037] Since the composition has the function of improving activity of lymphatic endothelial cells, it has important application value to be used as an active ingredient in preparation of a medicine or a cosmetic with the function of improving activity of lymphatic endothelial cells. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 Figure for the experiment result of the composition in enhancing mitochondrial function.

[0039] Figure 2 Figure for the experiment result of the composition in promoting autophagy of cells. DETAILED DESCRIPTION

[0040] The application will be further described in detail below in combination with specific examples, but the examples do not limit the application in any form.

[0041] Example 1: Preparation of the composition The composition is obtained by uniformly mixing Schisandra chinensis fruit extract and aescine at a weight ratio of 1:0.2. The Schisandra chinensis fruit extract is prepared by the following method: Dry Schisandra chinensis fruit is heated to 100℃ with water to reflux extract for 1.5 hours; after the extraction is completed, the extract is separated, and the extract is concentrated and dried to obtain the Schisandra chinensis fruit extract; wherein the weight ratio of dry Schisandra chinensis fruit to water is 1 kg:8 L.

[0042] Example 2 Preparation of the composition The Schisandra chinensis fruit extract, the Passiflora edulis extract and the aescin are mixed in a weight ratio of 1:1:0.2 to obtain the composition. The Schisandra chinensis fruit extract is prepared by the following method: The dried Schisandra chinensis fruit is heated to 100℃ with water to reflux extract for 1.5h. After the extraction is completed, the extract is separated. The extract is concentrated and dried to obtain the Schisandra chinensis fruit extract. The ratio of the dried Schisandra chinensis fruit to water is 1kg:8L.

[0043] The Passiflora edulis extract is prepared by the following method: The dried Passiflora edulis is crushed and passed through a 100 mesh sieve. Then the crushed Passiflora edulis is heated to 100℃ with water to reflux extract for 1.5h. After the extraction is completed, the extract is separated. The extract is concentrated and dried to obtain the Passiflora edulis water extract. The Passiflora edulis extract is obtained by taking the Passiflora edulis water extract. The ratio of the dried Passiflora edulis to water is 1kg:8L.

[0044] Example 3 Preparation of the composition The Schisandra chinensis fruit extract, the Passiflora edulis extract and the aescin are mixed in a weight ratio of 1:1:0.2 to obtain the composition. The Schisandra chinensis fruit extract is prepared by the following method: The dried Schisandra chinensis fruit is heated to 100℃ with water to reflux extract for 1.5h. After the extraction is completed, the extract is separated. The extract is concentrated and dried to obtain the Schisandra chinensis fruit extract. The ratio of the dried Schisandra chinensis fruit to water is 1kg:8L.

[0045] The Passiflora edulis extract is prepared by the following method: (1) The dried Passiflora edulis is crushed and passed through a 100 mesh sieve. Then the crushed Passiflora edulis is heated to 100℃ with water to reflux extract for 1.5h. After the extraction is completed, the extract is separated. The extract is concentrated and dried to obtain the Passiflora edulis water extract. The ratio of the dried Passiflora edulis to water is 1kg:8L; (2) The Passiflora edulis water extract is loaded onto a D101 macroporous resin column. First, 5 times the column volume of 32% ethanol aqueous solution is used to elute and remove impurities. Then, 5 times the column volume of 47% ethanol aqueous solution is used to elute. The eluate eluted by the 47% ethanol aqueous solution is collected, concentrated and dried to obtain the Passiflora edulis extract.

[0046] Comparative Example 1 Preparation of the composition The Schisandra chinensis fruit extract, the Passiflora edulis extract and the aescin are mixed in a weight ratio of 1:1:0.2 to obtain the composition. The Schisandra chinensis fruit extract is prepared by the following method: The dried Schisandra chinensis fruit is extracted by refluxing with water heated to 100℃ for 1.5h. After the extraction is completed, the extract is separated, and the extract is concentrated and dried to obtain the Schisandra chinensis fruit extract. The ratio of the dried Schisandra chinensis fruit to water is 1kg:8L.

[0047] The Passiflora edulis extract is prepared by the following method: (1) The dried Passiflora edulis is crushed and passed through a 100-mesh sieve, and then extracted by refluxing with water heated to 100℃ for 1.5h. After the extraction is completed, the extract is separated, and the extract is concentrated and dried to obtain the Passiflora edulis water extract. The ratio of the dried Passiflora edulis to water is 1kg:8L. (2) The Passiflora edulis water extract is loaded onto a D101 macroporous resin column. First, 5 column volumes of 10% ethanol aqueous solution are used to elute and remove impurities. Then, 5 column volumes of 25% ethanol aqueous solution are used to elute. The eluate eluted by the 25% ethanol aqueous solution is collected, concentrated and dried to obtain the Passiflora edulis extract.

[0048] Preparation of the composition of Comparative Example 2 The Schisandra chinensis fruit extract, the Passiflora edulis extract and the aescin are mixed in a ratio of 1:1:0.2 to obtain the composition. The Schisandra chinensis fruit extract is prepared by the following method: The dried Schisandra chinensis fruit is extracted by refluxing with water heated to 100℃ for 1.5h. After the extraction is completed, the extract is separated, and the extract is concentrated and dried to obtain the Schisandra chinensis fruit extract. The ratio of the dried Schisandra chinensis fruit to water is 1kg:8L.

[0049] The Passiflora edulis extract is prepared by the following method: (1) The dried Passiflora edulis is crushed and passed through a 100-mesh sieve, and then extracted by refluxing with water heated to 100℃ for 1.5h. After the extraction is completed, the extract is separated, and the extract is concentrated and dried to obtain the Passiflora edulis water extract. The ratio of the dried Passiflora edulis to water is 1kg:8L. (2) The Passiflora edulis water extract is loaded onto a D101 macroporous resin column. First, 5 column volumes of 45% ethanol aqueous solution are used to elute and remove impurities. Then, 5 column volumes of 60% ethanol aqueous solution are used to elute. The eluate eluted by the 60% ethanol aqueous solution is collected, concentrated and dried to obtain the Passiflora edulis extract.

[0050] Experimental Example 1: Cell viability experiment Collect human immortalized lymphatic endothelial cells (HLECs) in logarithmic growth phase, at a cell density of 1×10⁻⁶. 5 Cells were seeded per well in 24-well plates. The experimental group was incubated with culture medium containing 100 μg / mL of the test sample and cultured at 37℃ (5% CO2) for 48 h. The blank control group was incubated with culture medium without the test sample and cultured at 37℃ (5% CO2) for 48 h. Each group was divided into three replicates. After incubation, the supernatant was discarded, and 500 μL of culture medium containing 0.5 mg / mL MTT was added. The plates were incubated at 37℃ in the dark for 4 h. After incubation, the supernatant was discarded, and 300 µL of DMSO was added to each well. The OD value was read at 490 nm. Cell viability was calculated. The results are shown in Table 1.

[0051] The culture medium used in the above experiment was a special culture medium for the immortalization of human lymphatic endothelial cells; the specific types of the samples to be tested are shown in Table 1.

[0052] Cell viability = [OD of sample wells - OD of dead cells wells] / (OD of solvent control wells - OD of dead cells wells) * 100% Table 1. Results of cell viability assay Test sample Cell viability Blank control group - 100.000% Experimental group 1 Composition prepared in Example 1 112.321% Experimental group 2 Composition prepared in Example 2 123.415% Experimental group 3 Composition prepared in Example 3 168.653% Experimental group 4 Composition prepared in Comparative Example 1 129.782% Experimental group 5 Composition prepared in Comparative Example 2 134.135% As can be seen from the experimental results in Table 1, the cell viability of experimental group 1 was higher than that of the blank control group; this indicates that the combination of Schisandra chinensis fruit extract and aescin has the effect of improving the viability of lymphatic endothelial cells.

[0053] As can be seen from the experimental results in Table 1, the cell viability of experimental group 2 was higher than that of experimental group 1. This indicates that the addition of passion fruit extract to the composition of Schisandra chinensis fruit extract and Aescin further enhances the effect of improving the viability of lymphatic endothelial cells.

[0054] As can be seen from the experimental results in Table 1, the cell viability of experimental group 3 was significantly higher than that of experimental group 2. This indicates that the addition of passion fruit extract obtained by elution through the macroporous resin column of this invention to the composition composed of Schisandra chinensis fruit extract and Aescin further significantly enhances the effect of improving the viability of lymphatic endothelial cells compared to the addition of passion fruit extract obtained by water extraction alone.

[0055] As can be seen from the experimental results in Table 1, although the cell viability of experimental groups 4 and 5 was improved compared with that of experimental group 2, the improvement was not significant, and the improvement was much smaller than that of experimental group 3. This indicates that the macroporous resin column elution conditions of the present invention are crucial. In the composition composed of Schisandra chinensis fruit extract and Aesculin, it is necessary to further add passion fruit extract obtained by elution under the macroporous resin column elution conditions of the present invention. Compared with adding passion fruit extract obtained by water extraction alone, the effect of improving lymphatic endothelial cell viability can be further significantly improved. However, adding passion fruit extract obtained by elution under other macroporous resin column elution conditions does not result in a further significant improvement in the effect of improving lymphatic endothelial cell viability.

[0056] Example 2: Experiment to Enhance Mitochondrial Function Mitochondrial membrane potential (MMT) is a crucial indicator for evaluating normal mitochondrial function. It not only reflects the integrity of mitochondrial function but also significantly impacts ATP synthesis, ion transport, regulation of apoptosis, and antioxidant activity. Furthermore, it reflects cellular health and is vital for cell survival and development. Visualizing and quantifying MMT allows for the assessment of skin cell health and the evaluation of a sample's antioxidant capacity.

[0057] Collect human immortalized lymphatic endothelial cells (HLECs) in logarithmic growth phase, at a cell density of 1×10⁻⁶. 5 Inoculate 1 sample per well into a 24-well culture plate and incubate overnight in an incubator (37°C, 5% CO2); then, the experimental group, positive control group, and negative control group are incubated at 300 mJ / cm². 2 Expose to UVB light for 5 minutes; the blank control group received no UVB light. UVB irradiation; after UVB irradiation, the experimental group was added to culture medium containing 100 μg / mL of the test composition and cultured in an incubator (37℃, 5% CO2) for 24 h; the positive control group was added to culture medium containing 100 μg / mL VC and 7 μg / mL VE and cultured in an incubator (37℃, 5% CO2) for 24 h; the blank control group and negative control group were added to culture medium without the test sample and cultured in an incubator (37℃, 5% CO2) for 24 h; each group was set up with 3 replicates; after culture, the supernatant was discarded and the cells were washed 3 times with PBS. Then, Mito-Tracker Red CMXRos (mitochondrial red fluorescent probe) was used for staining; the mitochondrial membrane potential fluorescence staining results of each group are shown in Figure 1 below (red fluorescence represents membrane potential intensity).

[0058] The culture medium used in the above experiments was a special culture medium for the immortalization of human lymphatic endothelial cells; the test composition in the experimental group was the composition prepared in Example 3.Figure 1 The staining results show that the red fluorescence intensity of the experimental group is higher than that of the negative control group; this indicates that the composition described in this invention can also enhance mitochondrial function.

[0059] Example 3: Experiment to Promote Autophagy Autophagy is a crucial mechanism by which cells maintain homeostasis by degrading and recycling their own damaged or aging components (such as proteins and organelles). This process plays a key role in skin health, involving multiple aspects such as anti-aging, wound repair, inflammation regulation, and prevention and treatment of skin diseases. This study used human immortalized lymphatic endothelial cells (HLECs) and employed LC3B-II to evaluate the cells' autophagy capacity.

[0060] Immortalized lymphatic endothelial cells (HLECs) were obtained at a rate of 8 × 10⁻⁶. 4 Cells were seeded at a density of cells / well into 24-well plates and incubated overnight in an incubator (37℃, 5% CO2). The experimental group was then incubated with culture medium containing 100 μg / mL of the test composition for 24 h in an incubator (37℃, 5% CO2). The blank control group was incubated with culture medium without the test sample for 24 h in an incubator (37℃, 5% CO2). Each group had three replicates. After incubation, the supernatant was discarded, and the cells were washed three times with PBS before immunofluorescence staining. After staining, images were taken using a fluorescence microscope. The results are shown in the attached image. Figure 2 The red fluorescence represents LC3B-II.

[0061] The culture medium used in the above experiments was a special culture medium for the immortalization of human lymphatic endothelial cells; the test composition in the experimental group was the composition prepared in Example 3. Figure 2 The staining results showed that the red fluorescence in the blank control group was dim, while the red fluorescence in the experimental group was significantly enhanced, and the number of LC3B-II cells in the experimental group was significantly higher than that in the blank control group; this indicates that the composition described in this invention also has the effect of promoting autophagy.

Claims

1. A composition, characterized in that, It contains Schisandra chinensis fruit extract and aescin.

2. The composition according to claim 1, characterized in that, The weight ratio of Schisandra chinensis fruit extract to aescin is 1~3:0.1~0.

3.

3. The composition according to claim 1, characterized in that, The composition also contains passion fruit extract.

4. The composition according to claim 3, characterized in that, It contains Schisandra chinensis fruit extract, Passion fruit extract, and Aesculin.

5. The composition according to claim 4, characterized in that, The weight ratio of Schisandra chinensis fruit extract, Passion fruit extract, and aescin is 1~3:1~3:0.1~0.3; The most preferred embodiment is that the weight ratio of Schisandra chinensis fruit extract, Passion fruit extract and Aesculin is 1:1:0.

2.

6. The composition according to claim 1, characterized in that, The Schisandra chinensis fruit extract was prepared by the following method: Take dried Schisandra chinensis fruit, extract with water, separate the extract after extraction, concentrate and dry the extract to obtain the Schisandra chinensis fruit extract.

7. The composition according to claim 3, characterized in that, The passion fruit extract was prepared by the following method: Take dried passion fruit, crush it, and pass it through an 80-200 mesh sieve. Then extract it with water. After the extraction is completed, separate the extract, concentrate and dry the extract to obtain passion fruit water extract. Take passion fruit water extract to obtain the passion fruit extract.

8. Use of the composition according to any one of claims 1 to 7 in the preparation of a product having enhanced mitochondrial function.

9. Use of the composition according to any one of claims 1 to 7 in the preparation of a product having the function of promoting autophagy.

10. Use of the composition according to any one of claims 1 to 7 in the preparation of a product having the effect of improving the viability of lymphatic endothelial cells.

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