CN107158403B - Isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound, composition, preparation method and application - Google Patents
Isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound, composition, preparation method and application Download PDFInfo
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Abstract
The invention relates to an isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound, a composition, a preparation method and a new application thereof, wherein the isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound is used for preparing a medicament for improving or treating exercise-induced fatigue and the medicinal composition are characterized in that in the process of researching the isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound, through establishing an animal model, the change levels of hepatic glycogen, blood lactic acid and urea nitrogen in a test mouse sample are detected, the isoliquiritigenin is proved to have the exercise-induced fatigue resisting effect, and the exercise-induced fatigue resisting medicinal composition is prepared.
Description
Technical Field
The invention belongs to the technical field of application of deep processing of isoliquiritigenin, and particularly relates to an isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound, a composition, a preparation method and a new application thereof.
Background
Exercise fatigue is an eternal subject of research in sports medicine and sports physiology. As for the mechanism of development of exercise-induced fatigue, various hypotheses have been proposed, such as the theory of exhaustion of energy substances, the theory of free radical injury, the theory of neurotransmitters, the theory of accumulation of lactic acid and Ca2+Abnormal theory of homeostasis, etc.
The theory of exhaustion of energy substances suggests that the energy substances include various glycogen, ATP, creatine phosphate and the like, and when the body does exercise in a short time and at high intensity, a large amount of energy substances such as glycogen and the like are used for decomposition to provide energy, and if some content of the energy substances is reduced, the energy substances cannot be supplemented in time, so that exercise fatigue can be caused.
Isoliquiritigenin, molecular formula is (E) -1- (2, 4-dihydroxyphenyl) -3- (4-hydroxyphenyl) -2-propylene-1-ketone; 4,2',4' -trihydroxychalcone. The chemical structural general formula of the isoliquiritigenin is as follows:
the flavonoids in Glycyrrhrizae radix can be divided into water soluble flavone and fat soluble flavone, and have antiinflammatory, antivirus, antioxidant, antiaging, antiulcer, antitumor, and gastric mucosa protecting effects. In recent years, licoflavone has been found to inhibit the proliferation of cancer cells.
Isoliquiritigenin has pharmacological activity of eliminating free radicals and resisting oxidation, but has poor solubility in water and poor absorption after being taken, so that the problems of poor solubility and poor absorption of a human body of the isoliquiritigenin are required to be solved.
The preparation technology of isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound, and the research of the inclusion compound and the composition thereof for resisting exercise-induced fatigue are not reported yet.
Disclosure of Invention
The invention provides an isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound.
The invention also provides a preparation method of the isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound.
The invention also provides an isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound medicine composition.
The invention also provides a new application of the isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound.
An isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound contains isoliquiritigenin and cyclodextrin or cyclodextrin derivatives, the molar ratio of the isoliquiritigenin to the cyclodextrin or the cyclodextrin derivatives is 1: 1-1: 100, and the cyclodextrin derivatives are selected from one of hydroxyethyl-beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, methyl-beta-cyclodextrin, maltose-beta-cyclodextrin, carboxymethyl-beta-cyclodextrin, sulfoethyl-beta-cyclodextrin, sulfopropyl-beta-cyclodextrin and sulfobutyl-beta-cyclodextrin.
A preparation method of an isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound comprises the steps of taking cyclodextrin or cyclodextrin derivative, adding water for dissolving, adding isoliquiritigenin while stirring at room temperature, enabling the molar number of the added isoliquiritigenin to be 1: 1-1: 100, continuing stirring to enable the cyclodextrin or cyclodextrin derivative and the isoliquiritigenin to completely react, obtaining a solution after the reaction is completed, filtering the solution through a microporous membrane, pre-freezing the filtrate for 4 hours in a refrigerator at minus 80 ℃, and drying the filtrate overnight at minus 80 ℃ by using a freeze dryer to obtain a solid isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound;
the cyclodextrin derivative is selected from one of hydroxyethyl-beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, methyl-beta-cyclodextrin, maltose-beta-cyclodextrin, carboxymethyl-beta-cyclodextrin, sulfoethyl-beta-cyclodextrin, sulfopropyl-beta-cyclodextrin and sulfobutyl-beta-cyclodextrin.
A pharmaceutical composition comprises an isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound and pharmaceutically acceptable auxiliary materials, wherein the pharmaceutical composition takes the isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound as an active ingredient, the auxiliary materials are pharmaceutically acceptable excipients, and the weight ratio of the isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound to the auxiliary materials is 10: 90-90: 10;
the isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound contains isoliquiritigenin and cyclodextrin or cyclodextrin derivatives, wherein the molar ratio of the isoliquiritigenin to the cyclodextrin or the cyclodextrin derivatives is 1: 1-1: 100;
the cyclodextrin derivative is selected from one of hydroxyethyl-beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, methyl-beta-cyclodextrin, maltose-beta-cyclodextrin, carboxymethyl-beta-cyclodextrin, sulfoethyl-beta-cyclodextrin, sulfopropyl-beta-cyclodextrin and sulfobutyl-beta-cyclodextrin, and the cyclodextrin is beta-cyclodextrin.
An isoliquiritigenin-cyclodextrin or cyclodextrin derivative clathrate can be used for preparing medicine for treating and/or preventing exercise-induced fatigue.
According to the invention, through the inclusion behavior of the cyclodextrin or the cyclodextrin derivative, the water solubility of the isoliquiritigenin is improved, and the absorption capacity of a human body to the isoliquiritigenin is further improved;
the preparation method of the isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound can also adopt an ultrasonic method and a grinding method:
an ultrasonic method: accurately weighing isoliquiritigenin and cyclodextrin or cyclodextrin derivatives, adding water for dissolving, immediately carrying out ultrasonic treatment after adding isoliquiritigenin into a cyclodextrin or cyclodextrin derivative water solution until the solution is dissolved and transparent, wherein the ratio of the mole number of the added isoliquiritigenin to the mole number of the cyclodextrin or cyclodextrin derivatives is 1: 1-1: 100, filtering the solution through a microporous filter membrane after the ultrasonic treatment is finished, pre-freezing the filtrate for 4h in a refrigerator at minus 80 ℃, and drying the filtrate overnight at minus 80 ℃ by using a freeze dryer to obtain the solid isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound.
Grinding method:
precisely weighing cyclodextrin or cyclodextrin derivatives, adding appropriate amount of distilled water, uniformly grinding the cyclodextrin, slowly adding isoliquiritigenin in portions under grinding conditions, fully grinding, pre-freezing the obtained paste in a refrigerator at-80 ℃ for 4h, and drying overnight at-80 ℃ by using a freeze dryer to obtain a solid isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound, wherein the ratio of the mole number of the added isoliquiritigenin to the mole number of the cyclodextrin or cyclodextrin derivatives is 1: 1-1: 100.
The pharmaceutical composition comprises an isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound and pharmaceutically acceptable auxiliary materials, wherein the isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound is used as an active ingredient, the auxiliary materials are pharmaceutically acceptable excipients, and the excipients can be one or more of sucrose, lactose and glucose which are combined together to form a stabilizer. In specific embodiments, the weight ratio of the isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound to the auxiliary material can be 10:90, 20:80, 25:85, 30:70, 35:65, 40:60, 45:55, 50:50, 60:60, 70:30, 75:25, 80:20, 85:15, and 90: 10.
The application of the isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound in preparing the medicine for improving or treating exercise-induced fatigue and the medicine composition are that the inventor detects the change levels of liver glycogen, blood lactic acid and urea nitrogen in a test mouse sample through establishing an animal model in the process of researching the isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound, confirms that the isoliquiritigenin has the effect of resisting exercise-induced fatigue and prepares the medicine composition for resisting exercise-induced fatigue.
Drawings
FIG. 1 is an infrared spectrum of isoliquiritigenin, isoliquiritigenin-beta-cyclodextrin inclusion compound and isoliquiritigenin and beta-cyclodextrin mixture.
FIG. 2 shows the infrared spectra of isoliquiritigenin, isoliquiritigenin-hydroxypropyl-beta-cyclodextrin inclusion compound and isoliquiritigenin and hydroxypropyl-beta-cyclodextrin inclusion compound.
Detailed Description
The following examples of the preparation method of the isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound
The first embodiment is as follows: preparation method of isoliquiritigenin-beta-cyclodextrin inclusion compound
Dissolving 1mmol of beta-cyclodextrin in 60g of water, adding 1mmol of isoliquiritigenin while stirring at room temperature, continuously stirring to completely react the beta-cyclodextrin with the isoliquiritigenin, obtaining a solution after the reaction is finished, filtering the solution by a microporous membrane, pre-freezing the filtrate for 4h in a refrigerator at minus 80 ℃, and drying overnight at minus 80 ℃ by a freeze dryer to obtain the solid isoliquiritigenin-beta-cyclodextrin inclusion compound.
Example two: preparation method of isoliquiritigenin-beta-cyclodextrin inclusion compound
Taking 10mmol of beta-cyclodextrin, adding 600g of water for dissolving, adding 1mmol of isoliquiritigenin while stirring at room temperature, continuously stirring to enable the beta-cyclodextrin and the isoliquiritigenin to completely react, obtaining a solution after the reaction is completed, filtering the solution by a microporous membrane, pre-freezing the filtrate for 4h in a refrigerator at minus 80 ℃, and drying overnight by a freeze dryer at minus 80 ℃ to obtain the solid isoliquiritigenin-beta-cyclodextrin inclusion compound.
Example three: preparation method of isoliquiritigenin-hydroxypropyl-beta-cyclodextrin inclusion compound
Dissolving 1mmol of hydroxypropyl-beta-cyclodextrin in 10g of water, adding 1mmol of isoliquiritigenin while stirring at room temperature, continuously stirring to ensure that the hydroxypropyl-beta-cyclodextrin and the isoliquiritigenin completely react, obtaining a solution after the reaction is finished, filtering the solution by a microporous membrane, pre-freezing the filtrate for 4h in a refrigerator at minus 80 ℃, and drying overnight by a freeze dryer at minus 80 ℃ to obtain the solid isoliquiritigenin-hydroxypropyl-beta-cyclodextrin inclusion compound.
Example four: preparation method of isoliquiritigenin-hydroxypropyl-beta-cyclodextrin inclusion compound
Dissolving 10mmol of hydroxypropyl-beta-cyclodextrin in 100g of water, adding 1mmol of isoliquiritigenin while stirring at room temperature, continuously stirring to ensure that the hydroxypropyl-beta-cyclodextrin and the isoliquiritigenin completely react, obtaining a solution after the reaction is finished, filtering the solution by a microporous membrane, pre-freezing the filtrate for 4h in a refrigerator at minus 80 ℃, and drying overnight by a freeze dryer at minus 80 ℃ to obtain the solid isoliquiritigenin-hydroxypropyl-beta-cyclodextrin inclusion compound.
Example five: preparation method of isoliquiritigenin-hydroxypropyl-beta-cyclodextrin inclusion compound
Dissolving 100mmol of hydroxypropyl-beta-cyclodextrin in 500g of water, adding 1mmol of isoliquiritigenin while stirring at room temperature, continuously stirring to completely react the hydroxypropyl-beta-cyclodextrin with the isoliquiritigenin, obtaining a solution after the reaction is finished, filtering the solution by a microporous membrane, pre-freezing the filtrate for 4h in a refrigerator at-80 ℃, and drying overnight by a freeze dryer at-80 ℃ to obtain the solid isoliquiritigenin-hydroxypropyl-beta-cyclodextrin inclusion compound.
Example six: preparation method of isoliquiritigenin-carboxymethyl-beta-cyclodextrin inclusion compound
Dissolving 10mmol of carboxymethyl-beta-cyclodextrin in 100g of water, adding 1mmol of isoliquiritigenin while stirring at room temperature, continuously stirring to completely react the carboxymethyl-beta-cyclodextrin with the isoliquiritigenin, obtaining a solution after the reaction is finished, filtering the solution by a microporous membrane, pre-freezing the filtrate for 4h in a refrigerator at-80 ℃, and drying the filtrate overnight at-80 ℃ by a freeze dryer to obtain the solid isoliquiritigenin-carboxymethyl-beta-cyclodextrin inclusion compound.
Infrared spectroscopy is performed on the isoliquiritigenin-beta-cyclodextrin inclusion compound and the isoliquiritigenin-hydroxypropyl-beta-cyclodextrin inclusion compound prepared in the first and third examples, and whether the inclusion compound is generated is verified.
The infrared determination method comprises collecting trace amount of isoliquiritigenin, isoliquiritigenin- β -cyclodextrin clathrate, isoliquiritigenin and β -cyclodextrin mixture, isoliquiritigenin-HP- β -cyclodextrin clathrate, and isoliquiritigenin and HP- β -cyclodextrin mixture, adding appropriate amount of solid KBr, grinding, tabletting to obtain tablet with resolution of 2cm-1， 3500～500cm-1Scanning the full spectrum, respectively performing infrared measurement on isoliquiritigenin, isoliquiritigenin- β -cyclodextrin inclusion compound, isoliquiritigenin and β -cyclodextrin mixture, isoliquiritigenin-HP- β -cyclodextrin inclusion compound and isoliquiritigenin and HP- β -cyclodextrin mixture, and obtaining results shown in figures 1 and 2.
As can be seen from FIG. 1, isoliquiritigenin and beta-cyclodextrin have their own infrared characteristic absorption peaks. The spectrogram of the mixture has additivity and is represented by superposition of two components; the inclusion compound has different performances, and partial peaks of the inclusion compound are subjected to purple shift, partial peaks of the inclusion compound are subjected to red shift, and partial peaks of the inclusion compound disappear. Compared with the spectrum of cyclodextrin, the clathrate is mainly the spectrum of cyclodextrin, and the spectrum characteristic part of isoliquiritigenin disappears. This can indicate that the experimental results are almost consistent with theoretical expectations, the aperture of the cyclodextrin cavity is matched with the guest molecule, the isoliquiritigenin molecule is embedded in the cavity of beta-cyclodextrin, the molecular vibration is limited and the original infrared characteristics can not be fully shown. A supermolecular inclusion compound is formed between the isoliquiritigenin and the beta-cyclodextrin.
As can be seen from FIG. 2, isoliquiritigenin and HP-beta-cyclodextrin each have their own characteristic infrared absorption peak. The spectrogram of the mixture has additivity and is represented by superposition of two components; the inclusion compound has different performances, and partial peaks of the inclusion compound are subjected to purple shift, partial peaks of the inclusion compound are subjected to red shift, and partial peaks of the inclusion compound disappear. Compared with the spectrum of cyclodextrin, the clathrate is mainly the spectrum of cyclodextrin, and the spectrum characteristic part of isoliquiritigenin disappears. This indicates that the experimental results are almost consistent with theoretical expectations, the aperture of the cyclodextrin cavity is matched with the guest molecule, the isoliquiritigenin molecule is embedded in the cavity of HP-beta-cyclodextrin, the molecular vibration is limited and the original infrared characteristics cannot be fully shown. A supermolecular inclusion compound is formed between the isoliquiritigenin and HP-beta-cyclodextrin.
The pharmacological experiments of the isoliquiritigenin-beta-cyclodextrin inclusion compound and the isoliquiritigenin-hydroxypropyl-beta-cyclodextrin inclusion compound are as follows:
in the invention, the inventor verifies that the isoliquiritigenin-beta-cyclodextrin inclusion compound and the isoliquiritigenin-hydroxypropyl-beta-cyclodextrin inclusion compound have the effect of resisting exercise-induced fatigue by establishing an animal model and detecting the change levels of liver glycogen, blood lactic acid and urea nitrogen in a test mouse sample, and prepares the medicament composition for resisting exercise-induced fatigue.
Firstly, experimental animals: experimental mice were purchased from the center of laboratory animals of Ningxia medical university, male, four weeks old, body weight (22 + -2 g), SPF grade, and animal production license number SYXK (Ning). The feeding conditions comprise standard feed, tap water, room temperature of 24 days 2 deg.C, humidity of 50-60%, and daily illumination and dark time of 12 hr respectively. Animals were acclimated to the experimental environment for 3 days prior to experimental preparation.
Second, experimental medicine and instrument
Medicine preparation: the isoliquiritigenin-beta-cyclodextrin inclusion compound, isoliquiritigenin-hydroxypropyl beta-cyclodextrin inclusion compound, trichloroacetic acid (AR, Merlin reagent company), glucose (AR, Martin reagent company), sodium carboxymethylcellulose (AR, Merlin reagent company), anthrone (AR, Martin reagent company), thiourea (AR, Martin reagent company), sodium fluoride (AR, Martin reagent company), sodium tungstate (AR, Merlin reagent company), trichloroacetic acid (AR, Merlin reagent company), copper sulfate (AR, Martin reagent company), p-hydroxybiphenyl (AR, alatin reagent), calcium lactate (AR, alatin reagent), concentrated sulfuric acid (nixtamatin bio-reagent), ethanol (nixtamatin bio-reagent), NaOH (nixtamatin bio-reagent).
The instrument comprises the following steps: an analytical balance Model SPS202F (zhongzhou electronic weighing instrument ltd), a Model 1510 ultrasonic cleaner (wolong instrument equipment), a low-temperature centrifuge Model L-530 (sevela science and technology development), an ultraviolet-visible spectrophotometer Model UV-2901 (HITACHI), and a fully automatic biochemical analyzer Model BS-220 (wuhan heyday equipment).
Third, Experimental methods
1. Preparation of test drugs: isoliquiritigenin-beta-cyclodextrin inclusion compound solution and isoliquiritigenin-hydroxypropyl-beta-cyclodextrin inclusion compound solution.
Every three days according to the actual weight of the test mouse as 10mg kg-1Respectively preparing isoliquiritigenin- β -cyclodextrin inclusion compound solution and isoliquiritigenin-hydroxypropyl- β -cyclodextrin inclusion compound solution, and storing in a refrigerator at 4 deg.C for use;
the preparation operation of the isoliquiritigenin-beta-cyclodextrin inclusion compound solution is as follows: adding isoliquiritigenin-beta-cyclodextrin clathrate containing 1mg isoliquiritigenin into 0.5% sodium carboxymethylcellulose per 1mL for intragastric administration to the following group C experimental mice;
the preparation operation of the isoliquiritigenin-hydroxypropyl-beta-cyclodextrin inclusion compound solution is as follows: isoliquiritigenin-hydroxypropyl-beta-cyclodextrin inclusion compound containing 1mg isoliquiritigenin is added into 0.5% sodium carboxymethylcellulose per 1mL for intragastric administration to the following experimental mice in group D.
2. Experiment grouping
Experimental mice were 80, male, four weeks old, weight (22 ± 2g), SPF rating, normal rearing and adaptive swimming daily for 20 minutes, and three days later, randomly divided into four groups by weight:
a (quiet control group), B (endurance training group), C (endurance training + isoliquiritigenin-beta-cyclodextrin inclusion compound group), and D (endurance training + isoliquiritigenin-hydroxypropyl-beta-cyclodextrin inclusion compound group) 20 each.
Each group of experimental mice was 1 pm daily: 00 the dose is 10mg/kg per kg of mouse body weight-1；10mg·kg-1The amount of isoliquiritigenin in isoliquiritigenin- β -cyclodextrin inclusion compound solution per kilogram of body weight of mouse is 10mg, or 10mg/kg-1The amount of isoliquiritigenin in isoliquiritigenin-hydroxypropyl- β -cyclodextrin inclusion compound solution refilled for each kilogram of body of mice is 10mg, the mice in group A and group B are filled with equal volume of 0.5% sodium carboxymethylcellulose solution for stomach irrigation for 6 weeks continuously, the room temperature is 25 +/-2 ℃, cleaning pads are replaced daily, cleaning drinking bottles are replaced daily, and the weight of the mice is weighed once a day and recorded.
3. Endurance training of mice
The A group only drenches the stomach and does not carry out swimming training, B, C, D groups of mice respectively carry out daily progressive swimming exercise in an experimental animal swimming pool, the water depth is 40-50 cm, and the water temperature is 25 seconds and 2 ℃. Training was performed 5 days per week, 1 each in the morning and afternoon, starting at 9:00am, 3 pm: 00pm start training, the amount of training is as follows table 1:
TABLE 1 endurance training timetable
4. Exhaustive swimming exercise for mice
B, C, D groups of mice were swim twice a day on the schedule, and after the sixth week of training, exhaustive swimming movements were performed and the time to exhaustive swimming of the mice was recorded as follows in Table 2:
table 2 shows the exhaustive swimming time of the test mice
Note: p < 0.01 in the delta group compared with the B group.
As can be seen from Table 2, the exhaustion swimming time of the mice tested in the groups C and D is longer than that of the other groups, and therefore, the exhaustion swimming time of the mice can be obviously prolonged by the glycyrrhizin-beta-cyclodextrin inclusion compound solution and the glycyrrhizin-hydroxypropyl-beta-cyclodextrin inclusion compound, and the fatigue degree of the mice can be directly reflected by the swimming time, so that the isoliquiritigenin can improve the fatigue symptoms of the mice subjected to endurance training, improve the athletic ability of the mice, delay the occurrence of the athletic fatigue and has the effect of resisting the athletic fatigue.
5. Mouse selection
The apparatus used was sterilized before the mice were slaughtered. 10 mice in each group were randomly taken out without swimming, eyeball blood was directly taken, 80. mu.L of whole blood was immediately reserved, and serum was left.
The mice were sacrificed again, the skin of the mice was immediately sterilized with 75% alcohol, the livers were taken, the blood was washed with 4 ℃ physiological saline, the physiological saline was blotted with filter paper, weighed again, and quickly frozen in liquid nitrogen.
10 mice left in each group are subjected to swimming training for 100min, eyeball blood is taken after training, 80 mu L of whole blood is reserved after each mouse is taken, the remaining whole blood is immediately centrifuged for 15min at 3000rpm/min by a low-temperature centrifuge (4 ℃), and serum is carefully sucked and stored in a refrigerator at-80 ℃.
6. Detecting the index
This example mainly examined the relevant indicators of isoliquiritigenin-cyclodextrin inclusion compound on the resistance to exercise-induced fatigue in endurance-trained mice. Wherein the detection indexes mainly comprise: hepatic glycogen, Blood Lactic Acid (BLA), urea nitrogen (BUN).
(1) Method for measuring mouse liver glycogen by ketone-sulfuric acid method
Preparing a reagent:
preparing a glucose standard solution: 0.01g of glucose was weighed and dissolved in 10mL of distilled water.
Preparing 5% trichloroacetic acid (TCA): 5.0g of trichloroacetic acid was accurately weighed and dissolved in 100mL of distilled water.
Preparing an anthrone reagent: preparing a 72% concentrated sulfuric acid solution, when preparing the 72% concentrated sulfuric acid solution, firstly adding 140mL of distilled water into a beaker, slowly adding 360mL of concentrated sulfuric acid with the specific gravity of 1.84 while stirring until the concentration is H2SO4When the temperature is reduced to 80-90 ℃, 0.25g of anthrone and 5g of thiourea are added, shaken up, cooled and stored in a refrigerator.
A detection step: take group C as an example
Extraction calculation of hepatic glycogen from group C test mice: accurately weighing liver tissues of 10 non-swimming mice in group C, respectively weighing 100mg, respectively adding 8mLTCA, homogenizing for 1min, centrifuging at 3000 r/min for 15min, and transferring supernatant to another test tube; taking 1mL of the supernatant into a 10mL centrifuge tube, adding 4mL of 95% ethanol into each tube, fully and uniformly mixing, tightly covering the centrifuge tube, keeping the centrifuge tube upright, standing overnight at normal temperature, and centrifuging at the speed of 3000 r/min for 15 min; carefully discarding the supernatant, inverting the centrifuge tube for 10min until no liquid drops exist in the tube; adding 2mL of distilled water to ensure that glycogen on the tube wall is washed off, and oscillating the centrifugal tube to completely dissolve the glycogen; adding 10mL anthrone reagent into each tube, mixing thoroughly, cooling to cold water temperature, soaking in boiling water, heating for 15min, transferring to cold water bath, cooling, and measuring absorbance at 620nm wavelength to obtain a first group of absorbance values.
For more accurate data, three sets of parallel experiments were performed, i.e., the second set of parallel experiments was: continuously and accurately weighing 100mg of the rest liver tissues of 10 non-swimming mice in the group C, repeating the steps, and measuring the absorbance to obtain a second group of absorbance values; the third set of parallel tests was: accurately weighing 100mg of the rest liver tissues of the 10 non-swimming mice in the group C again, repeating the steps, and measuring the absorbance to obtain a third group of absorbance values;
and (3) respectively substituting the absorbance values of the three groups of the parallel tests into a liver glycogen formula to calculate to obtain the liver glycogen numbers of the three groups of the group C, and then calculating the average value of the liver glycogen numbers of the three groups to obtain the liver glycogen number of the group C.
As a comparative test protocol, a blank tube, a standard tube may also be prepared:
blank tube: 2mL of distilled water was taken in a clean test tube.
Standard tubes: 0.5mL of glucose standard solution and 1.5mL of distilled water were put in a standard tube and mixed well.
Then adding 10mL anthrone reagent into the blank tube and the standard tube respectively, mixing thoroughly, cooling to cold water temperature, soaking in boiling water, heating for 15min, transferring to cold water bath, cooling, and measuring blank tube absorbance and standard tube absorbance at 620nm wavelength respectively.
Similarly, the method for extracting and calculating liver glycogen of the experimental mice of the group A and the group B is the same as that of the group C, and the method for extracting and calculating liver glycogen of the liver tissue of 10 mice which do not swim is the same as that of the group C. As in table 4 below.
The hepatic glycogen formula is:
the number of milligrams of glycogen per 100g of liver (muscle) tissue is DU/DS × 0.5 × volume of extract/g of liver tissue × 100 × 0.9, wherein,
and (2) DS: absorbance of a standard tube;
DU: absorbance of the sample tube;
the volume of the extracting solution is 8 mL;
the gram number of the liver tissue is 0.1 g;
0.9 is a coefficient for converting glucose into glycogen;
0.5 is the glucose content in 0.5mL of glucose standard solution.
(2) Method for measuring blood lactic acid content of mouse by using self-prepared reagent
Preparing a reagent:
10% sodium tungstate: 10g of sodium tungstate was weighed and dissolved in 100mL of distilled water.
1/3mol/L sulfuric acid: measuring 0.8mL of concentrated sulfuric acid in a 50mL volumetric flask, and adding distilled water to a constant volume.
1% NaF solution: 1.125g NaF was accurately weighed and dissolved in 100mL of distilled water.
10% trichloroacetic acid: 9.9mL of trichloroacetic acid was measured and dissolved in 100mL of distilled water.
CuSO4Solution: accurately weighing 0.42g of CuSO4Dissolved in 10mL of distilled water.
Protein precipitant: 1/3mol/L sulfuric acid, 10% sodium tungstate and distilled water are mixed according to the volume ratio of 1:1: 1.
Protein precipitant-NaF mixed liquor: 10mL of 1% NaF and 30mL of protein precipitant were mixed by volume.
1.5% p-hydroxybiphenyl solution: 1.5g of p-hydroxybiphenyl was weighed out and dissolved in 100mL of hot 0.5% NaOH.
Lactic acid standard stock solution (1 g/L): 0.171g of calcium lactate is weighed and added to 10% trichloroacetic acid to a volume of 100 mL.
Lactic acid standard application liquid (0.01 g/L): sucking 1.0mL of lactic acid standard stock solution, dissolving in a 100mL volumetric flask, and fixing the volume with distilled water.
Detection of blood lactate in group C test mice:
0.48mL of 1% NaF solution was pipetted into a 5mL centrifuge tube and 20. mu.L of whole blood from group C swimming mice was added. Mixing, adding 1.5mL protein precipitant, shaking, centrifuging at 3000 r/min for 10min, collecting supernatant, heating in 30 deg.C water bath for 30min, placing in boiling water bath for 90s, cooling, and measuring absorbance at 560nm wavelengthSample tubeAnd then calculating according to a blood lactic acid content calculation formula to obtain the blood lactic acid content of the group C.
As a comparative test protocol, blank tubes and standard tubes can also be prepared separately, as shown in Table 3 below
TABLE 3 blood lactic acid determination protocol
Heating the standard tube in 30 deg.C water bath for 30min, placing in boiling water bath for 90s, cooling, and measuring absorbance value at 560nm wavelengthStandard tubeAnd calculating the blood lactic acid content of each group.
Similarly, the assay procedure for blood lactate was the same for A, B group mice as for group C, and A, B absorbance values were determined, i.e., group A for group ASample tubeGroup B, group ASample tubeAnd calculating to obtain the lactic acid content of A, B groups of blood. As in table 4 below.
The blood lactic acid content calculation formula is as follows:
blood lactic acid (mg/L) ═ ASample tube/AStandard tube×100×10
(3) Determination of biochemical index
In this experiment, serum of the test mice swimming in A, B, C groups and D groups was analyzed and measured by a full-automatic biochemical analyzer, and urea nitrogen (BUN) content of each group of test mice was measured. As in table 4.
7. Test results
TABLE 4 hepatic glycogen, urea nitrogen and blood lactic acid
Note: group # liver glycogen comparison P < 0.01; p < 0.01 for group B urea nitrogen; ■ compared to blood lactate in group B, P is < 0.01.
As can be seen from Table 4, the content of hepatic glycogen in groups C and D is higher than that in groups A and B, and it can be concluded from the energy metabolism that isoliquiritigenin-hydroxypropyl-beta-cyclodextrin inclusion compound and isoliquiritigenin-beta-cyclodextrin inclusion compound can reduce the decomposition of hepatic glycogen or promote the synthesis of hepatic glycogen, thereby improving exercise endurance and having good anti-exercise fatigue effect.
The blood lactic acid content of group C and D is lower than that of group B, so that the conclusion from the lactic acid accumulation theory shows that the isoliquiritigenin-hydroxypropyl-beta-cyclodextrin inclusion compound and isoliquiritigenin-beta-cyclodextrin inclusion compound can inhibit or reduce the generation of blood lactic acid or can decompose blood lactic acid, thereby improving exercise endurance and having good effect of resisting exercise fatigue.
The urea nitrogen content of the group C and the group D is lower than that of the group B, so that the data can conclude that the isoliquiritigenin-hydroxypropyl-beta-cyclodextrin inclusion compound and the isoliquiritigenin-beta-cyclodextrin inclusion compound can inhibit or reduce the generation of urea nitrogen or can decompose urea nitrogen, thereby improving exercise endurance and having good effect of resisting exercise fatigue.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.
Claims (3)
1. The application of the isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound in the preparation of the anti-exercise-induced fatigue drug is characterized in that the molar ratio of the isoliquiritigenin to the cyclodextrin or the cyclodextrin derivative is 1: 1-1: 100, and the cyclodextrin derivative is selected from one of hydroxyethyl-beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, methyl-beta-cyclodextrin, maltose-beta-cyclodextrin, carboxymethyl-beta-cyclodextrin, sulfoethyl-beta-cyclodextrin, sulfopropyl-beta-cyclodextrin and sulfobutyl-beta-cyclodextrin.
2. The use of the isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound of claim 1 in the preparation of an anti-exercise-induced fatigue medicament, wherein: the single-use amount of the isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound is 5-20 mg/kg.
3. The use of the isoliquiritigenin-cyclodextrin or cyclodextrin derivative inclusion compound of claim 1 in the preparation of an anti-exercise-induced fatigue medicament, wherein: the single application amount of isoliquiritigenin-cyclodextrin or cyclodextrin derivative clathrate is 10 mg/kg.
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