CN111357895A - Application mode of grapefruit extract in traditional Chinese medicine preparation or functional food - Google Patents
Application mode of grapefruit extract in traditional Chinese medicine preparation or functional food Download PDFInfo
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- A23L2/02—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation containing fruit or vegetable juices
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
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- 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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- A61K2236/50—Methods involving additional extraction steps
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Abstract
The invention relates to the technical field of grapefruit extracts, in particular to an application mode of grapefruit extracts in a traditional Chinese medicine preparation or functional food, which comprises a preparation method of the grapefruit extracts and application of the grapefruit extracts in the traditional Chinese medicine preparation or the functional food, wherein the traditional Chinese medicine preparation and the functional food have the effect of preventing and treating pneumonia, and the grapefruit extracts can be used for preventing and treating the pneumonia.
Description
Technical Field
The invention relates to the technical field of grapefruit extract, in particular to an application mode of the grapefruit extract in a traditional Chinese medicine preparation or a functional food.
Background
Huyou (Citrus changshanensis) is a filial generation of pomelo and lime, is a special hybrid Citrus fine breed in Changshan county in Quzhou city in Zhejiang province, and has a planting history for hundreds of years up to now. The fruit juice is unique and rich in fragrance, attractive in appearance, bright yellow, rich in luster, golden yellow in pulp, rich in juice, moderate in sweetness and sourness and slightly bitter, and is very tasty and refreshing after being eaten. The grapefruit has a plurality of pharmacological effects of cooling, fire removing, cough relieving, phlegm reducing, throat moistening, alcohol effect dispelling, skin nourishing, life prolonging and the like, and has high nutritional value. However, there is no report on whether grapefruit extract has an effect of treating pneumonia in a living body. .
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide an application mode of a grapefruit extract in a traditional Chinese medicine preparation or a functional food.
In order to achieve the purpose, the invention provides the following technical scheme: an application mode of a grapefruit extract in a traditional Chinese medicine preparation or a functional food, wherein the preparation method of the grapefruit extract comprises the following steps:
(1) selecting raw materials, namely selecting fresh grapefruit without mildew, rot, diseases, insect pests and black spot diseases;
(2) processing raw materials, namely cleaning fresh grapefruit, separating pulp from peel, and squeezing the pulp to obtain fruit juice and fruit residues; cutting pericarp, and mixing with crushed pomace;
(3) extraction: mixing the pomace and the pericarp, adding 6-12 times of purified water, adding calcium chloride powder and cellulase, heating to 30-36 deg.C, soaking for half an hour, heating and reflux-extracting, filtering, collecting the extractive solution, repeating for 2 times, and mixing the filtrates;
(4) concentration: vacuum decompression and low-temperature concentration are carried out on the leachate, and the temperature is not more than 80 ℃;
(5) and (3) drying: drying the concentrated extract at low temperature, wherein the temperature is controlled below 80 ℃, and the water content is controlled below 7%;
the Chinese medicinal preparation and the functional food have the functions of preventing and treating pneumonia,
the traditional Chinese medicine preparation is a lung moistening and protecting pomelo pear syrup formula, and the functional food is a grapefruit lung moistening and protecting functional beverage.
Preferably, in the step (3) of the preparation method of the grapefruit extract, the heating temperature is 95-120 ℃, and the reflux extraction time is 0.6-1 hour.
Preferably, the preparation method of the lung moistening and protecting pomelo pear syrup formula comprises the following steps:
(1) selecting fresh moldy and rotten-free snow pears;
(2) peeling and removing cores of snow pears, juicing and filtering to obtain filtered juice for later use;
(3) mixing the filtered juice with the fruit juice obtained in the step (2) of the preparation method of the grapefruit extract to obtain grapefruit pear juice;
(4) respectively carrying out vacuum decompression and low-temperature concentration on the leaching solution obtained in the step (3) in the preparation method of the grapefruit extract and the grapefruit pear juice at the temperature not higher than 80 ℃ to respectively obtain a grapefruit extract and a snow pear extract;
(5) mixing the grapefruit extract and the snow pear extract, adding crystal sugar and mint, heating, uniformly mixing and filling to obtain the paste formula.
Preferably, in the preparation method of the lung moistening and protecting pomelo-pear syrup formula, the pomelo-pear juice in the step (3) is prepared by mixing fruit juice and filtered juice in a ratio of 3: 7.
Preferably, in the preparation method of the lung moistening and lung protecting pomelo-pear syrup formula, the weight ratio of the mixed material of the pomelo extract and the snow pear extract in the step (5) is 4: 6-2: 8.
Preferably, the operation flow of the step (5) in the preparation method of the lung moistening and protecting pomelo-pear syrup prescription is to mix, fuse and convert materials in a vacuum stirring tank.
Preferably, the preparation method of the grapefruit lung moistening and protecting functional beverage comprises the following steps:
(1) respectively carrying out vacuum reduced pressure low-temperature concentration on the leachate obtained in the step (3) in the preparation method of the grapefruit extract and the juice obtained in the step (2) in the preparation method of the grapefruit extract, wherein the temperature is not higher than 80 ℃, and respectively obtaining a grapefruit extract and a grapefruit concentrated juice;
(2) mixing the grapefruit extract and grapefruit concentrated juice into beverage according to a predetermined ratio, and processing to obtain functional fruit juice;
(3) drying the extract at low temperature, controlling water content below 7%, mixing with solid beverage, granulating, and processing to obtain functional solid preparation.
Preferably, in the preparation method of the grapefruit lung moistening and protecting functional beverage, in the operation flow of the step (2), the adding proportion of the grapefruit extract and the grapefruit concentrated juice in the beverage is not more than 20%.
Preferably, in the operation flow of the step (3) in the preparation method of the grapefruit lung moistening and protecting functional beverage, the addition proportion of the grapefruit extract in the solid beverage is not more than 15%.
Compared with the prior art, the invention has the beneficial effects that: the grapefruit extract has a remarkable preventive and therapeutic effect on pneumonia, and the features, objects and advantages of the present invention will be more apparent from the explanation of the claims below.
Drawings
FIG. 1 shows the position of zebra fish swim bladder (inflammation part of analysis and statistical data) in the anti-inflammatory action of LPS-induced inflammatory neutrophils;
FIG. 2 is a table diagram showing the influence of each experimental group on the inflammation of zebra fish in the anti-inflammatory action of LPS-induced inflammatory neutrophils (the yellow dotted line frame is the position of the zebra fish swim bladder);
FIG. 3 is a graph showing the effect of grapefruit extract on inflammatory (neutrophil count) of zebrafish in the anti-inflammatory effect of LPS-induced inflammatory neutrophils in accordance with the present invention;
FIG. 4 is a graph showing the effect of grapefruit extract on the regression of inflammation in the anti-inflammatory effect of LPS-induced inflammatory neutrophils in accordance with the present invention;
FIG. 5 shows the position of the swim bladder of zebra fish (the inflammation part of the analysis and statistical data) in the anti-inflammatory action of LPS-induced inflammatory macrophages;
FIG. 6 is a table diagram showing the influence of each experimental group on the inflammation of zebra fish in the anti-inflammatory action of LPS-induced inflammatory macrophages (the yellow dotted line frame is the position of the zebra fish swim bladder);
FIG. 7 is a graph showing the effect of grapefruit extract on zebrafish inflammation (macrophages) in the anti-inflammatory effect of LPS-induced inflammatory macrophages according to the present invention;
FIG. 8 is a graph showing macrophage improving effect of grapefruit extract in anti-inflammatory effect on LPS-induced inflammatory macrophages according to the present invention;
FIG. 9 is a graph showing the relative expression level of TGF β -1 in the effect of the present invention on inflammation-associated genes;
FIG. 10 is a graph showing the relative expression level of IL-1 β in the effect of the present invention on inflammation-associated genes;
FIG. 11 shows the position of zebra fish swim bladder (yellow dotted line) in the histopathological analysis of the present invention;
FIG. 12 is a graph of the effect of grapefruit extract on swim bladder (yellow arrows are inflammatory cells) in the histopathological analysis of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a technical scheme that: an application mode of a grapefruit extract in a traditional Chinese medicine preparation or a functional food, wherein the preparation method of the grapefruit extract comprises the following steps:
(1) selecting raw materials, namely selecting fresh grapefruit without mildew, rot, diseases, insect pests and black spot diseases;
(2) processing raw materials, namely cleaning fresh grapefruit, separating pulp from peel, and squeezing the pulp to obtain fruit juice and fruit residues; cutting pericarp, and mixing with crushed pomace;
(3) extraction: mixing the pomace and the pericarp, adding 6-12 times of purified water, adding calcium chloride powder and cellulase, heating to 30-36 deg.C, soaking for half an hour, heating and reflux-extracting, filtering, collecting the extractive solution, repeating for 2 times, and mixing the filtrates;
(4) concentration: vacuum decompression and low-temperature concentration are carried out on the leachate, and the temperature is not more than 80 ℃;
(5) and (3) drying: drying the concentrated extractive solution at low temperature below 80 deg.C and water content below 7%.
The heating reflux extraction step of the step (3) in the preparation method of the grapefruit extract has the heating temperature of 95-120 ℃ and the reflux extraction time of 0.6-1 hour.
The Chinese medicinal preparation and the functional food have the functions of preventing and treating pneumonia,
the traditional Chinese medicine preparation is a lung moistening and protecting pomelo pear syrup formula, and the functional food is a grapefruit lung moistening and protecting functional beverage.
Example 1, an embodiment of a grapefruit extract in a cream formulation, a lung moistening and lung protecting grapefruit pear syrup formulation, is prepared by:
(1) selecting fresh moldy and rotten-free snow pears;
(2) peeling and removing cores of snow pears, juicing and filtering to obtain filtered juice for later use;
(3) mixing the filtered juice with the fruit juice obtained in the step (2) of the preparation method of the grapefruit extract to obtain grapefruit pear juice;
(4) respectively carrying out vacuum decompression and low-temperature concentration on the leaching solution obtained in the step (3) in the preparation method of the grapefruit extract and the grapefruit pear juice at the temperature not higher than 80 ℃ to respectively obtain a grapefruit extract and a snow pear extract;
(5) mixing the grapefruit extract and the snow pear extract, adding crystal sugar and mint, heating, uniformly mixing and filling to obtain the paste formula.
In the preparation method of the lung moistening and protecting pomelo-pear syrup formula, the pomelo-pear juice in the step (3) is prepared by mixing fruit juice and filtered juice in a ratio of 3: 7.
In the preparation method of the lung moistening and lung protecting pomelo-pear syrup formula, the weight ratio of the mixed material of the pomelo extract and the snow pear extract in the step (5) is 4: 6-2: 8.
The operation flow of the step (5) in the preparation method of the lung moistening and protecting pomelo-pear syrup prescription is to perform material mixing, fusion and transformation in a vacuum stirring tank.
Example 2, an embodiment of a grapefruit extract in a functional beverage, wherein the grapefruit lung-moistening and lung-protecting functional beverage is prepared by the following steps:
(1) respectively carrying out vacuum reduced pressure low-temperature concentration on the leachate obtained in the step (3) in the preparation method of the grapefruit extract and the juice obtained in the step (2) in the preparation method of the grapefruit extract, wherein the temperature is not higher than 80 ℃, and respectively obtaining a grapefruit extract and a grapefruit concentrated juice;
(2) mixing the grapefruit extract and grapefruit concentrated juice into beverage according to a predetermined ratio, and processing to obtain functional fruit juice;
(3) drying the extract at low temperature, controlling water content below 7%, mixing with solid beverage, granulating, and processing to obtain functional solid preparation.
In the preparation method of the grapefruit lung moistening and protecting functional beverage, in the operation flow of the step (2), the grapefruit extract and the grapefruit concentrated juice are added in the beverage in a proportion of not more than 20%.
In the operation flow of the step (3) in the preparation method of the grapefruit lung moistening and protecting functional beverage, the addition proportion of the grapefruit extract in the solid beverage is not more than 15%.
The beverage and solid beverage are grapefruit juice or dried powder.
Aiming at the prevention and treatment effects of the grapefruit extract on pneumonia, the following experimental demonstration is carried out:
experiment 1, experiment of anti-inflammatory action of grapefruit extract on LPS-induced inflammatory neutrophils:
(1) selecting 180-tailed 5dpf transgenic neutrophilic granulocyte fluorescent zebra fish;
(2) putting the zebra fish into a six-hole plate, and treating 30 zebra fish in each hole;
(3) all zebra fish are subjected to intravenous injection and LPS (low-pressure lipoprotein lipase) administration, and a zebra fish pneumonia model is established;
(4) the grapefruit extracts were administered to model zebrafish in 3 wells each dissolved in water in a six-well plate at concentrations of 889, 2667 and 8000 μ g/mL;
(5) in a six-well plate, one of the other 3 wells added with grapefruit extract is treated with standard dilution water to serve as a normal control group, the other one of the wells is taken as a model control group, and the remaining one of the wells is added with indometacin with the concentration of 60 mu M to serve as a positive control group;
(6) after the zebra fish in each hole is treated in a six-hole plate for 4 hours, selecting 10 zebra fish randomly, observing under a fluorescence microscope, taking a picture and storing the picture;
(7) analyzing the stored pictures by Nikon NIS-Elements D3.10 advanced image processing software, and calculating the number of neutrophils at the inflammatory part of the zebra fish;
(8) the anti-inflammatory effect of grapefruit extract on LPS-induced pneumonia of zebra fish was evaluated statistically.
The statistics of step (8) in the experimental mode of anti-inflammatory action of grapefruit extract on LPS-induced inflammatory neutrophils were statistically analyzed using analysis of variance and Dunnett's T-test, with p <0.05 indicating significant differences.
The calculation formula of the anti-inflammatory effect of the grapefruit extract in the step (8) in the experimental manner of the anti-inflammatory effect of the grapefruit extract on LPS-induced inflammatory neutrophils is as follows:
according to the experimental mode in experiment 1, the concentration comparison of multiple groups of extracts is carried out, the grapefruit extract does not cause death of zebrafish and does not see other obvious toxic phenotypes under the concentrations of 63, 125, 250, 500, 1000, 2000, 4000 and 8000 mug/mL, and the zebrafish is completely dead under the concentrations of 16000 and 32000 mug/mL, so that the maximum detection concentration (MTC) of the grapefruit extract on a zebrafish pneumonia model is determined to be 8000 mug/mL, and the concentration of the subsequent formal experiment is set as follows: 889 μ g/mL (1/9MTC), 2667 μ g/mL (1/3MTC) and 8000 μ g/ML (MTC). The alignment data are detailed in table 1.
Table 1 experimental results of "concentration-mortality" after treatment with grapefruit extract (n ═ 30)
After the experiment, the number of neutrophils (22) at the inflammatory part of the zebra fish in the model control group is compared with that of the neutrophils (3) in the normal control group, and p is less than 0.001, so that the success of LPS (low-temperature plasma) induced zebra fish pneumonia model is suggested. Compared with a model control group, the number of neutrophils (13) at the inflammatory part of the zebra fish in the 60 mu M indometacin group is less than 0.001, and the inflammation regression effect is 40.9 percent, which shows that the indometacin has obvious anti-inflammatory effect on the pneumonia zebra fish.
When the concentrations of the dichroa febrifuga extract are 889, 2667 and 8000 mu g/mL, the numbers of neutrophils at the inflammatory parts of the zebra fish are respectively 17, 14 and 15, and compared with a model control group (22), the p is less than 0.01 and less than 0.001, the inflammation resolution effects are respectively 22.7%, 36.4% and 31.8%, which indicates that the dichroa febrifuga extract has obvious anti-inflammatory effect on the pneumonia zebra fish under the experimental concentration condition.
The detailed data are shown in table 2, fig. 1, fig. 2, fig. 3 and fig. 4.
Table 2. effect of each experimental group on inflammation of zebrafish (neutrophil) (n ═ 10)
Comparison with model control group: p <0.01, p < 0.001;
in fig. 3, the effect of grapefruit antifebrile extract on inflammation (neutrophil count) in zebrafish compared to model control group: p <0.01, p < 0.001;
in fig. 4, the inflammation-resolving action of the grapefruit antifebrile extract, compared with the model control group: p <0.01, p < 0.001.
(1) selecting 180-tailed 5dpf transgenic macrophage green fluorescent zebra fish;
(2) putting the zebra fish into a six-hole plate, and treating 30 zebra fish in each hole;
(3) all zebra fish are subjected to intravenous injection and LPS (low-pressure lipoprotein lipase) administration, and a zebra fish pneumonia model is established;
(4) the grapefruit extracts were administered to model zebrafish in 3 wells each dissolved in water in a six-well plate at concentrations of 889, 2667 and 8000 μ g/mL;
(5) in a six-well plate, one of the other 3 wells added with grapefruit extract is treated with standard dilution water to serve as a normal control group, the other one of the wells is taken as a model control group, and the remaining one of the wells is added with indometacin with the concentration of 60 mu M to serve as a positive control group;
(6) after the zebra fish in each hole is treated in a six-hole plate for 4 hours, selecting 10 zebra fish randomly, observing under a fluorescence microscope, taking a picture and storing the picture;
(7) analyzing the stored picture through Image J Image processing software, and calculating the intensity of a fluorescence signal of the inflammatory part of the zebra fish;
(8) the improvement effect of grapefruit extract on LPS-induced inflammatory macrophages was evaluated statistically.
The statistics of step (8) in the experimental mode of anti-inflammatory action of grapefruit extract on LPS-induced inflammatory macrophages were statistically analyzed using ANOVA and Dunnett's T-test.
The improvement effect of macrophages of the grapefruit extract of step (8) in the experimental manner of the anti-inflammatory effect of LPS-induced inflammatory macrophages was calculated by the formula:
after the experiment, the fluorescence signal intensity (757860 pixels) of the inflammatory part of the zebra fish in the model control group is compared with that in the normal control group (71961 pixels) to ensure that the p is less than 0.001, which indicates that the model of the pneumonia of the zebra fish induced by LPS is successful. Compared with a model control group, the fluorescence signal intensity (371000 pixels) of the inflammatory part of the zebra fish in the 60 mu M indometacin group is less than 0.001, and the macrophage improvement effect of the indometacin group is 51.0%, which indicates that the indometacin has obvious macrophage improvement effect on the pneumonia zebra fish.
When the concentrations of the dichroa febrifuga extract are 889, 2667 and 8000 mu g/mL, the fluorescence signal intensities of inflammatory parts of the zebra fish are 520175, 310845 and 435642 pixels respectively, compared with a model control group (757860 pixels), the p is less than 0.001, the macrophage improvement effects are 31.4%, 59.0% and 42.5% respectively, and the dichroa febrifuga extract has obvious macrophage improvement effect on the pneumonia zebra fish under the condition of the experimental concentration.
The detailed data are shown in table 2, fig. 1, fig. 2, fig. 3 and fig. 4.
Table 3 effect of each experimental group on zebrafish inflammation (macrophages) (n ═ 10)
Comparison with model control group: p <0.001
In fig. 7, the effect of grapefruit antifebrile extract on zebrafish inflammation (macrophages), compared to model controls: p <0.001
In fig. 8, macrophage improving effect of grapefruit antifebrile extract, compared to model control group: p < 0.001.
Experiment 3, experiment of the effect of grapefruit extract on inflammation-related genes:
(1) selecting 180-tailed 5dpf transgenic neutrophilic granulocyte fluorescent zebra fish;
(2) putting the zebra fish into a six-hole plate, and treating 30 zebra fish in each hole;
(3) all zebra fish are subjected to intravenous injection and LPS (low-pressure lipoprotein lipase) administration, and a zebra fish pneumonia model is established;
(4) the grapefruit extracts were administered to model zebrafish in 3 wells each dissolved in water in a six-well plate at concentrations of 889, 2667 and 8000 μ g/mL;
(5) in a six-well plate, one of the other 3 wells added with grapefruit extract is treated with standard dilution water to serve as a normal control group, the other one of the wells is taken as a model control group, and the remaining one of the wells is added with indometacin with the concentration of 60 mu M to serve as a positive control group;
(6) after co-processing for 2 hours in a six-hole plate, extracting the total RNA of each group of zebra fishes by using a classical Trizol method for the zebra fishes in each hole;
(7) measuring the concentration and purity of the total RNA extracted from each group by using a Thermo ultramicro spectrophotometer;
(8) taking 2 mu g of zebra fish sample total RNA, synthesizing 20 mu LcDNA according to the specification operation of the cDNA first strand synthesis kit, and storing at-20 ℃;
(9) β -actin was used as an internal reference for gene expression, and the relative expression amounts of RNA of TGF β -1 and IL-1 β genes were calculated.
(10) Statistical analysis was performed using analysis of variance and Dunnett's T-test.
The calculation formula of the relative expression amount of RNA in the step (9) in the experimental mode of the effect of the grapefruit extract on the inflammation-related genes is as follows:
ΔC(t)＝C(t)target gene-C(t)β-actin；
RNA relative expression amount ═ 2 ^ Δ Δ c (t).
After the experiment is carried out, after the grapefruit dichroa febrifuga extract is treated, the total RNA of the zebra fish is extracted, the concentration of the RNA and the A260/A280 ratio (table 4) are measured by using a ultramicro spectrophotometer, the A260/A280 ratio is between 1.8 and 2.2, the quality of the extracted total RNA of the zebra fish is good, and the extracted total RNA can be used for a subsequent q-PCR experiment, the concentration of the total RNA is shown in table 4, the sequence information of a primer is shown in table 5, and the relative expression quantity of the grapefruit febrifuga extract on TGF β -1 and IL-1 β genes is shown in table 6.
TABLE 4 concentration of Total RNA
TABLE 5 primer sequence information
TABLE 6 Effect of grapefruit extract on TGF β -1 and IL-1 β Gene expression
P <0.05, p <0.01, p <0.001, compared to model control group
According to the calculation of a gene relative expression formula, the relative expression of the TGF β -1 gene of a model control group is 5.79, compared with a normal control group (1.03), p is less than 0.001, which indicates that the zebra fish pneumonia model induced by LPS succeeds, the relative expression of the TGF β -1 gene of a 60 mu M indometacin group is 2.48, compared with the p of the model control group (5.79), which indicates that the TGF β -1 gene of the 60 mu M indometacin group is significantly reduced, the TGF β -1 relative expression of the dichroa febrifuga extract groups at concentrations of 889, 2667 and 8000 mu g/mL are 3.27, 1.90 and 3.19 respectively, and compared with the model control group (5.79), the p is less than 0.001, which indicates that the TGF β -1 gene expression of the dichroa febrifuga extract is significantly inhibited.
According to the calculation of a gene relative expression formula, the relative expression level of the IL-1 β gene of the model control group is 11.91, compared with the normal control group (1.10), p is less than 0.001, which indicates that the LPS induces the zebrafish pneumonia model to be successful, the relative expression level of the IL-1 β gene of the 60 mu M indomethacin group is 1.83, compared with the model control group (11.91), p is less than 0.01, which indicates that the IL-1 β gene of the 60 mu M indomethacin group is significantly reduced, the relative expression levels of the IL-1 β of the dichroa febrifuga extract groups are respectively 4.28, 5.80 and 6.21, compared with the model control group (11.91), p is less than 0.01 and less than 0.05, which indicates that the dichroa febrifuga extract can significantly inhibit the IL-1 β gene expression, and detailed graphs of 9 and 10 are provided.
In FIG. 9, the expression of TGF β -1 was relative, as compared to model controls, <0.05, < 0.001;
in FIG. 10, the relative expression level of IL-1 β was <0.05 and < 0.01.
(1) selecting 180-tailed 5dpf transgenic neutrophilic granulocyte fluorescent zebra fish;
(2) putting the zebra fish into a six-hole plate, and treating 30 zebra fish in each hole;
(3) all zebra fish are subjected to intravenous injection and LPS (low-pressure lipoprotein lipase) administration, and a zebra fish pneumonia model is established;
(4) the grapefruit extracts were administered to model zebrafish in 3 wells each dissolved in water in a six-well plate at concentrations of 889, 2667 and 8000 μ g/mL;
(5) in a six-well plate, one of the other 3 wells added with grapefruit extract is treated with standard dilution water to serve as a normal control group, the other one of the wells is taken as a model control group, and the remaining one of the wells is added with indometacin with the concentration of 60 mu M to serve as a positive control group;
(6) after co-treatment in a six-well plate for 4 hours, the zebra fish in each well was fixed with 4% paraformaldehyde;
(7) and transferring the zebra fish into 70% ethanol after fixation, dehydrating, embedding, slicing, dyeing and sealing, and performing pathological analysis on the dyed zebra fish slices.
After the experiment, the normal control group swimming bladder tissue structure is regular, the cell morphology is complete, and the cytoplasm is uniform; obvious inflammatory infiltration can be seen in the swimming bladder of the model control group; the positive control group, the dichroa febrifuga extract 889, 2667 and the swimming bladder inflammatory infiltration group with the concentration of 8000 mug/mL are improved.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. An application mode of a grapefruit extract in a traditional Chinese medicine preparation or a functional food is characterized in that: the preparation method of the grapefruit extract comprises the following steps:
(1) selecting raw materials, namely selecting fresh grapefruit without mildew, rot, diseases, insect pests and black spot diseases;
(2) processing raw materials, namely cleaning fresh grapefruit, separating pulp from peel, and squeezing the pulp to obtain fruit juice and fruit residues; cutting pericarp, and mixing with crushed pomace;
(3) extraction: mixing the pomace and the pericarp, adding 6-12 times of purified water, adding calcium chloride powder and cellulase, heating to 30-36 deg.C, soaking for half an hour, heating and reflux-extracting, filtering, collecting the extractive solution, repeating for 2 times, and mixing the filtrates;
(4) concentration: vacuum decompression and low-temperature concentration are carried out on the leachate, and the temperature is not more than 80 ℃;
(5) and (3) drying: drying the concentrated extract at low temperature, wherein the temperature is controlled below 80 ℃, and the water content is controlled below 7%;
the Chinese medicinal preparation and the functional food have the functions of preventing and treating pneumonia,
the traditional Chinese medicine preparation is a lung moistening and protecting pomelo pear syrup formula, and the functional food is a grapefruit lung moistening and protecting functional beverage.
2. The mode of use of a grapefruit extract in a herbal formulation or a functional food according to claim 1, characterized in that: the heating reflux extraction step of the step (3) in the preparation method of the grapefruit extract has the heating temperature of 95-120 ℃ and the reflux extraction time of 0.6-1 hour.
3. The mode of use of a grapefruit extract in a herbal formulation or a functional food according to claim 1, characterized in that: the preparation method of the pomelo-pear syrup prescription for moistening and protecting lung comprises the following steps:
(1) selecting fresh moldy and rotten-free snow pears;
(2) peeling and removing cores of snow pears, juicing and filtering to obtain filtered juice for later use;
(3) mixing the filtered juice with the fruit juice obtained in the step (2) of the preparation method of the grapefruit extract to obtain grapefruit pear juice;
(4) respectively carrying out vacuum decompression and low-temperature concentration on the leaching solution obtained in the step (3) in the preparation method of the grapefruit extract and the grapefruit pear juice at the temperature not higher than 80 ℃ to respectively obtain a grapefruit extract and a snow pear extract;
(5) mixing the grapefruit extract and the snow pear extract, adding crystal sugar and mint, heating, uniformly mixing and filling to obtain the paste formula.
4. The mode of application of grapefruit extract in a Chinese medicinal preparation or a functional food according to claim 3, characterized in that: in the preparation method of the lung moistening and protecting pomelo-pear syrup formula, the pomelo-pear juice in the step (3) is prepared by mixing fruit juice and filtered juice in a ratio of 3: 7.
5. The mode of application of grapefruit extract in a Chinese medicinal preparation or a functional food according to claim 3, characterized in that: in the preparation method of the lung moistening and lung protecting pomelo-pear syrup formula, the weight ratio of the mixed material of the pomelo extract and the snow pear extract in the step (5) is 4: 6-2: 8.
6. The mode of application of grapefruit extract in a Chinese medicinal preparation or a functional food according to claim 3, characterized in that: the operation flow of the step (5) in the preparation method of the lung moistening and protecting pomelo-pear syrup prescription is to perform material mixing, fusion and transformation in a vacuum stirring tank.
7. The mode of use of a grapefruit extract in a herbal formulation or a functional food according to claim 1, characterized in that: the preparation method of the grapefruit lung moistening and protecting functional beverage comprises the following steps:
(1) respectively carrying out vacuum reduced pressure low-temperature concentration on the leachate obtained in the step (3) in the preparation method of the grapefruit extract and the juice obtained in the step (2) in the preparation method of the grapefruit extract, wherein the temperature is not higher than 80 ℃, and respectively obtaining a grapefruit extract and a grapefruit concentrated juice;
(2) mixing the grapefruit extract and grapefruit concentrated juice into beverage according to a predetermined ratio, and processing to obtain functional fruit juice;
(3) drying the extract at low temperature, controlling water content below 7%, mixing with solid beverage, granulating, and processing to obtain functional solid preparation.
8. The mode of use of a grapefruit extract in a herbal formulation or a functional food according to claim 7, characterized in that: in the preparation method of the grapefruit lung moistening and protecting functional beverage, in the operation flow of the step (2), the grapefruit extract and the grapefruit concentrated juice are added in the beverage in a proportion of not more than 20%.
9. The mode of use of a grapefruit extract in a herbal formulation or a functional food according to claim 7, characterized in that: in the operation flow of the step (3) in the preparation method of the grapefruit lung moistening and protecting functional beverage, the addition proportion of the grapefruit extract in the solid beverage is not more than 15%.
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