Veronica indica extract with anti-aging and wound scar formation inhibiting effects

Abstract

The invention discloses a waltheria indica extract with anti-aging and wound scar formation inhibiting effects. The waltheria indica extract is prepared by extracting waltheria indica plants through an enzymolysis-ultrasonic method. The waltheria indica extract prepared by the method disclosed by the invention has excellent anti-aging activity, and can be used for remarkably inhibiting light aging, oxidative stress and loss of collagen and elastin and reducing the activity of beta-galactosidase of cells; meanwhile, the waltheria indica extract can relieve inflammatory response of wounds, further inhibits excessive proliferation of fibroblasts and excessive deposition of collagen, and plays a role in inhibiting formation of scars on the wounds. The invention discovers that the waltheria indica extract has anti-aging and wound scar formation inhibiting effects for the first time, and the waltheria indica extract can be used as an anti-aging and wound scar formation inhibiting effective component to be applied to the fields of cosmetics, foods and medicines.

Description

Technical Field

[0001] The invention belongs to the technical field of plant extract application, and particularly relates to a coleus oleifera extract having the effects of resisting aging and inhibiting wound scar formation. Background Art

[0002] Aging is a process of cell and tissue structural damage and functional degeneration caused by many internal and external factors. Its occurrence mechanism is extremely complex and is accompanied by the occurrence of many related diseases. Therefore, how to delay aging and reduce the harm of aging-related diseases to human health has gradually become one of the hot research issues in recent years. Finding substances with anti-aging effects from natural plants is a hot topic in current research. Natural plants have attracted attention in the field of anti-aging research due to their own advantages such as wide sources and low toxicity and side effects.

[0003] Scars are the product of the human body's wound repair process. When the skin is damaged, the body will initiate a series of complex physiological processes to repair the wound, and scars may form during this process. Scar formation mainly includes the following three stages: (1) trauma and inflammatory response stage; (2) cell proliferation and tissue repair stage; (3) tissue remodeling and scar formation stage. If abnormal conditions occur at any stage, it may induce the formation of scar tissue. Among them, inflammation has a significant impact on scar formation. The wound surface can promote scar formation in the early stage of inflammation, and prolonged inflammation will further aggravate scar formation. Inflammation stimulates abnormal proliferation and migration of fibroblasts and continuous synthesis of a large amount of collagen, resulting in excessive deposition and disordered arrangement of collagen, promoting fibrosis of scar tissue, and causing scars. Scars not only affect appearance and bring psychological effects, but are also accompanied by symptoms such as itching and pain, affecting people's daily lives. At present, the main treatment for wound scars is drug therapy, but it has adverse reactions such as allergies, nausea, and vomiting. In recent years, finding effective substances from natural plants to inhibit wound scar formation has become a research hotspot. Natural plant active substances not only have a good effect in inhibiting scar formation on wounds, but are also highly safe.

[0004] Snake woman Waltheria indica L. It is a plant of the genus Snakewort of the family Sterculiaceae. Its aliases include Heta grass, Man carpet, and Cao Wutong. It is mainly distributed in Guangdong, Fujian, Hainan, Guangxi and other places in my country. It is a Chinese medicinal material with its roots and stems used as medicine. "Chinese Herbs" and "National Compilation of Chinese Herbal Medicines" record that Snakewort has the effects of anti-inflammatory, clearing away heat and detoxification, and removing dampness and expelling wind. At present, there are no research reports on the anti-aging and scar inhibition of Snakewort extracts. Summary of the invention

[0005] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for preparing a snakewort extract having anti-aging and wound scar formation inhibiting effects.

[0006] Another object of the present invention is to provide a Herba Coleopterae extract obtained by the above-mentioned preparation method, which has the effects of anti-aging and inhibiting wound scar formation.

[0007] Another object of the present invention is to provide the application of the above-mentioned snakewort extract having the effects of anti-aging and inhibiting wound scar formation.

[0008] The purpose of the present invention is achieved by the following technical scheme: a method for preparing a snakewort extract having anti-aging and wound scar formation inhibiting effects, comprising the following steps:

[0009] (1) Washing, drying, crushing and sieving the snakewort plants in sequence to obtain the snakewort raw material;

[0010] (2) The raw material of the snakewort obtained in step (1) is subjected to enzymatic hydrolysis and ultrasonic extraction in an extraction solvent, and then the extracted snakewort extract is centrifuged, filtered, and freeze-dried in sequence to obtain a snakewort extract with anti-aging and wound scar formation inhibition effects.

[0011] The snakewort raw material in step (1) is derived from the whole plant or any part of the snakewort plant.

[0012] The drying method described in step (1) is vacuum drying, and the drying conditions are a temperature of 40 to 60°C and a time of 2 to 6 hours; more preferably a temperature of 50°C and a time of 3 hours.

[0013] The mesh size of the sieve in step (1) is at least one of 40, 60, and 80, and more preferably 60 mesh.

[0014] The extraction solvent in step (2) is at least one of water, ethanol, methanol, n-butanol, ethyl acetate, petroleum ether, or a combination thereof; more preferably, it is an ethanol-water solution.

[0015] The enzyme in step (2) is at least one of cellulase, hemicellulase, pectinase, and protease, or a combination thereof; more preferably, cellulase and pectinase.

[0016] The enzymatic hydrolysis-ultrasonic extraction conditions described in step (2) are: extraction solvent 30-80% ethanol, liquid-to-solid ratio 10-40 mL / g, 2-8% cellulase, 1-4% pectinase, pH 4-5.5, ultrasonic time 15-30 min, ultrasonic temperature 40-60°C, and ultrasonic power 100-300 W; more preferably, the extraction solvent is 50% ethanol aqueous solution, liquid-to-solid ratio 20 mL / g, 6% cellulase, 3% pectinase, pH 4.6, ultrasonic time 20 min, ultrasonic temperature 50°C, and ultrasonic power 200 W.

[0017] The centrifugation conditions described in step (2) are 4°C and 10,000 rpm for 10 min.

[0018] The filtration method described in step (2) is a Buchner funnel.

[0019] The freeze-drying conditions described in step (2) are as follows: pre-freeze the snakewort extract at -80°C for 12 h, then quickly transfer it to a freeze dryer and freeze-dry it for 2 days.

[0020] A snakewort extract with the effects of anti-aging and inhibiting wound scar formation is prepared by the method.

[0021] The application of the snakewort extract with anti-aging and wound scar formation inhibition effects in the preparation of anti-aging or scar formation inhibition preparations.

[0022] The preparation can be used in the field of cosmetics or medicine.

[0023] Compared with the prior art, the present invention has the following advantages and effects:

[0024] (1) The present invention first discovered that the extract of Coleus chinensis has anti-aging and wound scar formation inhibiting effects. The present invention adopts enzymatic hydrolysis-ultrasound method to extract the Coleus chinensis extract with anti-aging and wound scar formation inhibiting effects. The extraction method is simple in process, high in efficiency, environmentally friendly, and suitable for industrial large-scale extraction.

[0025] (2) The snakewort extract prepared by the method of the present invention has good anti-aging activity, can significantly inhibit photoaging, oxidative stress, collagen and elastin loss, and reduce cell β-galactosidase activity; at the same time, the snakewort extract can reduce the inflammatory response of the wound surface, thereby inhibiting the excessive proliferation of fibroblasts and the excessive deposition of collagen, and playing a role in inhibiting the formation of scars on the wound surface. It can be used as an effective ingredient for anti-aging and inhibiting the formation of scars on the wound surface in the fields of beauty, cosmetics, and medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the result of the DPPH antioxidant test of the snakewort extract;

[0027] Figure 2 This is the experimental result diagram of the effect of snakewort extract on β-galactosidase in fibroblasts;

[0028] Figure 3 This is a graph showing the experimental results of the effect of snake plant extract on wound inflammation. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0030] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the present invention can be obtained from commercial sources.

[0031] Example 1

[0032] A method for preparing a snakewort extract comprises the following steps:

[0033] (1) Preparation of raw materials of snakewort: The collected whole plant samples of snakewort were washed twice with clean water, drained, and then placed in a vacuum drying oven at 50°C for 3 h. The dried snakewort was crushed in a grinder, and the crushed snakewort powder was passed through a 60-mesh sieve to obtain the raw materials of snakewort. The prepared raw materials of snakewort were stored in a refrigerator at 4°C.

[0034] (2) Preparation of the extract of snakewort: The prepared snakewort raw material was mixed with 50% ethanol aqueous solution at a liquid-to-solid ratio of 20:1 (mL / g), 6% cellulase and 3% pectinase were added, and the pH was adjusted to 4.6 for enzymatic hydrolysis for 30 min. After the enzymatic hydrolysis was completed, the extract was placed in an 85°C water bath for 5 min to inactivate the enzyme. Then, the extract was placed in an ultrasonic cleaner at an ultrasonic power of 200 W and a temperature of 50°C for 20 min. The ultrasonicated snakewort extract was placed in a 4°C refrigerator for 1 h, and then centrifuged at 10,000 rpm in a 4°C refrigerated centrifuge for 15 min. The supernatant was filtered with a Buchner funnel, concentrated by rotary evaporation, and freeze-dried to obtain the snakewort extract.

[0035] Comparative Example 1

[0036] This comparative example is to prepare the Herba Coleopterae extract under the condition of adjusting the ethanol concentration of Example 1. The operation is carried out according to Example 1, and the difference from Example 1 is that the 50% ethanol aqueous solution in Example 1 is replaced by distilled water.

[0037] Comparative Example 2

[0038] This comparative example is a method for preparing a Herba Coleopterae extract under the condition of adjusting the ethanol concentration of Example 1. The operation is carried out according to Example 1, but the difference from Example 1 is that the 50% ethanol aqueous solution in Example 1 is replaced with anhydrous ethanol.

[0039] Example 2

[0040] This example is a test of the in vitro DPPH antioxidant effect of the Herba Coleopterae extract obtained in Example 1, Comparative Example 1, and Comparative Example 2, and the steps are as follows:

[0041] DPPH free radical scavenging ability determination: 100 μL samples of different concentrations (5, 10, 20, 40, 80, 160, 320 μg / mL) were accurately pipetted into a 96-well plate, and 100 μL 0.2 mmol / L DPPH ethanol solution was added. The reaction was carried out at room temperature in the dark for 30 minutes, and the absorbance A of each sample solution was measured at a wavelength of 517 nm. 2 . 100 μL of anhydrous ethanol was used instead of DPPH ethanol solution to measure the sample background absorbance A 1 . 100 μL of distilled water was used instead of the sample solution to measure the absorbance of the blank solution. 0 Each concentration was repeated 3 times, and the average value was taken. Vc was used as a positive control and repeated 3 times. The calculation formula for DPPH free radical scavenging rate (%) is as follows:

[0042] DPPH free radical scavenging rate (%) = [1-(A 2 -A 1 ) / A 0 ]×100

[0043] The scavenging effect of snakewort extract on DPPH free radicals is as follows Figure 1 As shown, the experimental results show that as the sample concentration increases, the effect of DPPH free radical scavenging rate gradually increases. Among them, the DPPH free radical scavenging effect is Vc>Example 1>Comparative Example 1>Comparative Example 2. Compared with the control group Vc, both Example 1 and Comparative Example 1 reached DPPH antioxidant balance within 100 μg / mL, and the free radical scavenging rate when reaching DPPH antioxidant balance was not significantly different from that of the control group Vc, indicating that the snake lady extract has a good antioxidant effect.

[0044] Example 3

[0045] Since the in vitro antioxidant effect of the Coleus chinensis extract obtained in Example 1 is the best, this example is an in vivo zebrafish anti-photoaging experiment on the Coleus chinensis extract obtained in Example 1, and the steps are as follows:

[0046] AB wild-type zebrafish (purchased from Wuhan Zebrafish Resource Center, China) were used in the experiment. The zebrafish breeding method was carried out in accordance with DB32 / T 3979-2021 "Technical Conditions for Experimental Zebrafish Breeding". AB wild-type zebrafish fry 3 days after fertilization were selected and placed in a six-well plate, with 15 fish per well. The water in the 6-well plate was removed without harming the fry, and then 3 mL of snake mother sample solution (100 μg / mL) was quickly added to each well. At the same time, 3 mL of distilled water was added to the blank group and model group, and incubated at 28 ℃ in the dark for 4 h, followed by ultraviolet irradiation for 20 min (except the blank group). After irradiation, the zebrafish were weighed and homogenized with PBS, and the supernatant was collected by centrifugation. The fish collagen II (HCBⅡ) ELISA kit and fish elastin (ELN) ELISA kit were used for reaction (Gilead Biotechnology) to determine the content of collagen and elastin.

[0047] The experimental results are shown in Table 1. As can be seen from the table, compared with the control group, the content of collagen and elastin in the model group irradiated by ultraviolet light decreased, indicating that the model was successfully established. Compared with the model group, the content of collagen and elastin in the sample group increased, indicating that the extract of Coptis chinensis has a good anti-photoaging effect.

[0048] Table 1 Anti-aging effect of Coptis chinensis extract

[0049] Group Collagen (%) Elastin (%) Blank group 0.45±0.03 0.23±0.06 Model Group 0.12±0.02 0.06±0.01 Snake Lady Group 0.36±0.06 0.17±0.04

[0050] Example 4

[0051] This example is an in vivo zebrafish antioxidant stress experiment on the snakewort extract obtained in Example 1, and the steps are as follows:

[0052] The experiment used AB wild-type zebrafish (purchased from Wuhan Zebrafish Resource Center, China), and the zebrafish breeding method was carried out in accordance with DB32 / T 3979-2021 "Technical Conditions for Experimental Zebrafish Breeding". AB wild-type zebrafish larvae 3 days after fertilization were selected and placed in a six-well plate, with 15 fish per well. The water in the 6-well plate was removed without harming the larvae, and then 3 mL of snake mother sample solution (100 μg / mL) was quickly added to each well. At the same time, 3 mL of distilled water was added to the blank group and the model group, and the cells were incubated at 28 °C in the dark for 4 h, followed by the addition of 10 μM H 2 O 2The solution was incubated at 28 °C in the dark for 30 min (except for the blank group). After the incubation, the zebrafish homogenate supernatant was taken and reacted with a glutathione peroxidase detection kit (Biyuntian), a superoxide dismutase (SOD) detection kit (Merck), and a malondialdehyde (MDA) content detection kit (Solebo) to determine the contents of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and malondialdehyde (MDA).

[0053] The experimental results are shown in Table 2. As shown in the table, compared with the control group, 2 O 2 The GSH-Px and SOD contents in the model group decreased, and the MDA content increased, indicating that the model was successfully established. Compared with the model group, the GSH-Px and SOD contents in the sample group increased, and the MDA content decreased, and there was a certain dose effect, indicating that the extract of Coleus chinensis has a good anti-oxidative stress effect.

[0054] Table 2 Antioxidant stress effect of the extract of Coptis chinensis.

[0055] Group GSH-Px (ng / mL) SOD(ng / mL) MDA (nmol / mL) Blank group 11.32±1.16 5.33±0.46 1.32±0.12 Model Group 3.65±0.98 1.04±0.21 3.33±0.77 Snake Lady Group 7.34±1.06 3.36±0.36 2.12±0.34

[0056] Example 5

[0057] This example is an experiment on the effect of the Herba Corydalis extract obtained in Example 1 on β-galactosidase in fibroblasts, and the steps are as follows:

[0058] β-galactosidase is a marker related to cell senescence. The degree of cell senescence can be evaluated by staining β-gal with a β-galactosidase staining kit (Biyuntian). In this example, β-Gal is used as a marker of senescent cells and is stained with a chemical reagent X-Gal to make it blue-green.

[0059] Human embryonic lung fibroblasts (MRC-5, purchased from the cell bank of the Chinese Academy of Sciences) were cultured in MEM medium (containing 10% FBS, 1% sodium pyruvate, 1% non-essential amino acids, and 1% double antibody). After culture to the 42nd generation (P42), the degree of cell senescence was detected by preliminary experiments. The experiment could be carried out when the cell senescence rate was 60-80%. The blank control group, the snake lady extract group (100 μM) and the metformin group (100 μM, positive control group) were set up in the experiment. The drugs were co-incubated with the cells for 3 days, and then the cells were stained with β-galactosidase staining kit (Biyuntian) (marking senescent cells, blue-green), and DAPI (Sigma) was used to stain the cell nucleus (marking all cells, the cell nucleus was blue), and the number of senescent cells and the total number of cells were counted to calculate the cell senescence rate.

[0060] Cell senescence rate (%) = (number of senescent cells / total number of cells) × 100

[0061] The experimental results are shown in Figure 2 As shown in the table, compared with the blank control group, the cell senescence rate of the group treated with the snakewort extract was significantly reduced, and the ability to reduce the cell senescence rate was equivalent to that of the positive group, indicating that the snakewort extract has a good anti-aging effect.

[0062] Example 6

[0063] This example is an experiment on the effect of the Herba Coleopterae extract obtained in Example 1 on wound inflammation and scar formation, and the steps are as follows:

[0064] Forty 8-week-old mice (purchased from Guangdong Medical Experimental Animal Center) were randomly divided into 4 groups, 10 mice in each group, namely, blank group, wound group, topical application group of Coleoptera extract (100 μg / mL), and oral group of Coleoptera extract (50 mg / kg·BW). Except for the blank group, a wound with a diameter of 0.5 cm was made on the ventral side of the ear of each mouse using a puncher in the other groups. The topical application group of Coleoptera extract was 12 h / time, 4 times / month, and the oral group of Coleoptera extract was once every 2 days / time, with a treatment cycle of two months. At the same time, samples were collected 4 days after injury (peak period of inflammatory response), and tissue fluid around the wound was sampled by puncture, centrifuged, and the supernatant was taken. The level of inflammatory factor IL-6 was detected using a mouse IL-6 ELISA kit (Sigma).

[0065] The experimental results are shown in Figure 3 , Table 3. As can be seen from the table, compared with the wound group, the level of inflammatory factors in the group treated with the snakewort extract was significantly reduced, and the wound healing was good, indicating that the snakewort extract can inhibit the inflammatory response of the wound, promote wound healing, and inhibit scar formation.

[0066] Table 3 Comparison of scar formation effects

[0067] Group Wound condition Blank group Skin intact Wound group The color is reddish, the wound surface is irregular in shape, and the texture is hard Snakewort external application group The color is light, the texture is soft, and the wound heals well. Snakewort oral group The color is light, the texture is soft, and the wound heals well.

[0068] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a Herba Codonopsis pilosulae extract having anti-aging and wound scar formation inhibiting effects, characterized in that The steps include: (1) Washing, drying, crushing and sieving the snakewort plants in sequence to obtain the snakewort raw material; (2) The raw material of the snakewort obtained in step (1) is subjected to enzymatic hydrolysis and ultrasonic extraction in an extraction solvent, and then the extracted snakewort extract is centrifuged, filtered, and freeze-dried in sequence to obtain a snakewort extract with anti-aging and wound scar formation inhibition effects.

2. The preparation method according to claim 1, characterized in that: The snakewort raw material in step (1) is derived from the whole plant or any part of the snakewort plant.

3. The preparation method according to claim 1, characterized in that: The drying method described in step (1) is vacuum drying, and the drying conditions are a temperature of 40 to 60°C and a time of 2 to 6 hours; The mesh number of the mesh sieve in step (1) is at least one of 40, 60, and 80.

4. The preparation method according to claim 1, characterized in that: The extraction solvent in step (2) is at least one of water, ethanol, methanol, n-butanol, ethyl acetate, petroleum ether, or a combination thereof; The enzyme described in step (2) is at least one of cellulase, hemicellulase, pectinase, protease or a combination thereof.

5. The preparation method according to claim 1, characterized in that: The enzymatic hydrolysis-ultrasonic extraction conditions described in step (2) are: extraction solvent 30-80% ethanol, liquid-to-solid ratio 10-40 mL / g, 2-8% cellulase, 1-4% pectinase, pH 4-5.5, ultrasonic time 15-30 min, ultrasonic temperature 40-60 °C, and ultrasonic power 100-300 W.

6. The preparation method according to claim 1, characterized in that: The centrifugation conditions described in step (2) are 4°C, 10,000 rpm for 10 min; The filtration method described in step (2) is a Buchner funnel.

7. The preparation method according to claim 1, characterized in that: The freeze-drying conditions described in step (2) are as follows: pre-freeze the snakewort extract at -80°C for 12 h, then quickly transfer it to a freeze dryer and freeze-dry it for 2 days.

8. A Herba Coleopterae extract having anti-aging and wound scar formation inhibiting effects, characterized in that: It is prepared by the method according to any one of claims 1 to 7.

9. Use of the Herba Coleopterae extract having anti-aging and wound scar formation inhibiting effects as claimed in claim 8 in the preparation of anti-aging or scar formation inhibiting preparations.

10. The use of the extract of Coptis chinensis with anti-aging and wound scar formation inhibition effects according to claim 9, characterized in that: Used for preparing cosmetics or medicines for anti-aging or inhibiting scar formation.

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