CN111358868A

CN111358868A - Use of extract of Rubus obovatus Thunb for preparing pharmaceutical composition for inhibiting gene expression in retinal cells

Info

Publication number: CN111358868A
Application number: CN201910082722.9A
Authority: CN
Inventors: 林咏翔
Original assignee: TCI Co Ltd
Current assignee: TCI Co Ltd
Priority date: 2018-12-26
Filing date: 2019-01-28
Publication date: 2020-07-03
Also published as: TWI706786B; TW202023598A

Abstract

The invention relates to the field of plant extracts, in particular to application of a Rubus chingii extract in preparing a pharmaceutical composition for inhibiting gene expression in retinal cells. The gene expression is gene expression comprising vascular endothelial growth factor a (vegfa), cysteamine protease-3 (CASP3), cysteamine protease-8 (CASP8), or a combination thereof. The extract of the fruit of Rubus parvifolius is obtained by heating a Rubus parvifolius to remove water contained therein. The extract reduces retinal cell damage by inhibiting the expression of genes associated with angiogenesis, apoptosis, and inflammatory responses in retinal cells, thereby increasing the resistance of retinal cells to ultraviolet radiation and reducing the incidence of ocular diseases.

Description

Use of extract of Rubus obovatus Thunb for preparing pharmaceutical composition for inhibiting gene expression in retinal cells

Technical Field

The invention relates to the field of plant extracts, in particular to application of a Rubus chingii extract in preparing a pharmaceutical composition for inhibiting gene expression in retinal cells.

Background

The eye is the organ of vision responsible for receiving light from the environment and producing vision. In the process of forming vision, light firstly passes through the transparent cornea and the crystal in the eye, and by means of the light-gathering function of the two structures, the light can be focused on the retina to form an image, and the light-sensitive cells in the retina are stimulated to convert light stimulation into electric signals, and the electric signals reach the brain vision area through the conduction of optic nerves, and the vision is formed under the signal processing of the nerve cells.

Although the eye receives light as normal, the sunlight not only includes visible light, but also includes ultraviolet light with shorter wavelength and larger energy, which can be divided into ultraviolet light A (UVA; wavelength about 315-. The retina is easily damaged by long-term exposure to such short-wavelength light, which is caused by the fact that the high energy of the light itself destroys the structure of the molecules in the cell, and when the retinal cell generates energy by oxidation reaction during operation, many Reactive Oxygen Species (ROS) including superoxide radical, hydroxyl radical, hydrogen peroxide and peroxide are easily generated by the exposure to the short-wavelength light, thereby causing oxidative damage to the cell, and finally causing retinal related diseases such as macular degeneration, and even possible blindness of the serious patient.

To reduce uv damage to the retina, common protection methods include parasol, or the wearing of a wide-edged hat or sunglasses. In addition, the purpose of vision health care can be achieved by taking the food beneficial to the retina by the individual. Therefore, there is a need for a composition that can enhance the resistance of retinal cells to uv light irradiation.

Disclosure of Invention

Accordingly, an object of the present invention is to provide a use of a prunus catapult (Pandanus conoides) extract for preparing a pharmaceutical composition for inhibiting gene expression in retinal cells, wherein the prunus catapult extract is obtained by heating a prunus catapult to remove moisture contained therein, and the gene expression is a gene expression comprising Vascular Endothelial Growth Factor A (VEGFA), cysteine protease-3 (caspase-3, CASP3), cysteine protease-8 (caspase-8, CASP8), or a combination thereof.

In one embodiment of the invention, the gene expression further comprises gene expression of interleukin-1 β (interleukin-1 β -1 β), interleukin-8 (interleukin-8, IL-8), or any combination thereof.

In one embodiment of the present invention, the heating step is performed at 50 ℃ to 100 ℃.

In one embodiment of the present invention, the concentration of the extract of Rubus Suavissimus fruit ranges from 0.25 to 10 mg/mL.

In one embodiment of the present invention, the extract of Rubus obuensis reduces UV-induced retinal damage, such as death of a retinal pigment epithelium or cell membrane damage.

The present invention discloses that the extract of Rubus obovatus Linn can improve the ultraviolet light resistance of retinal cells, and achieve the effects of reducing retinal cell damage and preventing eye diseases by including but not limited to inhibiting gene expression related to angiogenesis, apoptosis and inflammatory reaction, thereby being helpful for vision health care. Therefore, the extract of the red babuya can be used for preparing a pharmaceutical composition for reducing the retinal damage caused by ultraviolet light. The pharmaceutical composition may be in the form of a powder, granule, solution, gel or paste for oral or topical administration to a subject.

The following embodiments are provided to illustrate the features and applications of the present invention, rather than to limit the scope of the invention, and it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention.

Drawings

FIG. 1 shows the cell viability of retinal pigment epithelial cells under UV light irradiation, either pretreated with or without the Rubus obuquifolius extract;

FIG. 2A shows a bright field micrograph (magnification 100X) of retinal pigment epithelial cells pre-treated with or without the Rubus obuensis extract under UV irradiation;

FIG. 2B shows a fluorescence micrograph (magnification 100X) of retinal pigment epithelial cells pre-treated with or without the Rubus obuensis extract under UV irradiation;

FIG. 3 shows the relative expression of the VEGFA gene under UV irradiation of retinal pigment epithelial cells pretreated with or without the Rubus bracteatum extract; controlling cells not to be irradiated by ultraviolet light;

FIG. 4 shows the relative expression levels of CASP3 and CASP8 genes in UV-irradiated retinal pigment epithelial cells pretreated with or without the Rubus bayanus extract; controlling cells not to be irradiated by ultraviolet light;

FIG. 5 shows the relative expression of IL-1 β and IL-8 gene in retinal pigment epithelial cells pre-treated with or without the Bambusia red fruit extract under UV irradiation, and control cells without UV irradiation.

Detailed Description

The present invention provides a use of a Rubus coreanus extract for preparing a pharmaceutical composition for inhibiting gene expression in retinal cells, the gene expression comprising VEGFA, CASP3, CASP8, or a combination thereof, and further comprising IL-1 β, IL-8, or any combination thereof, the Rubus coreanus extract of the present invention is obtained by heating a Rubus coreanus to remove water contained therein, and the heating step is performed at 50 ℃ to 100 ℃, the following examples disclose that the Rubus coreanus extract at a concentration of 0.25 to 10mg/mL significantly inhibits the increase in expression of genes associated with angiogenesis, apoptosis, and inflammatory response due to ultraviolet light irradiation, and reduces retinal damage due to ultraviolet light such as death of retinal pigment epithelial cells and cell membrane damage.

Definition of

As used herein, the numerical values are approximations and all numerical data are reported to be within the 20 percent range, preferably within the 10 percent range, and most preferably within the 5 percent range.

As used herein, "retinal damage caused by ultraviolet light" includes retinal cell death, apoptosis, cell membrane damage, and other damage that impairs physiological function. These injuries may involve, but are not limited to, ocular angiogenesis and inflammatory reactions.

Materials and methods

Material

Minimal Essential Medium (MEM) containing 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, Inc. 3- (4, 5-dimethyl-2-thiazole) -2, 5-diphenyltetrazolium bromide salt (3- (4, 5-dimethylthiozol-2-y 1) -2, 5-diphenyltetrazolium bromide, MTT) was purchased from AMERSCO. Dimethyl sulfoxide (DMSO) was purchased from Echo chemical Co.

Cell culture

The following examples use the human retinal pigment epithelial cell strain ARPE-19(ATCC CRL-2302) purchased from American Type Culture Collection (ATCC). The cells were cultured in MEM medium supplemented with 0.1mM of an unnecessary amino acid, 1.5g/L of sodium hydrogencarbonate, 1mM of sodium pyruvate, and 10% FBS at 37 ℃ under 5% carbon dioxide, and hereinafter referred to as cell culture medium.

MTT assay

Cell viability was determined by MTT assay. Briefly, MTT solution (4mg/mL MTT in PBS) was added at 15. mu.L/well to the cells subjected to the designated treatment in a 96-well plate, and reacted at 37 ℃ for 4 hours. After removing the reaction solution, DMSO was added to the cells at 50. mu.L/well and the reaction was shaken for 10 minutes to dissolve the formed formazan

(formazan) crystallization finally, absorbance (O.D.570) at 570nm of the cell mixture was measured using ELISA plate reader (BioTek). cell viability was calculated according to the formula of O.D.570 for each group/O.D.570 for control group × 100% statistically significant difference was student t of Excel softwareAnd (6) determining the verification.

Cell damage assay

Cells with damaged cell membranes were detected using fluorescently labeled linker v (annexin v)/pyridine iodide. Briefly, cells subjected to the designated treatments in 24-well plates were washed with PBS solution, and 10 μ L/well of Alexa Fluor 488-linker V conjugate (Invitrogen) and 200 μ L/well of linker V binding buffer (Invitrogen) were added to the 24-well plates and reacted for 20 minutes at room temperature in the absence of light to stain the damaged cell membranes. Thereafter, the cells were washed with PBS solution, and the cell nuclei were stained with pyridine iodide staining solution (purchased from BD pharmingen) at room temperature for 10 minutes. After the stained cells are washed by PBS solution, the green fluorescence distribution is observed by a ZEISS fluorescence microscope, and the more the green fluorescence quantity is, the more the cells with damaged cell membranes are.

Analysis of Gene expression

The expression levels of genes involved in angiogenesis, apoptosis, and inflammatory response in cells were determined based on quantitative polymerase chain reaction (qPCR), and the procedure is briefly described below. RNA was isolated from the cells using an RNA Extraction Kit (RNA Extraction Kit; Geneaid) according to the manufacturer's instructions and reverse transcriptase was used at 37 deg.C

III Reverse Transcriptase 2000ng RNA was Reverse transcribed to cDNA (Invitrogen). Subsequently, the cDNA was subjected to qPCR using a qPCR Kit (KAPA CYBR FAST qPCR Kit (2X); KAPA Biosystems) in a PCR reactor (Step One plus real-Time PCR system; Applied Biosystems) using a primer set (Table 1) of the target gene and a Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as an internal control to obtain a melting curve (melting curve).

TABLE 1

Finally, use 2^-ΔΔCTThe method determines the relative expression of the target gene. The method uses GAPDH geneCycle threshold (C)_T) The cycle threshold of the reference gene as an internal control was calculated as the relative fold change according to the following formula:

ΔC_Tc of target gene in experimental group or control group_TC of internal control_T

ΔΔC_TΔ C of experimental group_TΔ C of control groups_T

Multiple change 2^{Average value of- Δ Δ Ct}

Statistical analysis was performed by calculating the standard deviation of relative expression of each gene using the STDEV function in Excel software and calculating the statistical difference using the single-tailed student t test (TTEST).

Example 1

Preparation of babusha red fruit extract

Paphia fruit (Pandanus conoides) is a shrub of the genus Pandanus (Pandanus), and is distributed mainly in tropical and subtropical zones. The term babusha fruit as used herein refers to the fruit of the plant, which is cylindrical and red in shape, has a length of about 50 to 100 cm and a cross-sectional diameter of about 15 to 20 cm on average.

To obtain the extract of the fruit of Rubus Suavissimus, the fruit of Rubus Suavissimus is first washed and cut into pieces or slices, and then sterilized at 95 deg.C for a period of time, for example, about 30 minutes to 1 hour. Thereafter, the seeds in the chunkia mangrove blocks were removed and coarsely crushed using a crusher for extraction. During the extraction process, the crushed red babuya is heated continuously to remove the water in the crushed red babuya, the heating temperature is 50-100 ℃, preferably 60-80 ℃, and the heating time is 6-10 hours. The water-removed fructus Rubus Suberbae coarse powder is cooled to room temperature, and can be further filtered with 400 mesh filter screen to remove residual solid substance, to obtain fructus Rubus Suberbae extract.

Example 2

Reduction of retinal cell death by ultraviolet light by extracts of the fruit of Rubus obuensis

To examine the effect of the Rubus bracteatum extract on protecting retina from UV light, human retinal pigment epithelial cell line ARPE-19 was evaluated using cell survival assay (MTT assay)Cell viability under UV B irradiation after treatment with the Babuya red fruit extract described in example 1 briefly, ARPE-19 cells were treated with 5 × 10³Each cell/well was seeded in a 96-well culture plate containing 200 μ L of cell culture medium per well. After culturing the cells at 37 ℃ for 24 hours, the medium was removed and each well cell was treated in the following manner: (a) cultured in 200. mu.L of cell culture medium for 24 hours (control); (b) cultured in 200. mu.L of cell culture medium for 24 hours, and then placed in an ultraviolet light irradiation chamber (Vilber) to receive 12J/cm²UV B radiation for 1 hour (UV group), which resulted in half cell death; or (c) culturing in 200 μ L of cell culture medium containing 0.25mg/mL of Rubus Suavissimus extract for 24 hr, and further receiving 12J/cm²Uv light B was irradiated for 1 hour (uv light + pabuia mangostana extract group). Thereafter, MTT analysis and cell viability calculation were performed on each group of cells (four replicates).

FIG. 1 shows the survival rates of the retinal pigment epithelial cells of the aforementioned groups; in the figure, p < 0.01 and p < 0.001, respectively, are indicated. According to fig. 1, the uv group had a significantly reduced cell survival rate (about 21% reduction) compared to the control group, indicating that uv B irradiation caused massive death of retinal pigment epithelial cells. However, the UV-irradiated cells treated with the Bambusia red fruit extract had similar survival rates as the non-irradiated control cells. The results show that the extract of the red fruit of pabuia has a protective effect on retinal cells, thus avoiding massive cell death caused by ultraviolet light.

Example 3

Reduction of ultraviolet light-induced cell membrane damage in retinal cells by extracts of the fruit of the Rubus brevifolius

To examine whether the extract of Rubus Suavissimus fruit affects the damage of UV light on the cell membrane of retinal cells, this example uses fluorescence staining technique to observe the cell membrane integrity of human retinal pigment epithelial cell line ARPE-19 under UV light B after being treated with the extract of Rubus Suavissimus fruit as described in example 1. briefly, ARPE-19 cells were treated with 5 × 10⁴Each cell/well was seeded in a 24-well culture plate containing 500 μ L of cell culture medium per well. Culturing at 37 deg.CAfter 24 hours of cells, the medium was removed and each well cell was treated in the following manner: (a) cultured in 500. mu.L of cell culture medium for 24 hours (control); (b) culturing in 500 μ L cell culture medium for 24 hr, and placing in ultraviolet irradiation box to receive 12J/cm²Ultraviolet B irradiation for 1 hour (uv group); or (c) culturing in 500. mu.L of cell culture medium containing 0.25mg/mL of the extract of Rubus obovatus, for 24 hours, and further receiving 12J/cm²Uv light B was irradiated for 1 hour (uv light + pabuia mangostana extract group). Thereafter, cell damage assays were performed on each group of cells (four replicates) to observe the extent of cell membrane damage.

FIGS. 2A and 2B show photomicrographs and fluorescence photomicrographs of the aforementioned groups of retinal pigment epithelial cells, respectively. According to fig. 2B, the uv group had significantly more green fluorescence area than the control group, indicating that uv B irradiation caused damage to the membrane of retinal pigment epithelial cells, but pre-administration of 0.25mg/mL babu fruit extract reduced the damage to cells similar to the control group. This result demonstrates that the extract of Rubus obovatus protects retinal cells from uv-induced cell membrane damage and is therefore beneficial for vision maintenance.

Example 4

The extract of red fruit of Babuya inhibits the expression of genes related to angiogenesis, apoptosis and inflammatory reaction in retinal cells under ultraviolet irradiation

To investigate whether the extract of Rubus Suavissimus fruit affects the gene expression in retinal cells under UV irradiation, this example utilized qPCR to determine the amount of expression of genes involved in angiogenesis, apoptosis, and inflammatory response of human retinal pigment epithelial cell line ARPE-19 after being treated with the extract of Rubus Suavissimus fruit described in example 1 and irradiated with UV B.briefly, ARPE-19 cells were treated with 5 × 10⁴Each cell/well was seeded in a 24-well culture plate containing 500 μ L of cell culture medium per well. After culturing the cells at 37 ℃ for 24 hours, the medium was removed and each well cell was treated in the following manner: (a) cultured in 500. mu.L of cell culture medium for 48 hours (control group); (b) culturing in 500 μ L cell culture medium for 48 hr, and placing in ultraviolet irradiation box to receive 12J/cm²Ultraviolet B irradiation for 1 hour (uv group); or (c) culturing in 500. mu.L of cell culture medium containing 0.25mg/mL of the extract of Rubus obovatus, for 48 hours, and further receiving 12J/cm²Uv light B was irradiated for 1 hour (uv light + pabuia mangostana extract group). Thereafter, each set of cells (four replicates) was used for gene expression analysis to determine the relative expression, which is a multiple of the specific gene expression relative to the control set of cells.

4.1 inhibition of VEGFA Gene expression

FIG. 3 shows the relative expression amounts of Vascular Endothelial Growth Factor A (VEGFA) gene associated with angiogenesis in the aforementioned groups of retinal pigment epithelial cells; water in the figure indicates p < 0.01 compared to the uv group. According to fig. 3, the uv group showed more expression of VEGFA gene than the control group, suggesting that uv B-irradiated retinal pigment epithelial cells might promote peripheral angiogenesis, but pre-administration of 0.25mg/mL of babusha fruit extract significantly inhibited the increased expression of VEGFA gene. In view of the previous studies indicating that abnormal proliferation of ocular vessels is related to the progression of eye diseases such as age-related macular degeneration, diabetic retinopathy, etc., which may cause retinal pigment epithelium detachment, subretinal hemorrhage, and macular edema to affect vision, the results in fig. 3 indicate that the extract of rubus baphicaca may reduce the ocular angiogenesis probability by inhibiting the increase of VEGFA gene expression caused by ultraviolet light in retinal cells, and thus may be applied to prevent vision from being impaired by ultraviolet light irradiation.

4.2 inhibition of CASP3 and CASP8 Gene expression

FIG. 4 shows the relative expression levels of the genes for cysteamine protease-3 (CASP3) and cysteine protease-8 (CASP8) involved in apoptosis in the aforementioned groups of retinal pigment epithelial cells; in the figure, p < 0.01 compared to the uv group. According to FIG. 4, the UV group showed slightly more CASP8 gene expression than the control group, suggesting that UV B irradiation may induce apoptosis of retinal pigment epithelial cells, but pre-administration of 0.25mg/mL of the Rubus obuquifolius extract inhibited the increased CASP8 gene expression and inhibited CASP3 gene expression. This result suggests that the extract of red fruit of Bambusia could reduce the apoptosis of retinal cells due to ultraviolet light by inhibiting the expression of CASP8 and CASP3 genes.

4.3 inhibition of IL-1 β and IL-8 Gene expression

FIG. 5 shows the relative expression of proinflammatory interleukin-1 β (IL-1 β) and interleukin-8 (IL-8) genes in the retinal pigment epithelial cells of the previous groups, wherein p is < 0.05 compared to the UV group, according to FIG. 5, the UV group has a slightly greater expression of IL-8 gene compared to the control group, suggesting that retinal pigment epithelial cells irradiated with UV B may cause inflammatory reactions to damage retina and peripheral tissues, but the pre-application of 0.25mg/mL of the extract of Rubus obuensis inhibits the increased expression of IL-8 gene and inhibits the expression of IL-1 β gene, previous studies indicate that IL-1 β is still associated with apoptosis and IL-8 also has angiogenesis promoting effect, and the results of FIG. 5 indicate that the extract of Rubus obuensis may reduce the inflammatory and apoptotic eye inflammation and retinal cell death caused by UV light by inhibiting the expression of IL-1 β and IL-8 genes, thus avoiding UV-induced eye cell damage.

In summary, the extract of Rubus obovatus can improve the resistance of retinal cells to UV radiation, and reduce retinal cell damage and prevent ocular diseases by inhibiting the expression of genes related to angiogenesis, apoptosis, and inflammatory response, thereby facilitating vision care. Therefore, the extract of the red babuya can be used for preparing a pharmaceutical composition for reducing the retinal damage caused by ultraviolet light. The pharmaceutical composition may be in the form of a powder, granule, solution, gel or paste for oral or topical administration to a subject.

Claims

1. Use of a prunus babu extract obtained by heating a prunus babu to remove water contained therein for the preparation of a pharmaceutical composition for inhibiting gene expression in retinal cells, wherein the gene expression is a gene expression comprising vascular endothelial growth factor a (VEGF-a), cysteine protease-3 (CASP3), cysteamine protease-8 (CASP8), or a combination thereof.

2. The use of claim 1, wherein said gene expression further comprises gene expression of interleukin-1 β (IL-1 β), interleukin-8 (IL-8), or any combination thereof.

3. Use according to claim 1, characterized in that the heating step is carried out at 50 to 100 ℃.

4. Use according to claim 1, wherein the concentration of the rubus corchorifolius extract ranges from 0.25 to 10 mg/mL.

5. The use according to claim 1, wherein the extract of rubus bayanus reduces retinal damage caused by uv light.

6. The use according to claim 5, wherein the extract of Rubus bayanus reduces the death of a retinal pigment epithelium.

7. The use of claim 5, wherein the extract of Rubus parvifolius reduces cell membrane damage of a retinal pigment epithelial cell.

8. Use according to claim 1, characterized in that the pharmaceutical composition has the form of a powder, granules, a solution, a colloid, or a paste.