CN107823244B - Preparation method and antioxidant use of soybean taro leaf ethanol extract - Google Patents
Preparation method and antioxidant use of soybean taro leaf ethanol extract Download PDFInfo
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- CN107823244B CN107823244B CN201711175738.1A CN201711175738A CN107823244B CN 107823244 B CN107823244 B CN 107823244B CN 201711175738 A CN201711175738 A CN 201711175738A CN 107823244 B CN107823244 B CN 107823244B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
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- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
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Abstract
本发明公开了一种豆芋叶乙醇提取物的制备方法，包括以下步骤：将豆芋叶和含0.1mol/L盐酸的85％乙醇混合后打浆、超声浸提、离心，得上清液；将上清液在旋转蒸发仪进行旋转蒸发浓缩；浓缩液采用大孔树脂进行分离纯化，以含有0.05％甲酸的甲醇水溶液作为洗脱剂；洗脱液旋转蒸发浓缩，所得的浆状浓缩液先预冻，然后用真空冷冻干燥机将其干燥成粉末状，得到豆芋叶乙醇提取物。本发明还同时公开了该豆芋叶乙醇提取物在制备神经细胞氧化损伤抑制剂中的应用。
The invention discloses a preparation method of taro leaf ethanol extract, comprising the following steps: mixing the bean taro leaf and 85% ethanol containing 0.1 mol/L hydrochloric acid, beating, ultrasonic extraction and centrifugation to obtain a supernatant; The supernatant was concentrated by rotary evaporation on a rotary evaporator; the concentrated solution was separated and purified by macroporous resin, and the methanol aqueous solution containing 0.05% formic acid was used as the eluent; the eluent was concentrated by rotary evaporation, and the obtained slurry concentrate was first. It is pre-frozen, and then dried into powder with a vacuum freeze dryer to obtain the ethanol extract of taro leaves. The invention also discloses the application of the soybean taro leaf ethanol extract in the preparation of nerve cell oxidative damage inhibitor.
Description
Technical Field
The invention relates to the field of medicines, and particularly relates to a preparation method of a bean taro leaf ethanol extract and an antioxidant application thereof.
Background
Soy taro (Apios americana Medikus) is native to the southern region of canada to florida, usa in eastern north america and belongs to the perennial family of legumes (subfamily Papilionaceae). The edible part is underground tuber, and the protein content is higher than that of other plant tubers, so that the edible part is a crop which is both nutritional and economic, and is often used as staple food by Indian people. The introduction of the yam beans is successful in 2009 domestically and is gradually popularized and planted in areas such as Fuyang, Jinhua, Longyou and Wenzhou. Research shows that different parts (flowers, vines, leaves and tubers) of the yam beans contain rich free amino acids, soluble proteins, polysaccharides, saponins, flavones, isoflavones, vitamin C, vitamin E, mineral substances and other components. The bean taro leaves have the highest vitamin E content, and also contain high-content polysaccharides, total saponins and isoflavones, so that the bean taro leaves have great development potential.
The American yam bean has obvious improvement effect on modern life habit diseases such as hypertension, diabetes, hyperlipidemia and the like.
The free radical is an atom or a group generated by a substance after being stimulated by an external condition, has very active unpaired electrons, and is easily combined with other atoms or groups to form a stable structure. During metabolism, the human body can generate free radicals, mainly including superoxide anion free radicals, hydroxyl free radicals, peroxy free radicals, nitrogen peroxide, nitric oxide and the like, to cause a series of biological reactions. When a human body is in a normal physiological condition, a free radical scavenging system (also called an antioxidant system) in the body can scavenge excessive free radicals, so that cells and tissues are prevented from being harmfully attacked by the free radicals; however, under pathological conditions, the production system and the scavenging system of endogenous free radicals in the human body are out of balance, so that the free radicals are excessive. The excess free radicals induce oxidative stress phenomena, which can cause oxidative damage to biological macromolecules (such as lipids, proteins and DNA), causing a series of biological reactions, resulting in the development of chronic degenerative diseases such as inflammation, cancer, cardiovascular diseases, Alzheimer's disease and aging.
Antioxidants have the ability to scavenge reactive oxygen species and oxygen radicals, are substances that prevent or retard autoxidation, and play a very important role in maintaining the normal vital activities of the body. The antioxidant can scavenge excessive free radicals in vivo, and keep the production system and scavenging system of free radicals in vivo in dynamic balance. Epidemiology has shown that antioxidants play a crucial role in the prevention of chronic diseases. Free radicals can induce oxidative stress reaction, sufficient antioxidants are supplemented to effectively prevent or delay the reaction, and human cells can be effectively protected from being damaged by oxidative stress, so that the prevalence rate of chronic diseases such as diabetes, cardiovascular diseases and cancers is reduced. Increasing research has shown that antioxidants in cereals, fruits and vegetables can prevent or reduce this potential oxidative damage. Ingestion of grains, fruits and vegetables is highly relevant for reducing the risk of diseases such as cancer. Therefore, many researchers of nutrition and health food are concerned with research and topics on anti-oxidation and chronic disease occurrence and prevention, but the anti-oxidation activity of the extract of the taro leaves is reported to a small extent.
Biochemical characteristic component analysis of different parts of American yam bean reports:
when measuring free amino acids, soluble proteins and total sugars: pulverizing, sieving with 0.42mm sieve, adding distilled water at 100 deg.C at a ratio of 3:500(m/v), and extracting in constant temperature water bath at 100 deg.C.
When the contents of the total saponins, the total flavonoids and the isoflavones are measured: adding absolute ethyl alcohol at a ratio of 1:100(m/v), and carrying out constant-temperature water bath at 60 ℃ for 3 h.
When VC measurement is carried out: a sample (1 g) was taken and soaked in 40ml of 0.01mol/L HCl and sonicated at 60 ℃ for 40 min.
When VE is measured, 3g of sample is taken, 20ml of absolute ethyl alcohol is added, and the mixture is shaken for 50min at a constant temperature of 50 ℃.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of a bean taro leaf ethanol extract and an antioxidant application thereof, and providing a new resource for developing natural antioxidants in the fields of medicine, food science and the like.
In order to solve the technical problems, the invention provides a preparation method of a bean taro leaf ethanol extract, which comprises the following steps:
1) mixing the bean taro leaves and 85% ethanol containing 0.1mol/L hydrochloric acid according to a material-liquid ratio of 1g: 5-10 ml (preferably 1g:8ml), pulping, performing ultrasonic extraction on the obtained pulp at 45 +/-5 ℃ for 240 +/-10 min, and centrifuging the extracting solution (for 30min at 4000 revolutions/min) to obtain a supernatant;
2) carrying out rotary evaporation concentration on the supernatant in a rotary evaporator until the volume of the supernatant is 5-10% of the original volume;
3) separating and purifying the concentrated solution obtained in the step 2) by adopting macroporous resin; collecting eluent by using 1% formic acid aqueous solution with volume concentration as eluent and using methanol aqueous solution containing 0.05% formic acid as eluent;
the preparation method of the methanol aqueous solution containing 0.05 percent of formic acid comprises the following steps: adding 0.05ml of formic acid into 100ml of 90% methanol aqueous solution with volume concentration;
4) carrying out rotary evaporation and concentration on the eluent obtained in the step 3) to obtain 5-10% of the original volume, and obtaining a slurry concentrated solution;
5) pre-freezing the slurry-like concentrated solution obtained in the step 4) at-70 to-90 ℃ for 5 to 7 hours, and then drying the slurry-like concentrated solution into powder by using a vacuum freeze dryer to obtain the ethanol extract of the bean taro leaves.
The improvement of the preparation method of the soybean taro leaf ethanol extract of the invention comprises the following steps:
the step 3) is as follows:
firstly, activating macroporous resin by using methanol with twice column volume, and balancing by using double distilled water with three column volumes; then, sampling at a flow rate of 0.4-0.6 mL/min (preferably 0.5mL/min), after the extracting solution is sufficiently adsorbed, eluting with a formic acid aqueous solution with a volume concentration of 1% as an eluting agent (the eluting agent is used for removing substances such as proteins, sugars and acids), wherein the amount of the eluting agent is twice the column volume, and the flow rate is 0.4-0.6 mL/min (preferably 0.5 mL/min); finally, using a methanol water solution containing 0.05% formic acid as an eluent for elution, wherein the dosage of the eluent is three times of the column volume, and the flow rate is 0.4-0.6 mL/min (preferably 0.5 mL/min); collecting the eluent.
As a further improvement of the preparation method of the soybean taro leaf ethanol extract of the present invention: the macroporous resin in the step 3) is macroporous resin AB-8 (Tianjin south allows high molecular technology Co., Ltd.).
As a further improvement of the preparation method of the soybean taro leaf ethanol extract of the present invention: replacing the soybean taro leaves in the step 1) with the filter residues obtained by centrifugation in the step 1) to repeatedly carry out pulping, ultrasonic leaching and centrifugation in the step 1); the repetition times are 2-3 times;
all supernatants from the centrifugation were combined and subjected to step 2).
The invention also provides application of the soybean taro leaf ethanol extract in preparation of a nerve cell oxidative damage inhibitor.
In the present invention:
the rotary evaporation in the step 2) and the step 4) is carried out at the temperature of 38 +/-2 ℃ and the vacuum degree of-0.09 MPa.
The vacuum freeze drying of the step 5) is carried out at the temperature of minus 40 +/-2 ℃ and under the vacuum degree of 1.2Pa until the water content of the obtained ethanol extract of the bean taro leaves is less than or equal to 0.1 percent.
The invention also provides application of the soybean taro leaf ethanol extract prepared by any one of the methods in preparation of a nerve cell oxidative damage inhibitor.
The invention adopts PC12 cells (rat nerve cells) to carry out experiments, determines the influence of the treatment of the ethanol extract of the Doudouye on the proliferation activity of the PC12 cells by a thiazole blue (MTT) method, and adds hydrogen peroxide (H) after the ethanol extract of the Doudouye is adopted and protected for 24 hours in advance2O2) Inducing for 4h to construct a cell oxidative stress model, and discussing the protective effect of the soybean taro leaf ethanol extract on cell oxidative damage. Namely, the present invention provides a method for preparing a soybean taro leaf ethanol extract from hydrogen peroxide (H)2O2) Use of an inhibitor of oxidative damage to induced nerve cells.
The ethanol extract of the taro leaves can be developed into natural antioxidant drugs and foods to replace artificially synthesized antioxidants to treat diseases caused by excessive free radicals in human bodies.
The usage of the soybean taro leaf ethanol extract is oral administration, and the dosage is about 150-250 mg each time and three times a day.
The ethanol extract of the taro leaves prepared by the method can effectively protect the hydrogen peroxide (H)2O2) The induced nerve cell oxidative damage reduces the generation of active oxygen, and has good development prospect. The invention provides a new medical application for the ethanol extract of the bean taro leaves and expands a new application field.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 shows the ratio of ethanol extract of Doudouye to hydrogen peroxide (H) at different concentrations2O2) A graph comparing the effects of induced neural cell proliferation activity;
FIG. 2 shows the leaves of Doudou taro in different concentrationsEthanol extract to hydrogen peroxide (H)2O2) A map of the effects of induced neuronal cell morphology;
FIG. 3 shows the ratio of ethanol extract of Doudouye to hydrogen peroxide (H) at different concentrations2O2) Map of the effect of induced neuronal cell nuclear damage;
FIG. 4 is a graph showing the results of quantitative analysis of the fluorescence intensity in FIG. 3;
FIG. 5 shows the ratio of ethanol extract of Doudouye to hydrogen peroxide (H) at different concentrations2O2) The effect of induced reactive oxygen radicals (DHE fluorescence) of nerve cells;
FIG. 6 is a graph showing the results of quantitative analysis of the fluorescence intensity in FIG. 5;
FIG. 7 shows the ratio of ethanol extract of Doudouye to hydrogen peroxide (H) at different concentrations2O2) A graph of the effect of induced mitochondrial membrane potential in nerve cells;
FIG. 8 is a graph showing the results of quantitative analysis of the fluorescence intensity in FIG. 7;
FIG. 9 shows the concentration of ethanol extract of Doudouye versus hydrogen peroxide (H)2O2) A graph comparing the effects of induced mitochondrial membrane lipid peroxidation in nerve cells;
FIG. 10 is a graph showing the results of quantitative analysis of the fluorescence intensity in FIG. 9;
specifically, the following description is provided: in fig. 2, 3, 5, 7, 9, a. control; b.H2O2A model group; c.H2O2+ low concentration of the ethanol extract of the soybean taro leaves group (L); d.H2O2+ medium concentration of the ethanol extract group of the Doudouye (M); e.H2O2+ high concentration ethanol extract of taro leaves group (H), some technical indicators may be lost due to the gray scale processing of color pictures. In FIGS. 4, 6, 8 and 10, Control represents a Control group, H2O2Representing a set of models, L represents H2O2+ low concentration of ethanol extract of Doudouye (Doudouye Aromatica), H represents H2O2+ medium concentration of ethanol extract of Doudouye (Doudouye Aromatica), M represents H2O2+ high concentration ethanol extract of Doudouye.
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.
Example 1, a method for preparing an ethanol extract of taro leaves, sequentially comprising the following steps:
1) mixing the bean taro leaves with 85% ethanol containing 0.1mol/L hydrochloric acid in a ratio of 1g to 8ml, and pulping by using a juicer; ultrasonic leaching the obtained slurry at 45 deg.C (ultrasonic frequency of 40,000 Hz) for 240min, centrifuging the extractive solution at 4000 rpm for 30min,
replacing the bean taro leaves in the above steps with filter residues obtained by centrifugation, and repeatedly carrying out pulping, ultrasonic leaching and centrifugation in the above steps; the number of repetitions was 3;
the supernatants from the 3 centrifugations were combined and subjected to the following step 2).
2) Carrying out rotary evaporation concentration on the obtained supernatant in a rotary evaporator (the set process parameters of the rotary evaporation are 38 ℃ and the vacuum degree is-0.09 MPa) until the volume of the supernatant is 10 percent of the original volume;
3) separating and purifying the concentrated solution obtained by rotary evaporation by adopting macroporous resin, wherein the adopted macroporous resin is macroporous resin AB-8 (Technok Nanyu polymer technology Co., Ltd.), and the following steps are sequentially carried out:
firstly, activating macroporous resin by using methanol with twice column volume, and balancing by using double distilled water with three column volumes;
then, the sample is loaded at a flow rate of 0.5mL/min, and the loading amount is 200mL (10% of the column volume); after the extract is fully adsorbed, eluting with 1% formic acid (namely 1% formic acid aqueous solution with volume concentration) to remove substances such as protein, sugar, acid and the like, wherein the dosage of the eluting agent is twice of the column volume, and the flow rate is 0.5 mL/min; finally, using methanol water solution containing 0.05 percent of formic acid as eluent to fully elute, wherein the dosage of the eluent is three times of the column volume, and the flow rate is 0.5 mL/min; collecting the eluent.
The preparation method of the methanol aqueous solution containing 0.05 percent of formic acid comprises the following steps: adding 0.05ml of formic acid into 100ml of 90% methanol aqueous solution with volume concentration;
4) performing rotary evaporation and concentration on the eluent obtained by separating and purifying the macroporous resin until the concentration is 10 percent of the original volume by rotary evaporation (the set process parameters of the rotary evaporation are 38 ℃ and the vacuum degree is-0.09 MPa) to obtain slurry-like concentrated solution;
5) pre-freezing the concentrated solution at-80 deg.C for 6h, and drying with vacuum freeze drier for 48h (the vacuum freeze drier has set technological parameters of-40 deg.C and 1.2Pa) to obtain powder (water content is less than or equal to 0.1%) to obtain ethanol extract of folium Colocasiae Esculentae.
Experiment I, ethanol extract of Doudouye to hydrogen peroxide (H)2O2) Effect of induced neural cell proliferation Activity
Inoculating cultured PC12 cells into 96-well plate with 5000 cells per well, standing at 37 deg.C and 5% CO2After incubation for 24H in an incubator, the groups were divided into blank control group and H2O2Group, 10, 20, 40, 80, 160, 320, 640, 1280ug/mL of ethanol extract of Doudouye, repeating 8 wells for each group, placing at 37 deg.C and 5% CO2After incubation for 24H in the incubator, the residual liquid in the wells was aspirated, rinsed twice with Phosphate Buffered Saline (PBS) solution and added to the control group, H2O2Adding H into group, 10, 20, 40, 80, 160, 320, 640 and 1280ug/mL2O2The culture broth with a final concentration of 150uM was cultured for 2 h. The residual liquid was then aspirated from each well, rinsed twice with Phosphate Buffered Saline (PBS) solution, and 100ul of MTT (0.5mg/mL in serum free medium) was added at 37 deg.C with 5% CO2The cultivation was continued in the incubator for 4 hours. Subsequently, the liquid was removed from each well, 150ul of dimethyl sulfoxide (DMSO) was added to each well, shaken on a horizontal shaker for 10 minutes, and the absorbance was measured at 570nm with a microplate reader.
Cell proliferation activity (%). assay well OD value/control well OD value 100%.
As shown in FIG. 1, the results of the MTT experiment showed that the reaction was carried out at H2O2Adding the ethanol extracts of the bean taro leaves with different concentrations on the basis of induction injury for pre-culture, finding that the cell survival rates are superior to those of an oxidative injury model, wherein the cell survival rates are remarkably superior to those of the injury model under the concentrations of 320 ug/mL and 640ug/mL, and have no remarkable difference with the cell survival rate of an undamaged control group, so that the ethanol extracts of the bean taro leaves are proved to be 0-1280uThe concentration range of g/mL has no toxicity to PC12 cells, and is beneficial to repairing cell damage.
Experiment II, the ethanol extract of the Doudouye is used for treating hydrogen peroxide (H)2O2) Effect of induced neuronal cell morphology
Selecting PC12 cells in logarithmic growth phase, digesting monolayer cells with 0.25% pancreatin, preparing single cell suspension with RPIM1640 medium containing 10% new bovine serum, and culturing at 1 × 106Inoculating to 6-well plate, culturing in incubator for 24 hr, sucking out culture medium, treating with ethanol extract of folium Colocasiae Esculentae with concentration of 50, 100, and 200ug/mL (respectively corresponding to low concentration, medium concentration, and high concentration) for 24 hr, and treating with H2O2(final concentration of hydrogen peroxide 150. mu.M) treatment of cells for 2h induced oxidative stress cell model, followed by microscopic observation of cell morphology and photography.
As can be seen from FIG. 2, the number of cells in the visual field was decreased, the cytoplasm was dehydrated and concentrated, the nucleus was increased, the cell volume was decreased, and the apoptosis was exhibited as compared with the control group in the model group, indicating that H2O2The successful construction of the model induces cells to generate oxidative damage, thereby causing cell apoptosis. Cells added with the extract of the leaves of the bean taro with different concentrations for pre-culture show a relatively normal cell shape under the condition of oxidative stress, and the cell shape of a high-concentration culture group is basically similar to that of a control group along with the continuous increase of the concentration, so that the extract of the leaves of the bean taro with ethanol has the functions of repairing and protecting the oxidative damage of the cells.
Experiment III, the ethanol extract of the Doudouye is used for treating hydrogen peroxide (H)2O2) Effect of induced neuronal cell Nuclear Damage
Selecting PC12 cells in logarithmic growth phase, digesting monolayer cells with 0.25% pancreatin, preparing single cell suspension with RPIM1640 medium containing 10% new bovine serum, and culturing at 1 × 106Inoculating to 6-well plate, culturing in incubator for 24 hr, sucking out culture medium, treating with ethanol extract of folium Dolichos taro at concentration of 50, 100, and 200ug/mL for 24 hr, and treating with H2O2(final concentration of hydrogen peroxide 150. mu.M) treatment of cells for 2hAnd (3) guiding the oxidative stress cell model, adding Hoechst 33258 with the final concentration of 10 mu M respectively, dyeing for 30min, washing the dye, observing by using a fluorescence microscope, photographing, and calculating the average optical density value.
As shown in FIG. 3, the nuclear brightness of the model group was significantly increased compared to the control group, and dense and bright blue fluorescence was formed, and the blue mass was fragmented, and chromatin shrinkage was highlighted. This is particularly evident in FIG. 3b, indicating the occurrence of apoptosis. In the group pretreated by the bean taro leaf extract, the nuclear fluorescence intensity is reduced to different degrees compared with that of the model group, the nuclear shape is regular, the nuclear membrane is complete, the blue fluorescence is uniformly dispersed, and the trend of shrinkage reduction is consistent with that of the cell form shown in figure 2, which shows that the bean taro leaf ethanol extract has H pair2O2The induced oxidative damage of cell nucleus has protective effect. The quantitative results for each treatment group are shown in figure 4.
Experiment four, ethanol extract of Doudouye (Doudouye taro) was used for hydrogen peroxide (H)2O2) Effect of induced active oxygen free radicals (DHE fluorescence) in nerve cells
Selecting PC12 cells in logarithmic growth phase, digesting monolayer cells with 0.25% pancreatin, preparing single cell suspension with RPIM1640 medium containing 10% new bovine serum, and culturing at 1 × 106Inoculating to 6-well plate, culturing in incubator for 24 hr, sucking out culture medium, treating with ethanol extract of folium Dolichos taro at concentration of 50, 100, and 200ug/mL for 24 hr, and treating with H2O2(final concentration of hydrogen peroxide 150. mu.M) cells were treated for 2h to induce oxidative stress cell model, 10. mu.M DHE was added to each cell to stain for 30min, the dye was washed and observed with a fluorescence microscope and photographed, and the average optical density value was calculated.
As can be seen from fig. 5, the fluorescence intensity of the model group was significantly increased compared to the control group, and the semi-quantitative value was the highest value among the five groups. Whereas the fluorescence intensity of the group pre-cultured with the extract of Doudou leaves was significantly reduced, and as the concentration of the extract increased, H was observed2O2The content of induced ROS is reduced, and the fact that the soybean taro leaf ethanol extract can effectively eliminate reactive oxygen Radical (ROS) is fully proved. The quantitative results for each treatment group are shown in FIG. 6.
Experiment five, ethanol extract of Doudouye to hydrogen peroxide (H)2O2) Effect of induced mitochondrial Membrane potential in nerve cells
Selecting PC12 cells in logarithmic growth phase, digesting monolayer cells with 0.25% pancreatin, preparing single cell suspension with RPIM1640 medium containing 10% new bovine serum, and culturing at 1 × 106Inoculating to 6-well plate, culturing in incubator for 24 hr, sucking out culture medium, treating with ethanol extract of folium Dolichos taro at concentration of 50, 100, and 200ug/mL for 24 hr, and treating with H2O2The cells were treated for 2h (final hydrogen peroxide concentration 150. mu.M) to induce oxidative stress in the cell model, and then stained with a mitochondrial membrane potential probe rhodamine 123(RH123) fluorescent probe at 5. mu.g/mL for 30min, and after washing with the dye, the cells were observed with a fluorescence microscope and photographed, and the average optical density value was calculated.
As can be seen from fig. 7, the fluorescence intensity of the model group was significantly lower than that of the control group, and the number of cells visible in the visual field was also significantly reduced, indicating that oxidative stress caused a decrease in the mitochondrial membrane potential of the cells, a change in the mitochondrial matrix volume, a decrease in ATP, and finally a failure in maintenance of mitochondrial function, resulting in cell death. The cells added with the bean taro leaf ethanol extract for pre-culture still keep the mitochondrial membrane potential level and quantity superior to those of the model group under the oxidative stress condition, which shows that the bean taro leaf ethanol extract can effectively inhibit the mitochondrial damage caused by active oxygen, and the inhibition effect is positively correlated with the action concentration within the safe concentration range. The quantitative results for each treatment group are shown in FIG. 8.
Experiment six, ethanol extract of Doudouye to hydrogen peroxide (H)2O2) Effect of induced mitochondrial Membrane lipid peroxidation in nerve cells
Selecting PC12 cells in logarithmic growth phase, digesting monolayer cells with 0.25% pancreatin, preparing single cell suspension with RPIM1640 medium containing 10% new bovine serum, and culturing at 1 × 106Inoculating to 6-well plate, culturing in incubator for 24 hr, sucking out culture medium, treating with ethanol extract of folium Dolichos taro at concentration of 50, 100, and 200ug/mL for 24 hr, and treating with H2O2(final concentration of hydrogen peroxide 150. mu.M) treatment of cellsInducing oxidative stress cell model for 2h, adding 10 μ M NAO, staining for 30min, washing with dye, observing with fluorescence microscope, photographing, and calculating average optical density value.
As can be seen from fig. 9, compared with the control group, the numbers of mitochondria in the model group and the ethanol extract from the leaves of the legume taro in the visual field are reduced, but the average fluorescence intensity is increased with the increase of the extract concentration, and the average fluorescence intensity is significantly enhanced compared with the model group, and the trend is consistent with the staining result of the Rh123 probe, which proves that the ethanol extract from the leaves of the legume taro can effectively alleviate the lipid peroxidation condition of the mitochondrial membrane. The quantitative results for each treatment group are shown in FIG. 10.
Comparative example 1-1, the "85% ethanol with 0.1mol/L hydrochloric acid" in step 1) of example 1 was changed to "pure ethanol with 0.1mol/L hydrochloric acid", and the rest was the same as in example 1.
Comparative example 1-2, the 0.1 mol/L85% ethanol with hydrochloric acid in step 1) of example 1 was changed to "0.1 mol/L70% ethanol with hydrochloric acid", and the rest was identical to example 1.
Comparative example 2-1, the eluent in step 3) of example 1 was changed from "an aqueous methanol solution containing 0.05% formic acid" to "an aqueous methanol solution (90% aqueous methanol solution by volume)"; the rest is equivalent to embodiment 1.
Comparative example 2-2, the eluent in step 3) of example 1 was changed from "an aqueous methanol solution containing 0.05% formic acid" to "an aqueous methanol solution containing 0.1% formic acid (90% aqueous methanol solution by volume)"; the rest is equivalent to embodiment 1.
"90% aqueous methanol solution containing 0.05% formic acid" as an eluent in comparative example 3-1, example 1, step 3), the volume concentration of the aqueous methanol solution was changed from 90% to 80%; the rest is equivalent to embodiment 1.
"90% aqueous methanol solution containing 0.0.5% formic acid" as an eluent in comparative example 3-2, example 1, step 3), the volume concentration of the aqueous methanol solution was changed from 90% to 100% (i.e., pure methanol); the rest is equivalent to embodiment 1.
Comparative example 4, the macroporous resin is changed from macroporous resin AB-8 to macroporous adsorption resin D-101, and the rest is identical to example 1.
The ethanol extracts of the leaves of the legume taro obtained in all of the above comparative examples were examined according to the methods described in the first and fourth experiments, and the results are shown in table 1 below.
TABLE 1
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
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| CN107281258A (en) * | 2017-07-19 | 2017-10-24 | 浙江大学 | A kind of beans taro leaf flavonoid extract and its preparation method and application |
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| CN107281258A (en) * | 2017-07-19 | 2017-10-24 | 浙江大学 | A kind of beans taro leaf flavonoid extract and its preparation method and application |
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