CN114146124A - Application of Polymethoxyflavonoids of Citrus Citrus in the Preparation of Antioxidants - Google Patents
Application of Polymethoxyflavonoids of Citrus Citrus in the Preparation of Antioxidants Download PDFInfo
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- CN114146124A CN114146124A CN202111310247.XA CN202111310247A CN114146124A CN 114146124 A CN114146124 A CN 114146124A CN 202111310247 A CN202111310247 A CN 202111310247A CN 114146124 A CN114146124 A CN 114146124A
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Abstract
The invention belongs to the technical field of extraction and application of plant active ingredients, and discloses application of a phyllanthus emblica embryo polymethoxylated flavonoid compound in preparation of an antioxidant. The phyllanthus emblica embryo polymethoxylated flavone extract has a protective effect on cell oxidative damage, can remarkably reduce the intracellular ROS level and recover the membrane potential of mitochondria to recover the functions of the mitochondria, further relieves oxidative stress damage, shows a better clinical antioxidant effect, can be applied to various medicines, foods and skin care products with higher requirements on safety, and improves the antioxidant capacity of corresponding products; or as an antioxidant alone; can also be used as dietary supplement for scavenging excessive free radicals in vivo, and preventing or delaying aging; or used as medicine or adjuvant for treating neurodegenerative disease, tumor or inflammation related to oxidative damage.
Description
Technical Field
The invention belongs to the technical field of extraction and application of plant active ingredients, and particularly relates to an application of a phyllanthus emblica embryo polymethoxylated flavonoid compound in preparation of an antioxidant.
Background
In living organisms, there is a redox balance, which is destroyed and shifted to the oxidation direction when aging and disease occur, and various radicals and oxides such as Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) are accumulated in excess. These oxidizing substances are very easily combined with DNA, protein, phospholipid, etc. to cause oxidative damage of various organelles in cells, thereby accelerating the progress of diseases. It has been shown that the progression of diseases such as vascular injury, inflammation, and tumor is associated with oxidative damage of cells. Therefore, the use of antioxidants is important in the control of these diseases. However, the artificial antioxidants such as t-butyl p-hydroxyanisole, dibutyl hydroxytoluene, etc. have been reported to have potential acute toxicity and developmental toxicity, so the research on natural antioxidants is focused. In the last two decades, natural antioxidants are continuously found in animals and plants, and natural products have the characteristics of low toxicity, high efficiency and easy absorption, and have very wide development prospects.
Citrus reticulata (Citrus reticulata 'Chachi') is a cultivated variety of Citrus in the genus of Citrus of Rutaceae, is a basic source plant of Citrus reticulata Blanco (Citrus reticulata Blanco) and is named as "Citrus reticulata Blanco or Citrus reticulata Blanco" by adding medicine to pericarp of mature fruit of Citrus reticulata Blanco in Chinese medicine, but actually, dry young fruit of Citrus reticulata Blanco with undifferentiated fruit tissue can also be used as medicine, and is named as "Citrus reticulata Blanco embryo or Citrus reticulata Blanco of Citrus reticulata Blanco". The CHAZHIGAN can enter liver, gallbladder and stomach meridians, has effects of dispersing stagnated liver qi, relieving qi stagnation, resolving food stagnation, and resolving stagnation, and is used for food stagnation, qi stagnation, abdominal distention and pain, and contains flavones, volatile oils and amino acids as main chemical components. Modern pharmacological studies show that the camellia sinensis embryo has wide biological effects on digestive systems, cardiovascular systems, respiratory systems and the like, and the application prospect is worthy of attention. The tea branch citrus reticulata blanco embryo is different from the tangerine peel and the citrus reticulata blanco, the tissue of the tea branch citrus reticulata blanco embryo is not differentiated, and the tea branch citrus reticulata blanco embryo comprises undifferentiated pulp and pericarp; the latter (the citrus reticulata blanco peel and the citrus reticulata blanco peel) is dry mature peel, only refers to the peel with completely differentiated fruit tissues (the citrus reticulata blanco peel or the citrus reticulata blanco peel is stored for three years), does not include pulp, and has obvious difference in the aspects of chemical components, pharmacological activity, clinical application and the like. Pericarpium citri reticulatae enters lung and spleen channels, has the effects of regulating qi, tonifying spleen, stimulating appetite and reducing phlegm, is mainly used for preventing or treating anorexia, vomiting and diarrhea, cough and excessive phlegm and the like, and mainly comprises flavonoids, alkaloids, polysaccharides, volatile oil and the like. The pericarpium Citri Tangerinae is one kind of pericarpium Citri Tangerinae, is derived from one cultivated variety of Citrus reticulata (Citrus reticulata Blanco), and has milder action, more chemical components such as polymethoxylated flavone, and higher quality compared with pericarpium Citri Tangerinae. For a long time, many research reports are directed at the tangerine peel or the euryale peel, the developed related products are very abundant and various, but the research reports of the citrus reticulata blanco embryo are rarely seen. At present, the camellia sinensis embryo is not developed into an antioxidant drug, a health food or a cosmetic in the market, and the medicinal value of the camellia sinensis embryo is not fully exerted. And China has rich tea branch citrus embryo resources, which lays a favorable foundation for further enhancing the development and application of the tea branch citrus embryos.
Disclosure of Invention
The first aspect of the invention aims to provide the application of the phyllanthus emblica embryo polymethoxylated flavone extract in preparing an antioxidant.
The second aspect of the invention aims to provide the application of the chazu orange embryo polymethoxylated flavone extract in preparing the antioxidant additive.
In a third aspect, the invention aims at providing the application of the phyllanthus emblica embryo polymethoxylated flavone extract in the preparation of dietary supplements.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the invention, there is provided the use of an extract of chaga foetida in the preparation of an antioxidant.
Preferably, the antioxidant is used to protect cells from oxidative damage.
Preferably, the antioxidant is used to protect cells from oxidative damage caused by nitric oxide synthesis donors (SNPs).
In a second aspect of the invention, the application of the phyllanthus emblica embryo polymethoxylated flavone extract in preparing an antioxidant additive is provided.
Preferably, the antioxidant additive is used for preparing a medicine, a food or a skin care product.
Preferably, the medicament is for the treatment and/or prevention of diseases associated with oxidative damage.
Preferably, the diseases associated with oxidative damage include neurodegenerative diseases, inflammatory diseases and tumors.
In a third aspect of the invention, there is provided the use of an extract of chaga foetida in the manufacture of a dietary supplement.
Preferably, the dietary supplement is used for scavenging free radicals in the body and preventing or delaying aging.
According to the first, second and third aspects of the present invention, the preparation method of the chazuki citrus seed polymethoxylated flavone extract comprises the steps of:
(1) extraction: mixing the citrus reticulata blanco embryo with an organic solvent, extracting, carrying out solid-liquid separation, taking liquid, and concentrating to obtain an extract A1;
(2) and (3) extraction: mixing the extract A1 with water to obtain suspension, mixing the suspension with organic solvent, extracting, and collecting organic solvent phase, i.e. tea branch citrus fruit embryo polymethoxylated flavone extract.
Preferably, the citrus reticulata blanco embryo in the step (1) is crushed and sieved by a sieve of 12-16 meshes before being mixed.
Preferably, the organic solvent in step (1) is ethanol.
Preferably, the concentration of the ethanol is 20-100% (v/v); further 70 to 95% (v/v).
Preferably, the mass volume ratio of the citrus reticulata blanco embryo to the organic solvent in the step (1) is 1: (2-8); further 1: (3-5).
Preferably, the extraction mode in the step (1) is at least one of decoction extraction, ultrasonic-assisted extraction, reflux extraction and percolation extraction; further ultrasonic assisted extraction.
Preferably, the ultrasonic-assisted extraction is carried out under the conditions of 30-50 ℃, 200-400W and 30-50 kHz for 20-40 min.
Preferably, the extraction times in the step (1) are 1-6 times; further 1 to 5 times.
Preferably, the solid-liquid separation mode in the step (1) is filtration.
Preferably, the concentration in step (1) is concentration under reduced pressure.
Preferably, the mass-to-volume ratio of the extract A1 to water in the step (2) is 1 (0.5-1).
Preferably, the volume ratio of the suspension to the organic solvent in step (2) is 1: (2-5); further 1: (3-4).
Preferably, the organic solvent in the step (2) is at least one of petroleum ether, n-butane, dichloromethane, n-butanol and ethyl acetate; further, ethyl acetate.
Preferably, the extraction times in the step (2) are 1-6 times; further 2-5 times; further 3 to 4 times.
Preferably, the preparation method further comprises the following steps: the organic solvent phase is concentrated and dried.
Preferably, the concentration is concentration under reduced pressure.
Preferably, the drying method is freeze drying to constant weight.
Preferably, the content of polymethoxy flavonoids in the phyllanthus emblica embryo polymethoxy flavonoid extract is more than 50 percent; further, the content is 50 to 55%.
Preferably, the polymethoxylated flavonoids include isosinensetin, 5-hydroxy-6, 7,8,4' -tetramethoxyflavone, nobiletin, 3,5,6,7,8,3',4' -heptamethoxyflavone, hesperetin and 5-hydroxy-6, 7,8,3' -4' -pentamethoxyflavone.
Preferably, the mass ratio of the isosinensetin, 5-hydroxy-6, 7,8,4' -tetramethoxyflavone, nobiletin, 3,5,6,7,8,3',4' -heptamethoxyflavone, hesperetin and 5-hydroxy-6, 7,8,3' -4' -pentamethoxyflavone is (0.5-1.5): (2.5-4): (24-30): (0.5-1): (16-20): (2.5-4); further (1.29-1.5): (3.12-4): (27.45-30): (0.78-1): (18.15-20): (3.08-4).
The invention has the beneficial effects that:
the invention discloses the application of the chaga tire polymethoxylated flavone extract in the preparation of an antioxidant for the first time, the chaga tire polymethoxylated flavone extract has a protective effect on oxidative damage of cells, can obviously reduce the level of intracellular ROS (reactive oxygen species) and recover the membrane potential of mitochondria so as to recover the function of the mitochondria, further lighten oxidative stress damage, show better clinical antioxidant effect, can be applied to various medicines, foods and skin care products with higher requirements on safety, and improve the antioxidant capacity of corresponding products; or as an antioxidant alone; can also be used as dietary supplement for scavenging excessive free radicals in vivo, and preventing or delaying aging; or used as medicine or adjuvant for treating neurodegenerative disease, tumor or inflammation related to oxidative damage.
Drawings
FIG. 1 is a high performance liquid chromatogram of the chazu orange embryo polymethoxylated flavone extract obtained in example 1.
FIG. 2 is a graph of the effect of different concentrations of nitric oxide synthesis donors (SNPs) on cell viability of HUVECs.
FIG. 3 is a graph of the effect of different concentrations of the phyllanthus emblica embryo polymethoxylated flavone extract on SNP-induced oxidative damage of HUVECs cells: wherein p represents < 0.0001 compared to the blank control group; p < 0.001, compared to the blank control group; denotes p < 0.05 compared to the blank control group.
FIG. 4 is a graph of the effect of various concentrations of the phyllanthus emblica embryo polymethoxylated flavone extract on SNP-induced cell morphology of HUVECs.
FIG. 5 is a graph of the effect of various concentrations of the extract of the phyllanthus emblica embryo polymethoxylated flavones on the SNP-induced Reactive Oxygen Species (ROS) in HUVECs cells.
FIG. 6 is a graph of the effect of different concentrations of the phyllanthus emblica embryo polymethoxylated flavone extract on SNP induction of mitochondrial membrane potential in HUVECs cells.
Detailed Description
The present invention will be described in further detail with reference to specific examples.
The starting materials used in the examples were prepared by conventional means or purchased from commercial sources, except as otherwise specified.
Example 1A method for preparing a Polymethoxyflavone extract from Citrus reticulata Blanco embryos
(1) Crushing and sieving dried tea branch orange embryo (the tea branch orange embryo is collected from tea pit village in new meeting area of Jiangmen city, Guangdong province) by 14 meshes according to a solid-liquid ratio (mass-volume ratio) of 1: 4 adding 95% (v/v) ethanol water solution, performing ultrasonic-assisted extraction for 3 times (power 300W, frequency 40kHz, time 30min, temperature 40 ℃), filtering, combining filtrates, and concentrating under reduced pressure to obtain extract A1;
(2) dissolving the extract A1 with water (the mass-volume ratio (kg/L) of the extract A1 to the water is 1: 0.8) to obtain a suspension; adding ethyl acetate (volume ratio of ethyl acetate to suspension is 3: 1), extracting for 3 times, mixing ethyl acetate extractive solutions, and concentrating under reduced pressure to obtain extract A2;
(3) and (4) freeze-drying the extract A2 to constant weight to obtain the phyllanthus emblica polymethoxylated flavone extract.
Example 2A method for preparing a Citrus reticulata embryo polymethoxylated flavone extract
(1) Crushing and sieving dried tea branch orange embryo (the tea branch orange embryo is collected from tea pit village in new meeting area of Jiangmen city, Guangdong province) by 14 meshes according to a solid-liquid ratio (mass-volume ratio) of 1: 4, adding 80% (v/v) ethanol water solution, performing ultrasonic-assisted extraction for 4 times (power 300W, frequency 40kHz, time 30min, temperature 40 ℃), filtering, combining filtrates, and concentrating under reduced pressure to obtain extract A1;
(2) dissolving the extract A1 with water (the mass-volume ratio (kg/L) of the extract A1 to the water is 1: 0.8) to obtain a suspension; adding ethyl acetate (volume ratio of ethyl acetate to suspension is 5: 1), extracting for 2 times, mixing ethyl acetate extractive solutions, and concentrating under reduced pressure to obtain extract A2;
(3) and (4) freeze-drying the extract A2 to constant weight to obtain the phyllanthus emblica polymethoxylated flavone extract.
Example 3A method for preparing an extract of Polymethoxyflavone from Citrus chachiensis Koch
(1) Crushing and sieving dried tea branch orange embryo (the tea branch orange embryo is collected from tea pit village in new meeting area of Jiangmen city, Guangdong province) by 14 meshes according to a solid-liquid ratio (mass-volume ratio) of 1: 4 adding 70% (v/v) ethanol water solution, performing ultrasonic-assisted extraction for 5 times (power 300W, frequency 40kHz, time 30min, temperature 40 ℃), filtering, mixing filtrates, and concentrating under reduced pressure to obtain extract A1;
(2) dissolving the extract A1 with water (the mass-volume ratio (kg/L) of the extract A1 to the water is 1: 0.8) to obtain a suspension; adding ethyl acetate (volume ratio of ethyl acetate to suspension is 4: 1), extracting for 4 times, mixing ethyl acetate extractive solutions, and concentrating under reduced pressure to obtain extract A2;
(3) and (4) freeze-drying the extract A2 to constant weight to obtain the phyllanthus emblica polymethoxylated flavone extract.
Example 4A method for preparing an extract of Polymethoxyflavone from Citrus chachiensis Koch
(1) Crushing and sieving dried tea branch orange embryo (the tea branch orange embryo is collected from tea pit village in new meeting area of Jiangmen city, Guangdong province) by 14 meshes according to a solid-liquid ratio (mass-volume ratio) of 1: 4 adding 100% (v/v) ethanol solution, extracting with ultrasonic assistance for 1 time (power 300W, frequency 40kHz, time 30min, temperature 40 ℃), filtering, mixing filtrates, and concentrating under reduced pressure to obtain extract A1;
(2) dissolving the extract A1 with water (the mass-volume ratio (kg/L) of the extract A1 to the water is 1: 0.8) to obtain a suspension; adding ethyl acetate (volume ratio of ethyl acetate to suspension is 5: 1), extracting for 3 times, mixing ethyl acetate extractive solutions, and concentrating under reduced pressure to obtain extract A2;
(3) and (4) freeze-drying the extract A2 to constant weight to obtain the phyllanthus emblica polymethoxylated flavone extract.
Example 5A method for preparing an extract of Polymethoxyflavone from Citrus chachiensis Koch
(1) Crushing and sieving dried tea branch orange embryo (the tea branch orange embryo is collected from tea pit village in new meeting area of Jiangmen city, Guangdong province) by 14 meshes according to a solid-liquid ratio (mass-volume ratio) of 1: 4 adding 20% (v/v) ethanol water solution, performing ultrasonic-assisted extraction for 2 times (power 300W, frequency 40kHz, time 30min, temperature 40 ℃), filtering, combining filtrates, and concentrating under reduced pressure to obtain extract A1;
(2) dissolving the extract A1 with water (the mass-volume ratio (kg/L) of the extract A1 to the water is 1: 0.8) to obtain a suspension; adding ethyl acetate (volume ratio of ethyl acetate to suspension is 2: 1), extracting for 5 times, mixing ethyl acetate extractive solutions, and concentrating under reduced pressure to obtain extract A2;
(3) and (4) freeze-drying the extract A2 to constant weight to obtain the phyllanthus emblica polymethoxylated flavone extract.
Example 6 quantitative analysis of polymethoxyflavonoids in the extract of polymethoxyflavonoids from Citrus reticulata embryo
Quantitative analysis was performed on 6 polymethoxylated flavones in the citrus reticulata blanco polymethoxylated flavone extract obtained in example 1 by using a performance liquid chromatography (HPLC) method, which was repeated 3 times, and an analyzer used: shimadzu LC system charged with SPD-M20A detector; a chromatographic column: agilent Zorbax Eclipse XDB-C18(4.6 mm. times.250 mm, 5 μm); mobile phase: phase B is 0.1% (v/v) formic acid water solution, and phase B is acetonitrile; gradient elution procedure: 25-55% of B for 0-5 min; 55-68% of B for 5-15 min; 68-90% of B, 15-18 min; flow rate: 1 mL/min; detection wavelength: 283nm, 330 nm; sample introduction volume: 10 mu L of the solution; detecting the temperature: at 30 ℃. The HPLC results are shown in fig. 1 and table 1: the total content of polymethoxyflavone compounds in the extract of the citrus reticulata embryo obtained in example 1 is 50% or more, and the main 6 ingredients are isosinensetin, 5-hydroxy-6, 7,8,4' -tetramethoxyflavone, nobiletin, 3,5,6,7,8,3',4' -heptamethoxyflavone, hesperetin, and 5-hydroxy-6, 7,8,3' -4' -pentamethoxyflavone.
TABLE 1 measurement results of polymethoxyflavonoids in the polymethoxyflavone extract of Citrus reticulata embryo obtained in example 1
Example 7 antioxidant Activity of Citrus reticulata embryo polymethoxylated flavone extract
1. Laboratory instruments and materials
The experimental apparatus is shown in Table 2, and the experimental materials are shown in Table 3.
TABLE 2 Experimental instruments
TABLE 3 materials of the experiments
2. Experimental methods
(1) Establishment of HUVECs cell oxidative stress model
HUVECs cell suspension was diluted with complete medium (endothelial cell medium + 5% fetal bovine serum + 1% growth factor + 1% diabody) to a cell concentration of 6X 104cells/mL, 100. mu.L of cell fluid were inoculated into a 96-well plate using a discharge gun, after 24 hours of culture, the old cell fluid was discarded, and 100. mu.L of a nitric oxide synthetic donor (SNP) solution containing a series of concentrations (final concentrations of 2000. mu.M, 1500. mu.M, 1000. mu.M, 800. mu.M, 600. mu.M, 400. mu.M, 200. mu.M, and 100. mu.M, respectively) was added to each of the model groupsAdding 100 mu L of the culture medium into a blank control group, continuously culturing for 24h, adding 10 mu L/well MTT solution (5mg/mL) in a dark place, continuously culturing for 4h in an incubator, taking out, sucking the supernatant along the wall by using an injector, adding 100 mu L/well DMSO, shaking on a shaking table for 15min to completely dissolve the generated purple crystals, detecting the absorbance value A at 490nm by using a multifunctional microplate reader, calculating the survival rate of the cells according to the following formula (I), and determining the concentration of the inducer SNP.
Cell survival (%) ═ aModel set/ABlank control groupFormula (I);
wherein A isModel setAbsorbance of the model group (SNP administration group); a. theBlank control groupAbsorbance of blank control.
(2) Influence of phyllanthus emblica embryo polymethoxylated flavone extract on SNP (single nucleotide polymorphism) induced HUVECs cell oxidative damage
HUVECs cell suspension was diluted with complete medium (endothelial cell medium + 5% fetal bovine serum + 1% growth factor + 1% diabody) to a cell concentration of 4X 104cells/mL, 100. mu.L of cell broth was inoculated into a 96-well plate using a discharge gun, and after 24 hours of culture, the old cell broth was discarded, 100. mu.L of a medium containing a polymethoxyflavone (TB) solution obtained in example 1 at a series of concentrations (final concentrations of 25, 12.5 and 6.25. mu.g/mL, respectively, DMSO as a solvent) was added to the experimental group, 100. mu.L of a medium containing a sample dissolving solvent (DMSO) at a corresponding concentration was added to the blank control group, after 2 hours of further culture, the old cell broth was discarded, 100. mu.L of a medium containing an SNP solution at a concentration of 800. mu.M was added to the medium, and further 24 hours of culture was carried out in an incubator, 10. mu.L/well of MTT solution (5mg/mL) was added in the dark, and further culture was carried out for 4 hours in the incubator, and the supernatant was aspirated along the wall using a syringe, 100. mu.L/well of DMSO was added, and the resulting purple crystals were completely dissolved by shaking on a shaker for 15 minutes, detecting the absorbance value A at 490nm by using a multifunctional microplate reader, calculating the survival rate of cells according to a formula (I), and primarily evaluating the influence of the phyllanthus emblica embryo polymethoxylated flavone extract on the oxidative damage of the HUVECs induced by the SNP.
(3) Influence of phyllanthus emblica embryo polymethoxylated flavone extract on SNP (single nucleotide polymorphism) induced HUVECs cell morphology
Taking HUVECs in logarithmic phase, discarding old culture medium, gently washing twice with PBS, adding trypsin, digesting for 30-60 s, centrifuging, counting, and counting the number of cells according to 8 × 103One was inoculated in a Confocal dish and after the cells had grown to a confluency of about 80%, (24h), the experimental group was added with 100. mu.L of complete medium containing the phyllanthus emblica polymethoxylated flavone extract (TB) obtained in example 1 at a series of concentrations (final concentration 25, 12.5, 6.25. mu.g/mL, respectively, solvent DMSO) and SNP at a final concentration of 800. mu.M; adding 100 mu L of complete culture medium with final concentration of 800 mu M SNP into the model group to induce oxidative stress reaction; and adding 100 mu L of complete culture medium containing a sample dissolving solvent (DMSO) with corresponding concentration into the blank control group, culturing for 24h, removing the old culture medium after the intervention is finished, adding a Hoechst preparation solution containing 10 mu g/mL and a Rhodamin preparation solution containing 5 mu M, incubating for 30min in a dark place in an incubator, cleaning for 3 times by using a special culture medium for endothelial cells after the incubation is finished, adding a new complete culture medium, and observing the cell state under laser confocal conditions.
(4) Influence of phyllanthus emblica embryo polymethoxylated flavone extract on SNP (single nucleotide polymorphism) induction of Reactive Oxygen Species (ROS) in HUVECs (human serum endothelial cells)
Inoculating about 8X 10 in a special dish of a laser confocal scanning microscope3Cells were cultured for 24 hours, the medium was discarded, and 100. mu.L of complete medium containing the phyllanthus emblica polymethoxylated flavone extract (TB) obtained in example 1 at a series of concentrations (final concentrations of 25, 12.5, and 6.25. mu.g/mL, respectively, and DMSO as a solvent) and 800. mu.M SNP was added to the experimental group; adding 100 mu L of complete culture medium with final concentration of 800 mu M SNP into the model group to induce oxidative stress reaction; and adding 100 mu L of complete culture medium containing a sample dissolving solvent (DMSO) with a corresponding concentration into the blank control group, removing the old culture medium after culturing for 24h, diluting a reactive oxygen fluorescent probe (DCFH-DA) 1000 times by using a medium without FBS, adding the diluted solution into each well, simultaneously adding Hoechst 33258 dye to enable the final concentration to be 1 mu g/mL, incubating the solution in a cell culture box for 20min in a dark environment, adding a serum-free culture medium, slightly shaking and cleaning the cells for three times, removing the culture medium, adding the serum-free culture medium, and shooting the ROS condition in the cells by using a confocal laser scanning microscope.
(5) Influence of phyllanthus emblica embryo polymethoxylated flavone extract on SNP (single nucleotide polymorphism) induction of mitochondrial membrane potential in HUVECs (human hematopoietic stem cells)
And (3) detecting the change condition of the mitochondrial membrane potential by using a JC-1 fluorescent probe. About 8 x 10 seeds in special container for laser confocal scanning microscope3Cells, after the cells had grown to a confluency of about 80% (i.e., 24h of culture), the experimental group was added with 100. mu.L of complete medium containing the phyllanthus emblica polymethoxylated flavone extract (TB) obtained in example 1 at a series of concentrations (final concentrations of 25, 12.5, 6.25. mu.g/mL, respectively, DMSO as solvent) and SNP at a final concentration of 800. mu.M; adding 100 mu L of complete culture medium with final concentration of 800 mu M SNP into the model group to induce oxidative stress reaction; adding 100 mu L of complete culture medium containing sample dissolving solvent (DMSO) with corresponding concentration into the blank control group, culturing for 24h, discarding the old culture medium, dyeing by using JC-1 dye, namely adding 50 mu L of JC-1 (200X) into 8mL of ultrapure water, shaking up violently, adding 2mL of JC-1 dyeing buffer solution (5X), and mixing uniformly to obtain JC-1 dyeing working solution; the specific operation is as follows: removing the culture medium by suction, adding PBS, slightly shaking to wash the cells, discarding the PBS, adding JC-1 staining working solution, and incubating for 20min at 37 ℃; during the incubation period, 4 times of ultrapure water is added into JC-1 staining buffer (5 x), mixed evenly and placed on ice; after the incubation was completed, the supernatant was discarded and the cells were washed twice with JC-1 staining buffer (1 ×); the culture medium was added and observed under a confocal laser scanning microscope.
3. Results of the experiment
(1) Establishment of HUVECs cell oxidative stress model
The results of the effect of SNP concentration on the survival of HUVECs cells are shown in FIG. 2: when the concentration of SNP is reduced from 2000 mu M to 100 mu M, the survival rate of HUVECs cells shows a straight-rising trend, and when the concentration of SNP is 800 mu M, the cell survival rate of HUVECs is 64.19%, and the cell damage degree under the concentration is more suitable, so that 800 mu M SNP is selected as a modeling agent to establish an oxidative stress model of HUVECs.
(2) Influence of phyllanthus emblica embryo polymethoxylated flavone extract on SNP (single nucleotide polymorphism) induced HUVECs cell oxidative damage
The results of the effect of the phyllanthus emblica embryo polymethoxylated flavone extract on the oxidative damage of the HUVECs cells induced by SNP are shown in FIG. 3: compared with a model group (SNP 800 mu M), the phyllanthus emblica maxim polymethoxylated flavone extract can reduce the oxidative damage of SNP to HUVECs cells, so that the survival rate of the HUVECs cells induced by SNP is increased, and the effect is more obvious along with the improvement of the phyllanthus emblica maximhoxylated flavone extract.
(3) Influence of phyllanthus emblica embryo polymethoxylated flavone extract on SNP (single nucleotide polymorphism) induced HUVECs cell morphology
In the embodiment, the Hoechst 33342 and the Rhodamin method are adopted to observe the change condition of the cell nucleus and the cytoplasm, the Hoechst 33342 can emit indigo fluorescence at 461nm through the cell membrane of a normal cell, and the Rhodamin can pass through the cell membrane to selectively dye the fluorescent dye of living cell mitochondria, so that ATP in the cell can be detected, and the cell exhibits yellow green fluorescence at 507 nm; the results are shown in FIG. 4: the cell morphology of the blank control group is fusiform, the damaged cell morphology of the model group is circular, the number of cells with fusiform cell morphology in the experimental group (the citrus reticulata embryo polymethoxylated flavone extract (TB) with different concentrations) is larger, the number of cells with circular cell morphology is smaller, and the number of cells with fusiform cell morphology is increased along with the increase of the concentration of the citrus reticulata embryo polymethoxylated flavone extract (TB); compared with the model group, the staining quantity and fluorescence intensity of the blank control group and the experimental group are higher than those of the model group, which shows that: the phyllanthus emblica embryo polymethoxylated flavone extract (TB) can antagonize SNP-induced HUVECs cell damage and has a certain protection effect on SNP-induced HUVECs cells.
(4) Influence of phyllanthus emblica embryo polymethoxylated flavone extract on SNP (single nucleotide polymorphism) induction of Reactive Oxygen Species (ROS) in HUVECs (human serum endothelial cells)
The results of the effect of the phyllanthus emblica embryo polymethoxylated flavone extract on the induction of Reactive Oxygen Species (ROS) in HUVECs cells by SNP are shown in fig. 5: the placebo had almost no ROS signal; after HUVECs are induced by SNP, ROS are increased sharply; after the phyllanthus emblica embryo polymethoxylated flavone extract (TB) is subjected to pretreatment (an experimental group), the ROS is obviously lower than that of a model group, and the fact that the phyllanthus emblica embryo polymethoxylated flavone extract can reduce active oxygen (ROS) in HUVECs induced by SNP is shown.
(5) Influence of phyllanthus emblica embryo polymethoxylated flavone extract on SNP (single nucleotide polymorphism) induction of mitochondrial membrane potential in HUVECs (human hematopoietic stem cells)
In this example, JC-1 fluorescent probe was used to detect changes in mitochondrial membrane potential, where JC-1 forms polymers (J-aggregates) existing in the matrix of mitochondria and emitting red fluorescence when the mitochondrial membrane potential is high, and JC-1 is a monomeric compound and shows green fluorescence when the mitochondrial membrane potential collapses, as shown in FIG. 6: the blank control group only showed red fluorescence but no green fluorescence, indicating that the mitochondrial membrane potential was normal; after HUVECs are induced by SNP, green fluorescence is obviously enhanced, red fluorescence is obviously reduced, and the reduction of mitochondrial membrane potential and damage of mitochondria are proved; after the phyllanthus emblica embryo polymethoxylated flavone extract (TB) is subjected to pretreatment (experimental group), the mitochondrial membrane potential is reduced in a concentration-dependent manner, and the higher the concentration is, the more the reduction is, namely, the stronger the mitochondrial pre-protection effect is.
Example 8 clinical experiments with Camellia sinensis embryo polymethoxylated flavone extract
10 test volunteers were selected to perform the use effect test on the chazu orange embryo polymethoxylated flavone extract prepared in example 1. The test method comprises the following steps: after washing the face twice a day in the morning and at night, only the chaga foetida extract prepared in example 1 was applied, and the antioxidant effect test and the determination of the water content in the cuticle of the epidermis were carried out after 4 hours, 7 days, 14 days, 21 days and 28 days, respectively: the water content in the stratum corneum was measured 3 times and the average was taken.
The antioxidant effect test uses ORAC method (oxidizing free radical absorption capacity), according to the principle that the free radical destroys the fluorescent probe to change the fluorescence intensity, the vitamin E water-soluble analogue Trolox is used as the quantitative standard, the fluorescent microplate analyzer is used for analysis, the change of the fluorescence intensity reflects the damage degree of the free radical, when the antioxidant exists, the fluorescence change caused by the free radical can be inhibited, the inhibition degree reflects the antioxidant capacity of the antioxidant to the free radical, and the higher the ORAC content is, the stronger the antioxidant capacity of inhibiting the free radical is. The test results are shown in table 4: after 28 days of using the chaga yunnanensis extract prepared in example 1, the average epidermal stratum corneum water content of the skin increased by 9.44%, and the average total antioxidant free radical capacity value (relative to the ratio of the equivalent vitamin E water-soluble analogue Trolox) was about 36% (increased by 7.97%), indicating that the chaga yunnanensis extract has better antioxidant free radical capacity.
Table 4 test of effect of lyophilized powder
In conclusion, the phyllanthus emblica embryo polymethoxylated flavone extract provided by the invention has a protective effect on SNP-induced HUVECs cell damage, can remarkably reduce the intracellular ROS level of SNP-induced HUVECs, and recovers the membrane potential of mitochondria to recover the functions of the mitochondria, thereby relieving oxidative stress damage and showing a better clinical antioxidant effect. The phyllanthus emblica embryo polymethoxylated flavone extract can be applied to various medicines, foods and skin care products with higher requirements on safety, and the oxidation resistance of corresponding products is improved; or as an antioxidant alone for use in various biological experiments. The tea branch citrus embryo polymethoxylated flavone extract can also be used as dietary supplement for eliminating redundant free radicals in vivo and preventing or delaying aging; or used as medicine or adjuvant for treating neurodegenerative disease, tumor or inflammation related to oxidative damage.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
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| CN115887551A (en) * | 2022-12-16 | 2023-04-04 | 中山大学 | A kind of natural source TARC/CCL17 and MDC/CCL22 inhibitor and its preparation and application |
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