Application of andrographis paniculata water extract for promoting expression of cell growth factors

Abstract

According to the application of the andrographis paniculata water extract for promoting expression of the cell growth factors, a preparation method of the andrographis paniculata water extract comprises the following steps: taking andrographis paniculata, crushing, adding distilled water, soaking overnight, carrying out ultrasonication, filtering an extracting solution, carrying out rotary evaporation and concentration to prepare a concentrated solution, and carrying out vacuum freeze drying on the concentrated solution to prepare freeze-dried powder, namely the andrographis paniculata water extract for promoting expression of the cell growth factors. Dissolving the freeze-dried powder to prepare a common andrographis herb water extract mother solution; the andrographis paniculata water extract mother liquor is applied to the aspect of promoting expression of cell growth factors.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to an application of an Andrographis paniculata water extract for promoting the expression of cell growth factors. Background Art

[0002] Cell growth factors are a class of active proteins or polypeptides secreted by cells. They play key functions such as regulating cell division and tissue differentiation by binding with high affinity to specific cell membrane receptors. Cell growth factors can be divided into multiple categories, including platelet-derived growth factor (PDGF), osteosarcoma-derived growth factor (ODGF), epidermal growth factor (EGF), transforming growth factor (TGF-α and TGF-β), fibroblast growth factor (aFGF and bFGF), insulin-like growth factor (IGF-I and IGF-II), nerve growth factor (NGF), interleukin growth factors (such as IL-1, IL-2, IL-3, etc.) and erythropoietin (EPO).

[0003] A large number of studies have confirmed that cell growth factors have great application potential in the fields of medicine, beauty and biotechnology. In the medical field, cell growth factors can be used to promote wound healing, treat burns, fracture repair, and promote the recovery of healthy cells in certain cancer treatments. For example, epidermal growth factor (EGF) can accelerate skin and tissue repair; in the field of beauty, cell growth factors are used in anti-aging products to reduce wrinkles, improve skin texture and promote collagen production. For example, fibroblast growth factor (FGF) can promote fibroblast growth, improve the ability of the skin to synthesize collagen, and improve skin conditions; in the field of biotechnology, cell growth factors are key tools for stem cell research and tissue engineering. They can guide stem cell differentiation and tissue regeneration, providing new possibilities for regenerative medicine. With the deepening of research, the scope of application of cell growth factors has been continuously expanded, providing new solutions for the treatment of various diseases and improving the quality of human life. At present, many factors that promote cell growth can be produced by artificial synthesis or genetic engineering, but the cost of these methods is relatively high, resulting in expensive final products. In addition, if used improperly, growth factors may bring certain side effects, such as skin reactions, changes in skin quality, and abnormal tissue proliferation. Fortunately, current studies have shown that certain Chinese herbal ingredients can promote the secretion of cell growth factors, which provides new possibilities for reducing costs and reducing side effects.

[0004] Andrographis paniculata is the dried aerial part of the plant Andrographis paniculata of the Acanthaceae family. It has the medicinal effects of anti-inflammatory, detumescence, clearing away heat and detoxification. Modern scientific research has shown that Andrographis paniculata has anti-inflammatory, antibacterial, antioxidant and other efficacy activities.

[0005] However, there is no research result on the effect of Andrographis paniculata on promoting the expression of cell growth factors in the prior art. Summary of the invention

[0006] In view of the above problems, the present invention provides an application of Andrographis paniculata water extract for promoting the expression of cell growth factors. The effect of Andrographis paniculata water extract in promoting the expression of cell growth factors (such as KGF and FGF) has potential positive effects on clinical treatments and applications such as burns, tissue repair, and skin beauty.

[0007] In order to achieve the above invention object, the present invention adopts the following technical scheme:

[0008] An application of an Andrographis paniculata water extract for promoting the expression of cell growth factors, and an application of the Andrographis paniculata water extract in promoting the expression of cell growth factors.

[0009] Furthermore, the preparation method of the Andrographis paniculata water extract comprises the following steps: taking Andrographis paniculata, crushing it, adding distilled water, soaking it overnight, and ultrasonically crushing it; filtering the extract and then concentrating it by rotary evaporation to obtain a concentrated solution; vacuum freeze-drying the concentrated solution to obtain a lyophilized powder; and dissolving the lyophilized powder to prepare an Andrographis paniculata water extract mother solution; and using the Andrographis paniculata water extract mother solution in promoting the expression of cell growth factors.

[0010] Furthermore, in the preparation method of the Andrographis paniculata water extract, an appropriate amount of Andrographis paniculata is taken, crushed and then added with 10 times the amount of distilled water, soaked overnight at 4°C, ultrasonically crushed for 60 minutes, the extract is filtered and concentrated by rotary evaporation to obtain a concentrated solution; the concentrated solution is vacuum freeze-dried for 48 hours to obtain a lyophilized powder; the lyophilized powder is then added to PBS to dissolve and prepared into a mother solution of Andrographis paniculata water extract with a concentration of 100 mg / mL.

[0011] Furthermore, the water extract of Andrographis paniculata can upregulate the expression levels of cell growth factors such as TGF-β, VEGFA, KGF and bFGF in HaCaT cells in a concentration-dependent manner.

[0012] Furthermore, the water extract of Andrographis paniculata can upregulate the expression levels of matrix metalloproteinases such as MMP1, MMP3, and MMP9 in HaCaT cells in a concentration-dependent manner.

[0013] Furthermore, the water extract of Andrographis paniculata can significantly upregulate the phosphorylation levels of mTOR and ERK in a concentration-dependent manner.

[0014] Furthermore, the water extract of Andrographis paniculata can upregulate the expression levels of collagen COL1A1 and COL3A1 in HaCaT cells in a concentration-dependent manner.

[0015] Furthermore, the Andrographis paniculata aqueous extract can promote cell migration.

[0016] Furthermore, the water extract of Andrographis paniculata can upregulate the expression levels of cell growth factors FGF and KGF, and collagens COL1A1 and COL3A1 in the skin tissue near the wound, promoting skin wound repair and scab shedding.

[0017] Further, the Andrographis paniculata water extract is used in any one or more of the following:

[0018] 1) Application in the preparation of drugs for promoting the expression of cell growth factors;

[0019] 2) Application in the preparation of drugs for tissue repair;

[0020] 3) Application in the preparation of anti-aging beauty products;

[0021] 4) Application in the preparation of medicines for treating wounds.

[0022] By adopting the above technical scheme, the Chinese medicinal herb Andrographis paniculata water extract provided by the present invention can upregulate the expression of multiple cell growth factors and promote collagen synthesis at the cellular and tissue levels, and has a positive promoting effect on the clinical treatment and application of burns, tissue repair, skin beauty and other problems. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Effects of Andrographis paniculata aqueous extract on the expression of growth factors in HaCaT cells

[0024] Compared with the blank group, * p<0.05, ** p<0.01, *** p<0.001

[0025] Figure 2 Compared with the blank group, the effect of Andrographis paniculata water extract on the expression of matrix metalloproteinases in HaCaT cells * p<0.05, ** p<0.01, *** p<0.001

[0026] Figure 3 Compared with the blank group, the effect of Andrographis paniculata water extract on the phosphorylation levels of mTOR and ERK in HaCaT cells *** p<0.001

[0027] Figure 4 Effects of Andrographis paniculata aqueous extract on collagen expression in HaCaT cells

[0028] Compared with the blank group, * p<0.05, *** p<0.001

[0029] Figure 5 Effects of Andrographis paniculata aqueous extract on migration of HaCaT cells

[0030] Compared with the blank group, *** p<0.001

[0031] Figure 6 Effects of Andrographis paniculata water extract on skin wound repair in mice

[0032] Compared with the blank group, * p<0.05, ** p<0.01, *** p<0.001

[0033] Figure 7 Effect of the water extract of Andrographis paniculata on the expression of cell growth factors in the tissue near the skin wound of mice. Compared with the blank group, * p<0.05, *** p<0.001 DETAILED DESCRIPTION

[0034] The following is attached Figure 1-7 The present invention is further explained in the following specific embodiments, which only describe some implementation examples of the present invention and do not limit the content of the present invention. Other embodiments carried out by relevant researchers in this field without making any innovative contributions are within the scope of protection of the present invention.

[0035] Example 1 A method for preparing a water extract of Andrographis paniculata, a traditional Chinese medicine, for promoting the expression of cell growth factors.

[0036] An appropriate amount of Chinese herbal medicine Andrographis paniculata was crushed and added with 10 times the amount of distilled water, soaked at 4°C overnight, and ultrasonically crushed (Start 30s, Stop 60s, power 80%) for 60 min. The extract was filtered and concentrated by rotary evaporation (rotation speed 100 rpm, heating pot temperature 55°C) to obtain a concentrate; the concentrate was vacuum freeze-dried (freezing temperature -50°C, vacuum pressure 50Pa) for 48 hours to obtain a freeze-dried powder, which was kept at -20°C for use.

[0037] Example 2 Effect of the water extract of Andrographis paniculata on the expression of growth factors, etc. in HaCaT cells.

[0038] The Andrographis paniculata freeze-dried powder prepared in Example 1 was dissolved in PBS to prepare a mother solution of Andrographis paniculata water extract with a concentration of 100 mg / mL. The experiment was divided into a blank group and an Andrographis paniculata group. HaCaT fibroblasts in the logarithmic growth phase were taken at a rate of 1×10 6The amount of cells / well was inoculated into a 6-well plate and placed in a CO2 incubator (37°C, 5% CO2) for overnight culture. The Andrographis paniculata group was added with 100 mg / mL of Andrographis paniculata water extract mother solution to make the final concentrations of 10, 50 and 100 μg / mL respectively and continued to culture for 24 hours. After the cells were lysed and centrifuged, the total protein extract was obtained and the protein concentration was determined. Protein electrophoresis was performed using SDS-PAGE, and the protein was transferred to the PVDF membrane at a constant voltage of 100V for 1 hour. After the PVDF membrane was placed in TBST blocking solution containing 5% BSA at room temperature for 1 hour, different primary antibodies (1:3000) were added and incubated at 4°C overnight. After washing three times with TBST, the secondary antibody (1:5000) was added and incubated at room temperature for 1 hour. After washing three times with TBST, ECL luminescent solution was added, and the ChemiDoc XRS+ system was used for imaging to detect the expression levels of cell growth factors such as KGF and related proteins.

[0039] The experimental results are shown in Figure 1 In the figure, CON is the blank group. Compared with the blank group, the water extract of Andrographis paniculata can significantly upregulate the expression levels of cell growth factors such as TGF-β, VEGFA, KGF and bFGF in HaCaT cells in a concentration-dependent manner.

[0040] As a transforming growth factor, TGF-β can promote wound healing, shorten the healing time, and regulate the expression of other downstream growth factors. Vascular endothelial growth factor (VEGFA) has a significant promoting effect on angiogenesis. Keratinocyte growth factor (KGF) and fibroblast growth factor (FGF) can stimulate the metabolism of epithelial cells and promote the differentiation, proliferation and migration of epithelial cells.

[0041] The experimental results are shown in Figure 2 In the figure, CON is the blank group. Compared with the blank group, the water extract of Andrographis paniculata can significantly upregulate the expression levels of matrix metalloproteinases such as MMP1, MMP3, and MMP9 in HaCaT cells in a concentration-dependent manner.

[0042] Matrix metalloproteinases (MMPs) play a key role in the dynamic balance of the extracellular matrix and the complex process of wound healing. When growth factors are released, they can trigger the increased expression of MMPs, thereby participating in the regulation of extracellular matrix metabolism. The increased expression of MMP1 significantly enhances the migration ability of cells and accelerates the degradation of the cell matrix. MMP3, as a type of matrix lysin, can activate and release growth factors and cytokines stored in the extracellular matrix (ECM). This process helps to strengthen the connection between vascular endothelial cells and promote the increase of vascular permeability. MMP9 is mainly produced by epithelial cells and keratinocytes, and is also expressed in inflammatory cells, monocytes and macrophages, which helps to promote the inflammatory response and accelerate wound healing.

[0043] The experimental results are shown in Figure 3 , CON in the figure is the blank group. Compared with the blank group, the water extract of Andrographis paniculata can significantly upregulate the phosphorylation levels of mTOR and ERK in a concentration-dependent manner.

[0044] The mTOR / ERK signaling pathway has been reported to regulate angiogenesis and collagen synthesis. After phosphorylation, mTOR and ERK can promote the synthesis of growth factors, thereby further promoting the proliferation, migration and differentiation of endothelial cells, increasing the rate of angiogenesis and collagen deposition, and accelerating wound healing.

[0045] The experimental results are shown in Figure 4 Compared with the blank group, the water extract of Andrographis paniculata could significantly upregulate the expression levels of collagen COL1A1 and COL3A1 in HaCaT cells in a concentration-dependent manner.

[0046] Type I and III collagen (COL1A1 and COL3A1) are the main collagen components in skin tissue. When their content increases, they can promote skin wound repair, increase the elasticity and flexibility of new tissue, and avoid scar formation.

[0047] Example 3 Effect of Chinese herbal medicine Andrographis paniculata water extract on HaCaT cell migration

[0048] The Andrographis paniculata freeze-dried powder prepared in Example 1 was dissolved in PBS to prepare a mother solution of Andrographis paniculata water extract with a concentration of 100 mg / mL. The experiment was divided into a blank group and an Andrographis paniculata group. HaCaT cells in the logarithmic growth phase were taken at a rate of 5×10 5 The cells were inoculated into 12-well plates at the amount of 100 / well and placed in a CO2 incubator (37°C, 5% CO2) until the cell confluence was close to 100%. The cells were scratched vertically on the well plate using a 200 μL micropipette tip, washed once with PBS, and then the blank group was added with culture medium, and the Andrographis paniculata group was added with culture medium containing Andrographis paniculata water extract at final concentrations of 50 and 100 μg / mL, and the cells were observed and photographed under a microscope at 0 h, 24 h, 48 h, and 72 h, respectively.

[0049] The closer the distance between cells on both sides of the scratch, the higher the cell migration rate. Figure 5 In the figure, CON is the blank group. Compared with the blank group, the Andrographis paniculata water extract showed a significant effect of promoting cell migration at 24h, 48h, and 72h.

[0050] Example 4 Effect of the Chinese herbal medicine Andrographis paniculata water extract on the repair of mouse skin wounds.

[0051] The Andrographis paniculata freeze-dried powder prepared in Example 1 was dissolved in PBS to prepare an Andrographis paniculata water extract mother solution with a concentration of 100 mg / mL.

[0052] C57BL / 6 mice were adaptively fed for 3 days and randomly divided into a blank group and an Andrographis group. All mice were anesthetized by intraperitoneal injection (20% urethane), and the hair on the back of the mice was shaved. A 1cm×1cm full-thickness skin excision wound was made in the middle of the back spine of each mouse using a skin biopsy device and scissors. After modeling, the blank group was only treated with normal saline, and the Andrographis group mice were treated with low-concentration (50mg / kg / d) and high-concentration (200mg / kg / d) Andrographis water extract. Both groups were treated for 14 days (once / d). The wounds of each group were photographed on the day of surgery, 7 days and 14 days after surgery, and the wound healing rate was calculated to evaluate the wound repair.

[0053] The experimental results are shown in Figure 6 In the figure, CON is the blank group. Compared with the blank group, the water extract of Andrographis paniculata can significantly promote the repair of mouse skin wounds and scab shedding on the 7th and 14th days.

[0054] Example 5 Effect of the Chinese medicine Andrographis paniculata water extract on the expression of cell growth factors and other factors in the tissue near the skin wound of mice.

[0055] The skin tissue near the wound of mice on the 0th, 7th and 14th days in Example 4 was used, and the total protein extract was obtained after lysis and centrifugation, and the protein concentration was determined. Protein electrophoresis was performed using SDS-PAGE, and the protein was transferred to the PVDF membrane at a constant voltage of 100V for 1h. After the PVDF membrane was placed in a TBST blocking solution containing 5% BSA and blocked at room temperature for 1h, a primary antibody (1:3000) was added and incubated overnight at 4°C. After washing three times with TBST, a secondary antibody (1:5000) was added and incubated at room temperature for 1h. After washing three times with TBST, ECL luminescent liquid was added, and the ChemiDoc XRS+ system was used for imaging to detect the expression levels of proteins such as cell growth factors in the skin tissue near the wound.

[0056] The experimental results are shown in Figure 7 Compared with the blank group, low-concentration and high-concentration Andrographis paniculata water extracts could significantly upregulate the expression levels of cell growth factors EFG and KGF, and collagens COL1A1 and COL3A1 in the skin tissue near the wound surface at 7 days.

[0057] The above description shows and describes the preferred embodiments of the present invention. As mentioned above, it should be understood that the present invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the invention concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art do not depart from the spirit and scope of the present invention, and should be within the scope of protection of the claims attached to the present invention.

Claims

1. An application of an Andrographis paniculata water extract for promoting the expression of cell growth factors, characterized in that: Application of Andrographis paniculata water extract in promoting cell growth factor expression.

2. The use of an Andrographis paniculata water extract for promoting the expression of cell growth factors as described in claim 1, characterized in that: The preparation method of the Andrographis paniculata water extract comprises the following steps: taking Andrographis paniculata, crushing it, adding distilled water, soaking it overnight, and ultrasonically crushing it; filtering the extract and then concentrating it by rotary evaporation to obtain a concentrated solution; vacuum freeze-drying the concentrated solution to obtain a lyophilized powder; and dissolving the lyophilized powder to prepare a mother solution of the Andrographis paniculata water extract; and using the mother solution of the Andrographis paniculata water extract in promoting the expression of cell growth factors.

3. The use of an Andrographis paniculata water extract for promoting the expression of cell growth factors as described in claim 2, characterized in that: In the preparation method of the Andrographis paniculata water extract, an appropriate amount of Andrographis paniculata is taken, crushed, and then 10 times the amount of distilled water is added, soaked overnight at 4°C, ultrasonically crushed for 60 minutes, the extract is filtered and then concentrated by rotary evaporation to obtain a concentrated solution; the concentrated solution is vacuum freeze-dried for 48 hours to obtain a lyophilized powder; the lyophilized powder is then added to PBS to dissolve and prepared into a mother solution of Andrographis paniculata water extract with a concentration of 100 mg / mL.

4. The use of an Andrographis paniculata water extract for promoting the expression of cell growth factors as claimed in claim 1 or 2, characterized in that: Andrographis paniculata water extract can upregulate the expression levels of cell growth factors such as TGF-β, VEGFA, KGF and bFGF in HaCaT cells in a concentration-dependent manner.

5. The use of an Andrographis paniculata water extract for promoting the expression of cell growth factors as claimed in claim 1 or 2, characterized in that: Andrographis paniculata water extract can upregulate the expression levels of matrix metalloproteinases such as MMP1, MMP3, and MMP9 in HaCaT cells in a concentration-dependent manner.

6. The use of an Andrographis paniculata water extract for promoting the expression of cell growth factors as claimed in claim 1 or 2, characterized in that: Andrographis paniculata water extract can significantly upregulate the phosphorylation levels of mTOR and ERK in a concentration-dependent manner.

7. The use of an Andrographis paniculata water extract for promoting the expression of cell growth factors as claimed in claim 1 or 2, characterized in that: Andrographis paniculata water extract can upregulate the expression levels of collagen COL1A1 and COL3A1 in HaCaT cells in a concentration-dependent manner.

8. The use of an Andrographis paniculata water extract for promoting the expression of cell growth factors as claimed in claim 1 or 2, characterized in that: Andrographis paniculata aqueous extract can promote cell migration.

9. The use of an Andrographis paniculata water extract for promoting the expression of cell growth factors as claimed in claim 1 or 2, characterized in that: Andrographis paniculata water extract can upregulate the expression levels of cell growth factors FGF and KGF, and collagens COL1A1 and COL3A1 in the skin tissue near the wound, promoting skin wound repair and scab shedding.

10. The use of an Andrographis paniculata water extract for promoting the expression of cell growth factors as claimed in claim 1 or 2, characterized in that: Andrographis paniculata water extract is used in any one or more of the following: 1) Application in the preparation of drugs for promoting the expression of cell growth factors; 2) Application in the preparation of drugs for tissue repair; 3) Application in the preparation of anti-aging beauty products; 4) Application in the preparation of medicines for treating wounds.

Images