TWI706786B - Use of pandanus conoideus fruit extracts for suppressing gene expression in retina cells - Google Patents

Abstract

The present invention provides use of a Pandanus conoideus fruit extract for manufacture of pharmaceutical composition for inhibiting expression of genes including VEGFA, CASP3, CASP8, or combinations thereof. The Pandanus conoideus fruit extract is obtained by heating the fruit of Pandanus conoideus to remove water contained therein. Said extract reduces retinal cell impairment by inhibiting the expression of genes involved in angiogenesis, apoptosis, and inflammatory response in retinal cells, thereby increasing the resistance of retinal cells to ultraviolet irradiation and reducing the incidence of ocular diseases.

Description

Use of Papua Red Fruit Extract to inhibit gene expression in retinal cells

The present invention relates to the use of a plant extract for vision care, in particular to the use of a Papua red fruit extract for preparing a pharmaceutical composition for inhibiting gene expression in retinal cells.

The eye is an organ of vision, responsible for receiving light from the environment and producing vision. In the process of vision formation, light first passes through the transparent cornea and lens of the eye. With the light-gathering function of these two structures, the light will focus on the retina to form an image, and stimulate the photoreceptor cells in the retina to convert light stimulation into electrical signals Signal, through the optic nerve conduction, the electrical signal reaches the brain's visual area, where the nerve cell's signal processing forms vision.

Although the eyes receive light as its normal function, sunlight includes not only visible light, but also ultraviolet light with a shorter wavelength and greater energy, which can be divided into ultraviolet light A (UVA; wavelength about 315-400nm) and ultraviolet light according to the wavelength. B (UVB; wavelength is about 280-315nm). Long-term exposure to this type of short-wavelength light is likely to cause damage to the retina. On the one hand, the damage is caused by the high energy of the light itself, which destroys the structure of the molecules in the cell, and on the other hand, because the retinal cells undergo oxidation during operation. When generating energy, it is easy to produce many reactive oxygen species (ROS) due to the irradiation of short-wavelength light, including superoxide radicals, hydroxyl radicals, hydrogen peroxide, and peroxides, which cause damage to cells. Oxidative damage eventually leads to retinal related diseases, such as macular degeneration, and even blindness in severe cases.

In order to reduce the damage of ultraviolet light to the retina, common protection methods include holding a parasol, or wearing a wide-brimmed hat or sunglasses. In addition, individuals can still achieve vision by taking foods that are beneficial to the retina. The purpose of health care. Therefore, it is necessary to develop a composition that can enhance the resistance of retinal cells to ultraviolet light.

For this reason , one object of the present invention is to provide a use of Pandanus conoideus extract for the preparation of a pharmaceutical composition for inhibiting gene expression in retinal cells, wherein the Papua red fruit extract is heated by heating a Papua red fruit to remove its The water content is obtained, and the gene expression line includes vascular endothelial growth factor A (vascular endothelial growth factor A, VEGFA), cysteine protease-3 (caspase-3, CASP3), cysteine protease-8 ( caspase-8, CASP8), or a combination of gene expression.

In an embodiment of the present invention, the gene expression further comprises genes of interleukin-1β (IL-1β), interleukin-8 (IL-8), or any combination thereof which performed.

In an embodiment of the present invention, the heating step is performed at 50°C to 100°C.

In an embodiment of the present invention, the concentration of the Papua red fruit extract ranges from 0.25 to 10 mg/mL.

In one embodiment of the present invention, the Papua red fruit extract reduces retinal damage caused by ultraviolet light, such as the death of a retinal pigment epithelial cell or cell membrane damage.

The present invention discloses that Papua red fruit extract can improve the resistance of retinal cells to ultraviolet light. By including but not limited to inhibiting gene expression related to angiogenesis, apoptosis, and inflammation, it can reduce retinal cell damage and prevent eye diseases. The effect of this, therefore helps vision care. Therefore, the Papua red fruit extract of the present invention can be used to prepare a pharmaceutical composition for reducing retinal damage caused by ultraviolet light. The pharmaceutical composition can be powder, granule, solution, colloid or paste, which can be administered to a body by oral or topical application.

The following will further illustrate the implementation of the present invention in conjunction with the drawings. The following examples are used to illustrate the inventive features and applications of the present invention, rather than limiting the scope of the present invention. Anyone familiar with the art will not depart from Within the spirit and scope of the present invention, some changes and modifications can be made. Therefore, the protection scope of the present invention shall be subject to those defined by the appended patent scope.

Figure 1 shows the cell survival rate of retinal pigment epithelial cells pre-treated with or without papua red fruit extract under ultraviolet light irradiation.

Figure 2A shows a bright-field photomicrograph (magnification 100X) of retinal pigment epithelial cells pre-treated with Papua red fruit extract under ultraviolet light.

Figure 2B shows the fluorescence micrograph of retinal pigment epithelial cells pretreated with or without Papua red fruit extract under ultraviolet light (magnification 100X).

Figure 3 shows the relative expression of VEGFA gene in retinal pigment epithelial cells pre-treated with or without Papua red fruit extract under ultraviolet light; cells in the control group were not exposed to ultraviolet light.

Figure 4 shows the relative expression levels of CASP3 and CASP8 genes in retinal pigment epithelial cells pre-treated with or without Papua red fruit extract under ultraviolet light; cells in the control group are not exposed to ultraviolet light.

Figure 5 shows the relative expression levels of IL-1β and IL-8 genes in retinal pigment epithelial cells pre-treated with or without Papua red fruit extract under ultraviolet light; cells in the control group are not exposed to ultraviolet light.

The present invention provides a use of Papua red fruit extract for the preparation of a pharmaceutical composition for inhibiting gene expression in retinal cells. The gene expression system includes gene expression of VEGFA , CASP3 , CASP8 , or a combination thereof, and may further include IL-1β, Gene expression of IL-8 , or any combination thereof. The Papua red fruit extract of the present invention is obtained by heating a Papua red fruit to remove the water content, and the heating step is performed at 50°C to 100°C. The following examples reveal that the Papua red fruit extract at a concentration of 0.25 to 10 mg/mL significantly inhibits the increase in the expression of angiogenesis, apoptosis, and inflammation-related genes caused by ultraviolet light irradiation, and reduces the death of retinal pigment epithelial cells and cell membranes Damage to the retina caused by ultraviolet light.

definition

The numerical values used herein are approximate values, and all experimental data are expressed in the range of 20%, preferably in the range of 10%, and most preferably in the range of 5%.

The "retina damage caused by ultraviolet light" mentioned herein includes death, apoptosis, cell membrane damage, and other damages that impair physiological functions. These injuries may involve, but are not limited to, ocular angiogenesis and inflammation.

Materials and Methods material

Gibco Eagle's minimum essential medium (MEM) containing Earle's balanced salt solution (Gibco Earle's balanced salt solution, EBSS), fetal bovine serum (FBS), sodium bicarbonate (sodium bicarbonate), sodium pyruvate (sodium pyruvate) and phosphate buffered saline (PBS) were purchased from Thermo Fisher Scientific. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT) was purchased from AMERSCO. Dimethyl sulfoxide (DMSO) was purchased from Echo Chemical Company.

Cell culture

The following examples used the human retinal pigment epithelial cell line ARPE-19 (ATCC CRL-2302) purchased from the American Type Culture Collection (ATCC). The cell line was cultured in a MEM medium supplemented with 0.1 mM non-essential amino acid, 1.5 g/L sodium bicarbonate, 1 mM sodium pyruvate, and 10% FBS under the conditions of 37° C. and 5% carbon dioxide, which is hereinafter referred to as cell culture medium.

MTT analysis

(formazan)結晶。最終，使用ELISA讀盤機(enzyme-linked immunosorbent assay reader；BioTek)測量該細胞混合物在570nm的吸光值(O.D.570)。細胞存活率依下列公式計算：細胞存活率=各組的O.D.570/控制組的O.D.570×100%。統計上顯著差異係以Excel軟體之學生t檢定決定。 The cell survival rate was determined by MTT analysis. In short, 15 μL/well of MTT solution (4 mg/mL MTT dissolved in PBS solution) was added to the designated treated cells in a 96-well plate, and reacted at 37° C. for 4 hours. After removing the reaction solution, add DMSO 50μL/well to the cells and shake the reaction for 10 minutes to dissolve the formazan

(formazan) crystallization. Finally, an ELISA disk reader (enzyme-linked immunosorbent assay reader; BioTek) was used to measure the absorbance (OD570) of the cell mixture at 570 nm. The cell survival rate is calculated according to the following formula: cell survival rate = OD570 of each group/OD570 of the control group×100%. The statistically significant difference is determined by the student t test of Excel software.

Cell damage test

Fluorescently labeled annexin V/pyridine iodide is used to detect cells with damaged cell membranes. In short, wash the designated cells in a 24-well plate with PBS solution, and then mix 10 μL/well of Alexa Fluor 488-connexin V conjugate (Invitrogen) with 200 μL/well of connexin V binding buffer (Invitrogen) Add the 24-well plate and react for 20 minutes in the dark at room temperature to stain the damaged cell membrane. its After that, the cells were washed with PBS solution, and the nuclei were stained with pyridine iodide staining solution (purchased from BD pharmingen) at room temperature for 10 minutes. After staining, the cells are washed with PBS solution to observe the green fluorescence distribution with a ZEISS fluorescence microscope. The more green fluorescence, the more cells with damaged cell membrane.

Gene expression analysis

Based on quantitative polymerase chain reaction (qPCR) to determine the expression level of genes related to angiogenesis, apoptosis, and inflammation in cells, the steps are briefly described as follows. According to the manufacturer’s instructions, RNA extraction kit (Geneaid) was used to isolate RNA from cells, and then 2000ng of RNA was reverse-transcribed into cDNA with SuperScript® III Reverse Transcriptase (Invitrogen) at 37°C. Afterwards, using the primer pair of the target gene and the Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as an internal control (Table 1), using the qPCR kit (KAPA CYBR FAST qPCR Kit (2X) ); KAPA Biosystems) performs qPCR on the aforementioned cDNA in a PCR reaction machine (Step One Plus Real-Time PCR system; Applied Biosystems) to obtain a melting curve.

Finally, the 2- ^△△CT method was used to determine the relative expression of the target gene. The method is the threshold cycle (C _T) GAPDH gene as an internal control of the reference cycle threshold gene, calculated by the following formula relative fold change: C gene of △ C _T = the experimental group or the control group _T - internal control C _T

△△ C _T = test group △ C _T - △ C _T of the control group

Multiple change=2 ^{-△△Ct average}

The statistical analysis department uses the STDEV function in Excel software to calculate the standard deviation of the relative expression of each gene, and uses the one-tailed Student's t test (TTEST) to calculate the statistical difference.

Example 1 Preparation of Papua Red Fruit Extract

Papua red fruit ( Pandanus conoideus ) is a shrub of the genus Pandanus ( Pandanus ), mainly distributed in the tropics and subtropics. The red papua referred to herein refers to the fruit of the plant, which is cylindrical and red, with a length of about 50 to 100 cm, and an average cross-sectional diameter of about 15 to 20 cm.

In order to obtain the Papua red fruit extract, first, the Papua red fruit is washed and cut into blocks or slices, and then sterilized at 95°C for a period of time, for example, about 30 minutes to 1 hour. Afterwards, remove the seeds from the massive papua red fruit and use a grinder to coarsely crush them for extraction. During the extraction process, the crude red papua fruit is continuously heated to remove the moisture contained in the crude crush. The heating temperature is between 50°C and 100°C, preferably between 60°C and 80°C, and the heating time is between 6 and 10°C. hour. After removing the moisture, the crude red papua fruit is cooled to room temperature, and can be further filtered with a 400 mesh (mesh) filter to remove residual solids to obtain the red papua extract.

Example 2 Papua red fruit extract reduces retinal cell death caused by ultraviolet light

In order to test the effect of Papua red fruit extract in protecting the retina from ultraviolet light, cell survival analysis (MTT analysis) was used to evaluate the human retinal pigment epithelial cell line ARPE-19 after being treated with Papua red fruit extract described in Example 1. Cell survival rate under B irradiation. In brief, 5×10 ³ cells/well of ARPE-19 cells were seeded on a 96-well culture plate containing 200 μL of cell culture medium in each well. After culturing the cells at 37°C for 24 hours, the medium was removed, and the cells in each well were treated in the following manner: (a) culture in 200 μL cell culture medium for 24 hours (control group); (b) culture in 200 μL cell culture medium for 24 hours, Then place it in a UV light box (Vilber) and receive 12J/cm ² UV light B for 1 hour (UV light group), this radiation dose will cause half of the cells to die; or (c) when it contains 0.25mg/mL Papua Red Fruit Extract Cultured in 200μL of cell culture medium for 24 hours, then received 12J/cm ² ultraviolet light B for 1 hour (UV light + Papua red fruit extract group). After that, MTT analysis was performed on each group of cells (four repeated experiments) and the cell survival rate was calculated.

Figure 1 shows the survival rate of retinal pigment epithelial cells in the aforementioned groups; in the figure ** and *** indicate that compared with the ultraviolet light group, p <0.01 and p <0.001, respectively. According to Figure 1, the ultraviolet light group has a significantly lower cell survival rate (a decrease of about 21%) than the control group, indicating that ultraviolet light B irradiation can cause a large number of deaths of retinal pigment epithelial cells. However, the cells treated with Papua red fruit extract and then irradiated with ultraviolet light had similar survival rates as the unirradiated control cells. This result shows that Papua red fruit extract has a protective effect on retinal cells, thus avoiding a lot of cell death caused by ultraviolet light.

Example 3 Papua red fruit extract reduces cell membrane damage of retinal cells caused by ultraviolet light

In order to investigate whether the red papua extract affects the damage of ultraviolet light to the cell membrane of retinal cells, this example uses fluorescence staining to observe the human retinal pigment epithelial cell line ARPE-19 after being treated with the red papua extract described in Example 1. Cell membrane integrity under UV light B irradiation. In brief, 5×10 ⁴ cells/well of ARPE-19 cells were seeded on a 24-well culture plate containing 500 μL of cell culture medium in each well. After culturing the cells at 37°C for 24 hours, remove the medium and treat the cells in each well in the following manner: (a) culture in 500 μL cell culture medium for 24 hours (control group); (b) culture in 500 μL cell culture medium for 24 hours, Then place it in a UV light box and receive 12J/cm ² UV light B for 1 hour (UV light group); or (c) Cultivate in 500μL cell culture medium containing 0.25mg/mL Papua red fruit extract for 24 hours, then receive 12J/cm ² ultraviolet light B irradiation for 1 hour (ultraviolet light + Papua red fruit extract group). Thereafter, the cell damage test was performed on each group of cells (four repeated tests) to observe the degree of cell membrane damage.

Figures 2A and 2B respectively show the micrographs and fluorescence micrographs of the aforementioned groups of retinal pigment epithelial cells. According to Figure 2B, the ultraviolet light group has significantly more green fluorescent area than the control group, indicating that ultraviolet light B irradiation can cause cell membrane damage of retinal pigment epithelial cells, but pre-administration of 0.25mg/mL Papua red fruit extract can reduce The damage degree of this cell is similar to that of the control group. This result indicates that Papua red fruit extract can protect retinal cells from cell membrane damage caused by ultraviolet light, so it is beneficial for vision maintenance.

Example 4 Papua red fruit extract inhibits the expression of angiogenesis, apoptosis, and inflammation related genes in retinal cells under ultraviolet light irradiation

In order to investigate whether the Papua red fruit extract affects the gene expression in retinal cells under ultraviolet light irradiation, this example uses qPCR to determine the human retinal pigment epithelial cell line ARPE-19, which is treated with the Papua red fruit extract described in Example 1, and then treated with ultraviolet light B After irradiation, the expression level of genes related to angiogenesis, cell apoptosis, and inflammation response changes. In brief, 5×10 ⁴ cells/well of ARPE-19 cells were seeded on a 24-well culture plate containing 500 μL of cell culture medium in each well. After culturing the cells at 37°C for 24 hours, the medium was removed and the cells in each well were treated in the following manner: (a) cultured in 500 μL cell culture medium for 48 hours (control group); (b) cultured in 500 μL cell culture medium for 48 hours, Then place it in an ultraviolet light box and receive 12J/cm ² ultraviolet light B for 1 hour (ultraviolet light group); or (c) culture in 500 μL cell culture medium containing 0.25 mg/mL Papua red fruit extract for 48 hours, then accept 12J/cm ² ultraviolet light B irradiation for 1 hour (ultraviolet light + Papua red fruit extract group). Thereafter, each group of cells (four-repeat test) was used for gene expression analysis to determine the relative expression level, which is a multiple of the specific gene expression level of the control group of cells.

4.1 Inhibition of VEGFA gene expression

Figure 3 shows the relative expression levels of vascular endothelial growth factor A ( VEGFA ) genes related to angiogenesis in the aforementioned groups of retinal pigment epithelial cells; ** in the figure indicates p <0.01 compared with the ultraviolet light group. According to Figure 3, the ultraviolet light group has more VEGFA gene expression than the control group, suggesting that the retinal pigment epithelial cells irradiated by ultraviolet light B may promote peripheral angiogenesis, but pre-administration of 0.25 mg/mL Papua red fruit extract is significant Inhibition of this VEGFA gene expression increases. In view of the previous studies that the abnormal proliferation of blood vessels in the eye is related to the progression of age-related macular degeneration, diabetic retinopathy and other ocular diseases, it may cause retinal pigment epithelial cell detachment, subretinal hemorrhage, and macular edema, which may affect vision. It shows that Papua red fruit extract may reduce the chance of ocular angiogenesis by inhibiting the increase of VEGFA gene expression in retinal cells caused by ultraviolet light, so it can be used to prevent vision damage due to ultraviolet light.

4.2 Inhibition of CASP3 and CASP8 gene expression

Figure 4 shows the relative expression levels of cysteine protease-3 ( CASP3 ) and cysteine protease-8 ( CASP8 ) genes involved in apoptosis in the aforementioned groups of retinal pigment epithelial cells; ** in the figure indicates the relative expression level of Compared with the light group, p <0.01. According to Figure 4, the ultraviolet light group had slightly more CASP8 gene expression than the control group, suggesting that ultraviolet light B irradiation may promote the apoptosis of retinal pigment epithelial cells, but the pre-administration of 0.25 mg/mL Papua red fruit extract inhibits this CASP8 gene Increased performance and inhibited CASP3 gene performance. This result indicates that Papua red fruit extract may inhibit the expression of CASP8 and CASP3 genes and reduce the apoptosis of retinal cells due to ultraviolet light.

4.3 Inhibition of IL-1β and IL-8 gene expression

Figure 5 shows the relative expression levels of proinflammatory interleukin-1β ( IL-1β ) and interleukin-8 ( IL-8 ) genes in the aforementioned groups of retinal pigment epithelial cells; Compared with the group, p <0.05. According to Figure 5, the ultraviolet light group has slightly more IL-8 gene expression than the control group, suggesting that the retinal pigment epithelial cells irradiated by ultraviolet light B may trigger inflammation and damage the retina and surrounding tissues, but 0.25 mg/ mL Papua red fruit extract inhibits the increase in IL-8 gene expression and inhibits IL-1β gene expression. In view of previous studies that IL-1β is still related to cell apoptosis, and IL-8 also has the effect of promoting angiogenesis, the results in Figure 5 indicate that Papua red fruit extract may reduce UV by inhibiting the expression of IL-1β and IL-8 genes Inflammation of the eye, abnormal blood vessel proliferation and apoptosis of retinal cells caused by light, thus avoiding ocular cell damage caused by ultraviolet light.

In summary, Papua red fruit extract can enhance the resistance of retinal cells to ultraviolet light. By including but not limited to inhibiting the expression of genes related to angiogenesis, apoptosis, and inflammation, it can reduce retinal cell damage and The effect of preventing eye diseases, so it helps vision care. Therefore, the Papua red fruit extract of the present invention can be used to prepare a pharmaceutical composition for reducing retinal damage caused by ultraviolet light. The pharmaceutical composition can be powder, granule, solution, colloid or paste, which can be administered to a body by oral or topical application.

<110> Dajiang Biomedical Co., Ltd.

<120> The use of Papua red fruit extract to reduce retinal damage caused by ultraviolet light

<130> 107B0483-I1

<160> 12

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 1

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 2

<210> 3

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 3

<210> 4

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 4

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 5

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 6

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 7

<210> 8

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 8

<210> 9

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 9

<210> 10

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 10

<210> 11

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 11

<210> 12

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> PCR primer

<400> 12

Claims (6)

A red papua extract is used to prepare a pharmaceutical composition for reducing retinal cell damage caused by ultraviolet light, wherein the red papua extract inhibits the cysteine protease-3 (CASP3) gene and cysteine protease-8 (CASP8) Gene expression to reduce retinal cell apoptosis caused by ultraviolet light. The use as described in item 1 of the scope of patent application, wherein the Papua red fruit extract further inhibits the expression of vascular endothelial growth factor A (VEGFA) gene to reduce the abnormal proliferation of retinal blood vessels caused by ultraviolet light irradiation. The use described in item 1 of the scope of patent application, wherein the Papua red fruit extract further inhibits the expression of interleukin-1β (IL-1β) gene and interleukin-8 (IL-8) gene to reduce ultraviolet light exposure Inflammation of the retina caused. The use described in item 1 of the scope of the patent application, wherein the Papua red fruit extract is obtained by heating a Papua red fruit to remove the moisture contained therein, and the heating step is performed at 50°C to 100°C. The use according to any one of items 1 to 3 of the scope of the patent application, wherein the concentration of the Papua red fruit extract is in the range of 0.25 to 10 mg/mL. The use described in item 1 of the scope of patent application, wherein the pharmaceutical composition has a dosage form of powder, granule, solution, colloid, or paste.

Images