CN114306336A - Application method for enhancing learning and memory by using piperine - Google Patents
Application method for enhancing learning and memory by using piperine Download PDFInfo
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Abstract
The invention discloses an application method for enhancing learning and memory by using piperine, relating to the technical field of piperine application; specifically comprises establishing fruit fly sugar reward olfaction association type learning memory model, capillary-nanometer microemulsion body food medicine feeding. The method for establishing the fruit fly sugar reward olfactory associative learning and memory paradigm comprises the following steps: preparing and preparing smell, training sugar reward, testing fruit flies, and counting and calculating data. In the preparation and preparation of the odor, the odor used comprises benzaldehyde Benzaldehyde (BenZ) (Sigma, #100-52-7) and 3-Octanol (OCT) (Sigma, #589-98-0) at a concentration of 100uL/100 ml. The invention researches and searches piperine as a high-efficiency learning and memory enhancing medicament for the first time, and makes up the vacancy in the prior art.
Description
Technical Field
The invention relates to the technical field of piperine application, in particular to an application method for enhancing learning and memory by using piperine.
Background
Learning and memory have been one of the focuses of neuroscience research. Learning refers to the process by which an animal acquires new information about its surroundings and reacts by changing some aspect of its behavior, while memory stores and reads the information acquired. Both are high-level functions of the nervous system of animals and are important conditions under which humans and animals live. A classical conditioned reflex test (A review [ J ]. J. Chinese Compare medicine, 2003-68) using a traditional model of learning and memory animal, such as Bavlov's dog model (reference 1, Emilio Cartoni, Bernard Balllene, Gianluca Baldasarre.empirical Pavlovian-atomic Transfer: A review [ J ]. Neurosci Biobehav R,2016,71:829-848) has yielded a rich research result in learning and memory brain structure, synapse and neurotransmission mechanism, neurotransmitter and neuron membrane potential, and synthesis of intracerebral protein (reference 2, Ishikao bridge, Chengjun. learning and memory genetic engineering animal model development [ J ]. Chinese Compare medicine, 2003-65-68). However, the molecular mechanisms of the formation, maintenance and regression of learning and memory, and the decline of memory associated with aging and diseases are urgently needed to be elucidated. How to intervene in the memory decline associated with aging and neurodegenerative diseases is also pending.
Pepper is the most basic component of indian medicine and is used to treat a variety of diseases. Pharmacological studies have demonstrated many of their traditional effects such as: analgesic and antipyretic, antioxidant, antibacterial, etc. Piperine is an effective component of pepper. The piperine has wide pharmacological action, and has the functions of oxidation resistance, immunoregulation, tumor resistance and the like. The composition is also a broad-spectrum anticonvulsant, has good antagonistic action on experimental electroconvulsive of mice, and has antagonistic action of different degrees on pentaerythrine, tetrandrine, strychnine, and convulsive attacks and auditory attacks caused by intracerebroventricular injection of curarine, glutamic acid and the like. The clinical trial shows that the medicine also has curative effect on certain types of epilepsy.
Through search of Chinese patents, such as patent publication No. CN110141594A, a medicine for treating Alzheimer disease is disclosed, and patent publication No. CN101808635A discloses the application of piperine and derivatives thereof in treating neurological diseases, and the piperine is used for related medicine research.
However, in the prior art, no piperine is applied to learning and memory enhancement, and the invention provides an application method for enhancing learning and memory by using piperine, aiming at filling the gap of the prior art and solving the problems of study and application of the piperine to the learning and memory enhancement.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides an application method for enhancing learning and memory by using piperine.
In order to achieve the purpose, the invention adopts the following technical scheme:
s1: preparing a fruit fly solid culture medium;
s2: selecting and hybridizing virgins;
s3: establishing a fruit fly sugar reward olfaction association type learning and memory paradigm;
s4: feeding medicine with capillary-nanometer microemulsion liquid food;
in the S1, the components of the fruit fly solid culture medium are as follows: 100g of white sugar, 320g of corn flour, 40g of soybean flour, 260g of brown sugar syrup, 40g of agar, 72g of yeast powder and 5L of water;
the step of S3 includes the steps of:
s31: preparing and preparing smell;
s32: sugar reward training;
s33: learning and memory testing;
s34: and (6) counting and calculating data.
In the S4, a capillary tube administration mode is adopted, the nano microemulsion is used for dissolving the piperine into liquid food to feed the fruit flies, and the fed medicines are mixed with food-grade pigments.
Preferably: in the S1, the specific preparation method of the culture medium comprises the following steps:
s11: respectively weighing 40g of agar and 72g of yeast powder for later use;
s12: 100g of white sugar, 320g of corn flour and 40g of soybean flour are continuously weighed in a clean beaker with a range of 1L;
s13: 5L of water is added into the pot and heated to 40-50 ℃. Adding hot water into a beaker of about 600ml until the beaker is full, and uniformly stirring;
s14: sequentially adding weighed agar and yeast powder into hot water in a pot, and continuously stirring with a spoon in the period;
s15: add the mixture in the beaker and stir continuously. Then 260g of brown sugar syrup is weighed by a beaker and added into the pot, and the syrup retained on the inner wall of the beaker can be dissolved in cold water added into the beaker and placed in a microwave oven for about 1min and then added into the pot;
s16: continuously stirring the food in the pan until the food is viscous, and accelerating the stirring until the food is boiled for more than 6 min;
s17: after the food is boiled for 6min, the fire is turned off. Stirring every 30min to prevent food from coagulating and forming layer;
s18: adding antiseptic when the temperature of food is reduced to about 50 deg.C (the pan wall is not scalded when the naked hand touches), and packaging. The fruit fly tube and the fruit fly bottle are baked in an oven for more than 2 hours before being subpackaged with food to kill mites;
s19: preservatives should be added to 4L of the food: 28ml of methyl paraben solution +46.5ml of mixed acid, adjusted to 100ml with water, are added to the cooled food.
In the S19, the preservative is prepared from methyl p-hydroxybenzoate: ethanol 2 g: 7ml, mixed acid consisting of water: propionic acid: 540.5ml phosphoric acid: 418 ml: 41.5 ml.
In the S1, the fruit fly culture condition is that the temperature is constant at 25 ℃ and the humidity is 50-60%.
Further: in the S2, the selection method of the virgins comprises the following steps: removing all imagoes in the stock bottle, collecting female fruit flies which are just emerged every 8 hours, and putting the female fruit flies into a culture bottle for standby application, wherein in S2, the fruit fly hybridization method comprises the following steps:
s21: separately placing two drosophila melanogaster with different genotypes in CO2After anesthesia is carried out on the anesthesia flight board, male and female selection is carried out;
s22: collecting the selected virgins of one genotype and the male flies of the other genotype to a brand-new fruit fly tube for hybridization;
s23: filling the tube bottom into a 10cm fruit fly solid culture medium;
s24: after 4-6 days, when the eggs laid by a plurality of female flies are observed on the surface of the food at the bottom of the tube, the fruit flies in the tube can be completely removed;
s25: after the four stages of egg → larva → pupa → imago, the hybrid progeny drosophila melanogaster is obtained. The hybrid progeny drosophila can be used for experiments after phenotype observation and sorting operation.
On the basis of the scheme: in the S31, the smell includes BenZ and OCT with the concentration of 100uL/100ml, and the specific preparation steps are as follows:
s311: respectively dissolving BenZ and OCT in mineral oil;
s312: mounting the mineral oil with dissolved odor in a glass bottle corresponding to the diffuser at a rate of 10 ml/bottle;
s313: checking whether the bottle mouth is damaged or not to ensure the air tightness of the diffuser.
The better scheme in the scheme is as follows: in S32, the method includes the steps of:
s321: 50 fruit flies are transferred into a fruit fly tube with wet filter paper to be starved for 24 hours in advance;
s322: putting fruit flies into a training tube which is not soaked with cane sugar, connecting a T-Maze upper fly inlet, aligning an upper airflow port of the middle plate to the fly inlet, connecting an upper air suction port to an air pump, and measuring the airflow in the training tube to be 750 mL/min;
s323: waiting for 1min30s to ensure the fruit fly is adapted to the experimental environment;
s324: 1min30s-3min30s, the OCT (CS) smell was passed through the training tube-)2min, detecting the airflow size to 750 ml/min;
s325: 3min30s-4min, discharging OCT smell, introducing air into the training tube, detecting the size of air flow to 750ml/min, and resting the fruit flies;
s326: 4-6 min, transferring fruit fly into training tube soaked with 2M sucrose saturated solution (US), introducing BenZ odor (CS) into the training tube+) And detecting the size of the airflow to 750 ml/min.
As a further scheme of the invention: in S33, the learning ability testing method specifically includes the steps of:
s331: after the fruit flies are trained, lifting the lower cavity of the middle plate to a fly loading port, and slightly knocking the fruit flies in the training pipe into the cavity;
s332, mounting collecting pipes on the left side and the right side of the lower through hole, connecting the collecting pipes to the corresponding positions of the diffusers, and connecting an air pump to an air suction port on the lower side;
s333: the middle plate is pressed to the bottom. At this time, the fruit fly has two kinds of odors (CS)+&CS-) Selecting;
s334: after 2min, the middle plate is lifted to the state that the left and right collecting arms are completely separated, and the fruit flies are collected into the corresponding testing tubes after the smell and the airflow are removed.
Meanwhile, in the S34, the CS obtained in the S33 is added+And CS-Counting the total number of the fruit flies respectively according to the formula: PI (proportional integral)1/2＝(#CS+-#CS-)/(#total flies)；PI＝(PIBenZ with US+PIOCT with US) And/2 calculation.
As a preferable aspect of the present invention: in the S4, the medicine concentration is 2mg/mL, the medicine feeding time is 18h, and the hunger time before sugar reward training is 24h, and the method specifically comprises the following steps:
s41: collecting eclosion-1-2 d fruit fly, and performing mild CO2Anaesthetizing;
s42: sorting the fruit flies in 30-50 flies/tube;
s43: after sexual maturity of the fruit flies is carried out for 1-2 days, pure hungry is carried out for 4 hours;
s44: feeding piperine (experimental group)/nano microemulsion (control group) for 18 h;
s45: starving wet filter paper (containing water) for 24 h;
s46: carrying out sugar reward training on the fruit flies;
s46: after training, the fruit flies are transferred back to normal solid food for re-feeding for 6 h;
s47: starving the wet filter paper for 18h again;
s48: and (5) performing learning and memory tests on the fruit flies.
The invention has the beneficial effects that:
1. the invention researches and searches piperine as a high-efficiency learning and memory enhancing medicament for the first time, and makes up the defects of the prior art.
2. The piperine can enhance the long-term memory capacity of drosophila melanogaster and prolong the memory time limit of drosophila melanogaster by enhancing the activity of a transcription factor CREB, and has recovery effect on A beta 42 drosophila melanogaster and memory decline caused by aging.
3. The piperine can be used as a novel medicine for learning and memory in model organisms such as drosophila melanogaster and mice and clinically.
Drawings
FIG. 1 is a schematic representation of piperine's ability to enhance Drosophila learning and memory by enhancing CREB activity;
in the figure: CON, control; PIP, piperine.
FIG. 2 is a diagram showing the HPLC analysis result of piperine in the brain of Drosophila melanogaster.
FIG. 3 is a schematic of the memory decline of piperine in the rescue of disease model A β 42 fruit flies.
FIG. 4 is a schematic diagram of the ability of piperine to restore age-induced memory decline.
FIG. 5 is a schematic view of a fruit fly candy smell-awarding associative learning and memory device.
In the figure:
a-training tube (sugar tube + blank tube), b-testing tube, c-T-maze and d-bubbler.
Detailed Description
The technical solution of the present patent will be described in further detail with reference to the following embodiments.
Reference will now be made in detail to embodiments of the present patent, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are exemplary only for the purpose of explaining the present patent and are not to be construed as limiting the present patent.
In the description of this patent, it is to be understood that the terms "center," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like are used in the orientations and positional relationships indicated in the drawings for the convenience of describing the patent and for the simplicity of description, and are not intended to indicate or imply that the referenced devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and are not to be considered limiting of the patent.
In the description of this patent, it is noted that unless otherwise specifically stated or limited, the terms "mounted," "connected," and "disposed" are to be construed broadly and can include, for example, fixedly connected, disposed, detachably connected, disposed, or integrally connected and disposed. The specific meaning of the above terms in this patent may be understood by those of ordinary skill in the art as appropriate.
An application method for enhancing learning and memory by piperine is shown in fig. 1-4, and specifically comprises the following steps:
s1: preparing a fruit fly solid culture medium;
s2: selecting and hybridizing virgins;
s3: establishing a fruit fly sugar reward olfaction association type learning and memory paradigm;
s4: feeding medicine with capillary-nanometer microemulsion liquid food;
in the S1, the components of the fruit fly solid culture medium are as follows: 100g of white sugar, 320g of corn flour, 40g of soybean flour, 260g of brown sugar syrup, 40g of agar, 72g of yeast powder and 5L of water;
the step of S3 includes the steps of:
s31: preparing and preparing smell;
s32: sugar reward training;
s33: learning and memory testing;
s34: and (6) counting and calculating data.
In the S4, a capillary tube administration mode is adopted, the nano microemulsion is used for dissolving the piperine into liquid food to feed the fruit flies, and the fed medicines are mixed with food-grade pigments.
In the S1, the specific preparation method of the culture medium comprises the following steps:
s11: respectively weighing 40g of agar and 72g of yeast powder for later use;
s12: 100g of white sugar, 320g of corn flour and 40g of soybean flour are continuously weighed in a clean beaker with a range of 1L;
s13: 5L of water is added into the pot and heated to 40-50 ℃. Adding hot water into a beaker of about 600ml until the beaker is full, and uniformly stirring;
s14: sequentially adding weighed agar and yeast powder into hot water in a pot, and continuously stirring with a spoon in the period;
s15: add the mixture in the beaker and stir continuously. Then 260g of brown sugar syrup is weighed by a beaker and added into the pot, and the syrup retained on the inner wall of the beaker can be dissolved in cold water added into the beaker and placed in a microwave oven for about 1min and then added into the pot;
s16: continuously stirring the food in the pan until the food is viscous, and accelerating the stirring until the food is boiled for more than 6 min;
s17: after the food is boiled for 6min, the fire is turned off. Stirring every 30min to prevent food from coagulating and forming layer;
s18: adding antiseptic when the temperature of food is reduced to about 50 deg.C (the pan wall is not scalded when the naked hand touches), and packaging. The fruit fly tube and the fruit fly bottle are baked in an oven for more than 2 hours before being subpackaged with food to kill mites;
s19: preservatives should be added to 4L of the food: 28ml of methyl paraben solution +46.5ml of mixed acid, adjusted to 100ml with water, are added to the cooled food.
In the S19, the preservative is prepared from methyl p-hydroxybenzoate: ethanol 2 g: 7ml, mixed acid consisting of water: propionic acid: 540.5ml phosphoric acid: 418 ml: 41.5 ml.
In the S1, the fruit fly culture condition is that the temperature is constant at 25 ℃ and the humidity is 50-60%.
In the S2, the selection method of the virgins comprises the following steps: removing all imagoes in the stock bottle, collecting newly emerged female drosophila melanogaster every 8 hours, and putting the female drosophila melanogaster into a culture bottle for later use; since the newly emerged fruit fly is slender and tender and almost transparent, black food residues of the digestive tract in the abdominal cavity can be seen from the abdominal surface through the shell of chitin. Therefore, the female with dark spots in the abdomen was identified as virgins.
In the S2, the drosophila hybridization method comprises the following steps:
s21: separately placing two drosophila melanogaster with different genotypes in CO2After anesthesia is carried out on the anesthesia flight board, male and female selection is carried out;
s22: collecting the selected virgins of one genotype and the male flies of the other genotype to a brand-new fruit fly tube for hybridization;
s23: filling the tube bottom into a 10cm fruit fly solid culture medium;
s24: after 4-6 days, when the eggs laid by a plurality of female flies are observed on the surface of the food at the bottom of the tube, the fruit flies in the tube can be completely removed;
s25: after the four stages of egg → larva → pupa → imago, the hybrid progeny drosophila melanogaster is obtained. The hybrid progeny drosophila can be used for experiments after phenotype observation and sorting operation.
The test in said S3 uses Drosophila flies all from Blomington Drosophila Stock Center (BDSC) for growth at 12 h: 12h light dark, 25 ℃, 50% RH, and through at least six generations of genetic background control, the fruit fly tested was collected within 1-2d after eclosion, using mild CO2After anaesthesia, sorting was performed in a number of 30-50/tube. The fruit flies were then kept in the feed for 12 h: the test is carried out after 2-3 days at 25 ℃ and 50% RH in 12h light-dark environment (drug feeding/drug non-feeding). The experimental environment was 25 ℃, 70% RH, red darkroom. To reduce the influence of environment on the fruit fly to the maximum extent, the genes comprise:
(1)w1118 BDSC 6326
(2)CRE-LUC BDSC 30743
(3)elavGal4GS BDSC 43642
(4)UAS-Aβ42 BDSC 64216
in the S31, the smell includes BenZ and OCT with the concentration of 100uL/100ml, and the specific preparation steps are as follows:
s311: respectively dissolving BenZ and OCT in mineral oil;
s312: filling the mineral oil with dissolved odor in a corresponding glass bottle on a diffuser at a rate of 10 ml/bottle;
s313: checking whether the bottle mouth is damaged or not to ensure the air tightness of the diffuser.
In order to ensure that the odor is not polluted, all right positions (airflow channels and collecting pipes) in the diffuser and the T-Maze are specified to be BenZ odor in the experiment; all left positions are OCT scent.
In S32, the method includes the steps of:
s321: 50 fruit flies are transferred into a fruit fly tube with wet filter paper to be starved for 24 hours in advance;
s322: putting fruit flies into a training tube which is not soaked with cane sugar, connecting a T-Maze upper fly inlet, aligning an upper airflow port of the middle plate to the fly inlet, connecting an upper air suction port to an air pump, and measuring the airflow in the training tube to be 750 mL/min;
s323: waiting for 1min30s to ensure the fruit fly is adapted to the experimental environment;
s324: 1min30s-3min30s, the OCT (CS) smell was passed through the training tube-)2min, detecting the airflow size to 750 ml/min;
s325: 3min30s-4min, discharging OCT smell, introducing air into the training tube, detecting the size of air flow to 750ml/min, and resting the fruit flies;
s326: 4-6 min, transferring fruit fly into training tube soaked with 2M sucrose saturated solution (US), introducing BenZ odor (CS) into the training tube+) And detecting the size of the airflow to 750 ml/min.
The intermediate operating times are not taken into account in the above-described time step statistics.
In S33, the learning ability testing method specifically includes the steps of:
s331: after the fruit flies are trained, lifting the lower cavity of the middle plate to a fly loading port, and slightly knocking the fruit flies in the training pipe into the cavity;
s332, mounting collecting pipes on the left side and the right side of the lower through hole, connecting the collecting pipes to the corresponding positions of the diffusers, and connecting an air pump to an air suction port on the lower side;
s333: the middle plate is pressed to the bottom. At this time, the fruit fly has two kinds of odors (CS)+&CS-) Selecting;
s334: after 2min, the middle plate is lifted to the state that the left and right collecting arms are completely separated, and the fruit flies are collected into the corresponding testing tubes after the smell and the airflow are removed.
The residual fruit flies in the middle cavity are not collected, and by using the paradigm, the learning ability (1min), short-term memory (3h), long-term memory (24h) and other tests of the fruit flies can be carried out.
In the S34, the CS obtained in the S33 is added+And CS-Counting the total number of the fruit flies respectively according to the formula: PI (proportional integral)1/2＝(#CS+-#CS-)/(#total flies)；PI＝(PIBenZ with US+PIOCT with US) And/2 calculation.
In the S4, the medicine concentration is 2mg/mL, the medicine feeding time is 18h, and the hunger time before sugar reward training is 24h, and the method specifically comprises the following steps:
s41: collecting eclosion-1-2 d fruit fly, and performing mild CO2Anaesthetizing;
s42: sorting the fruit flies in 30-50 flies/tube;
s43: after sexual maturity of the fruit flies is carried out for 1-2 days, pure hungry is carried out for 4 hours;
s44: feeding piperine (experimental group)/nano microemulsion (control group) for 18 h;
s45: starving wet filter paper (containing water) for 24 h;
s46: carrying out sugar reward training on the fruit flies;
s46: after training, the fruit flies are transferred back to normal solid food for re-feeding for 6 h;
s47: starving the wet filter paper for 18h again;
s48: and (5) performing learning and memory tests on the fruit flies.
Example 1:
an application method for enhancing learning and memory by piperine comprises selecting ELAVGal4 as genotypeGS；UAS-CREBDNThe drosophila melanogaster is taken as an experimental group, and the genotype is selected as elavGal4GS(ii) a UAS-CD8, fruit fly with GFP as control group; utensil for cleaning buttockThe operation steps are as follows:
a1: collecting the 1-2d post-eclosion Drosophila melanogaster by mild CO2Anaesthetizing, and sorting the fruit flies by 30-50 flies/tube;
a2: after sexual maturity of the fruit flies for 1-2 days, the fruit flies are purely starved for 4h, and the fruit flies of the experimental group and the control group are respectively fed with piperine (+)/nano microemulsion (-) only;
a3: starving the fruit flies for 24h in a fruit fly tube containing wet filter paper;
a4: carrying out sugar reward training on the fruit flies;
a5: after training, the fruit flies are transferred back to normal solid food for re-feeding for 6 h;
a6: the drosophila were again starved for 18h in a drosophila tube containing wetted filter paper;
a7: and (5) performing learning and memory tests on the fruit flies.
When the application is carried out, the piperine can enhance the learning and memory of the fruit flies by enhancing the activity of the transcription factor CREB, and the long-term memory of the fruit flies in a control group is obviously enhanced by feeding the piperine; while over-expressing dominant negative CREB (CREB)DN) Can obviously inhibit the piperine-mediated long-term memory enhancement of drosophila melanogaster, and shows that the piperine plays a role depending on CREB.
Example 2:
an application method for enhancing learning and memory by piperine comprises selecting ELAVGal4 as genotypeGS(ii) a DrosophilA melanogaster of UAS-AbetA 42 is taken as an experimental group, and the genotype is selected as elavGal4GS(ii) a UAS-CD8, fruit fly with GFP as control group; the specific operation steps are as follows:
b1: collecting the 1-2d post-eclosion Drosophila melanogaster by mild CO2Anaesthetizing, and sorting the fruit flies by 30-50 flies/tube;
b2: after sexual maturity of the drosophila for 1-2d, inducing for 5d, 10d and 15d by 50uL Ru (+);
b3: after induction at different times, the drosophila were purely starved for 4h and fed with piperine (+)/nanomicroemulsion (-) alone to the experimental and control groups, respectively;
b4: starving the fruit flies for 24h in a fruit fly tube containing wet filter paper;
b5: carrying out sugar reward training on the fruit flies;
b6: after training, the fruit flies are transferred back to normal solid food for re-feeding for 6 h;
b7: the drosophila were again starved for 18h in a drosophila tube containing wetted filter paper;
b8: and (5) performing learning and memory tests on the fruit flies.
In this embodiment: after 5 days, Abeta 42 drosophila shows a decline but not significant of long-term memory initially, and the long-term memory is significantly enhanced after the piperine feeding rescue; after 10 days, the long-term memory of the Abeta 42 fruit fly is continuously reduced, and after the fruit fly is subjected to piperine feeding rescue, the long-term memory of the fruit fly is also improved, but compared with the fruit fly of 5 days, the rescue effect is reduced; after 15 days, the long-term memory of the Abeta 42 fruit fly is further reduced than that of 10 days, and the re-feeding of piperine can not save the learning memory.
Example 3:
an application method for enhancing learning and memory by piperine comprises selecting w as gene1118(ii) a The method comprises the following specific steps of using CRE-LUC fruit flies:
c1: collecting the 1-2d post-eclosion Drosophila melanogaster by mild CO2Anaesthetizing, and sorting the fruit flies by 30-50 flies/tube;
c2: after sexual maturity of the fruit flies for 1-2 days, aging in normal solid food;
c3: pure starvation of the drosophila was carried out for 4h after 20d, 40d and 60d of senescence, and the experimental and control groups were fed with piperine (+)/nano microemulsion (-) only, respectively;
c4: starving the fruit flies for 24h in a fruit fly tube containing wet filter paper;
c5: carrying out sugar reward training on the fruit flies;
c6: after training, the fruit flies are transferred back to normal solid food for re-feeding for 6 h;
c7: the drosophila were again starved for 18h in a drosophila tube containing wetted filter paper;
c8: and (5) performing learning and memory tests on the fruit flies.
In this embodiment: first, only the left side is observed, the long-term memory of the fruit flies is obviously reduced along with the aging, and the right side is combined to show that the long-term memory of the fruit flies aged 20d, 40d and 60d can be enhanced to a certain extent by feeding piperine, and the enhancement effect is gradually weakened along with the aging.
The following examples are given in combination with examples 1 to 3: the piperine can enhance the long-term memory capacity of drosophila melanogaster and prolong the memory time limit of drosophila melanogaster by enhancing the activity of a transcription factor CREB, and has recovery effect on A beta 42 drosophila melanogaster and memory decline caused by aging.
Example 4:
an application method for enhancing learning and memory by piperine is disclosed, and the experimental device is shown in FIG. 5, and uses a manual learning and memory device in T Tully, W G Quinn.Classification and termination in normal and mutant Drosophila melanogaster [ J ]. J Comp physical A.1985,157(2):263-277, and mainly comprises a training tube (sugar tube + blank tube) a; a test tube b; t-maze c; a bbler (odor storage device) d, etc.
In this embodiment, during training, the fruit flies are first put into a: in the blank training tube, d: bubbler introducing Cs-The gas is studied for the first 2 min; after 2min, disconnect d: babbler and c: connecting T-maze, and introducing air for 30s to allow the drosophila to rest; after 30s, the flies were transferred to another sucrose training tube, d: bubbler introducing Cs+The gas was studied for a second 2 min. After 2min training was complete, the flies were transferred to normal solid food.
At the time of testing, c: the lower cavity of the T-maze intermediate plate is lifted to a fly loading port, and the fruit flies are lightly knocked into the cavity. B is installed on the left side and the right side of the lower through hole: test tubes and d: and connecting the corresponding positions of the bubbler. The air pump is connected to press the middle plate to the bottom. At the moment, the fruit flies have two smells (CS)+&CS-) Is selected. After 2min, the middle plate is lifted to the state that the left and right collecting arms are completely separated, and the fruit flies are collected to the corresponding b after the smell and the airflow are unloaded: and carrying out statistics and calculation in the test tube.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (10)
1. Application of piperine in preparing medicine for enhancing learning and memory is provided.
2. The application of piperine as medicine for enhancing learning and memory in model organism (fruit fly, mouse).
3. An application method for enhancing learning and memory by piperine is characterized by comprising the following steps:
s1: preparing a fruit fly solid culture medium;
s2: selecting and hybridizing virgins;
s3: establishing a fruit fly sugar reward olfaction association type learning and memory paradigm;
s4: feeding medicine with capillary-nanometer microemulsion liquid food;
in the S1, the components of the fruit fly solid culture medium are as follows: 100g of white sugar, 320g of corn flour, 40g of soybean flour, 260g of brown sugar syrup, 40g of agar, 72g of yeast powder and 5L of water;
the step of S3 includes the steps of:
s31: preparing and preparing smell;
s32: sugar reward training;
s33: learning and memory testing;
s34: and (6) counting and calculating data.
In the S4, a capillary tube administration mode is adopted, the nano microemulsion is used for dissolving the piperine into liquid food to feed the fruit flies, and the fed medicines are mixed with food-grade pigments.
4. The method as claimed in claim 3, wherein the specific formulation of the culture medium in S1 comprises the following steps:
s11: respectively weighing 40g of agar and 72g of yeast powder for later use;
s12: 100g of white sugar, 320g of corn flour and 40g of soybean flour are continuously weighed in a clean beaker with a range of 1L;
s13: 5L of water is added into the pot and heated to 40-50 ℃. Adding hot water into a beaker of about 600ml until the beaker is full, and uniformly stirring;
s14: sequentially adding weighed agar and yeast powder into hot water in a pot, and continuously stirring with a spoon in the period;
s15: add the mixture in the beaker and stir continuously. Then 260g of brown sugar syrup is weighed by a beaker and added into the pot, and the syrup retained on the inner wall of the beaker can be dissolved in cold water added into the beaker and placed in a microwave oven for about 1min and then added into the pot;
s16: continuously stirring the food in the pan until the food is viscous, and accelerating the stirring until the food is boiled for more than 6 min;
s17: after the food is boiled for 6min, the fire is turned off. Stirring every 30min to prevent food from coagulating and forming layer;
s18: adding antiseptic when the temperature of food is reduced to about 50 deg.C (the pan wall is not scalded when the naked hand touches), and packaging. The fruit fly tube and the fruit fly bottle are baked in an oven for more than 2 hours before being subpackaged with food to kill mites;
s19: preservatives should be added to 4L of the food: 28ml of methyl paraben solution +46.5ml of mixed acid, adjusted to 100ml with water, are added to the cooled food.
In the S19, the preservative is prepared from methyl p-hydroxybenzoate: ethanol 2 g: 7ml, mixed acid consisting of water: propionic acid: 540.5ml phosphoric acid: 418 ml: 41.5 ml.
In the S1, the fruit fly culture condition is that the temperature is constant at 25 ℃ and the humidity is 50-60%.
5. The method as claimed in claim 3, wherein in S2, the method for selecting virgins comprises: removing all imagoes in the stock bottle, collecting female fruit flies which are just emerged every 8 hours, and putting the female fruit flies into a culture bottle for standby application, wherein in S2, the fruit fly hybridization method comprises the following steps:
s21: separately placing two drosophila melanogaster with different genotypes in CO2After anesthesia is carried out on the anesthesia flight board, male and female selection is carried out;
s22: collecting the selected virgins of one genotype and the male flies of the other genotype to a brand-new fruit fly tube for hybridization;
s23: filling the tube bottom into a 10cm fruit fly solid culture medium;
s24: after 4-6 days, when the eggs laid by a plurality of female flies are observed on the surface of the food at the bottom of the tube, the fruit flies in the tube can be completely removed;
s25: after the four stages of egg → larva → pupa → imago, the hybrid progeny drosophila melanogaster is obtained. The hybrid progeny drosophila can be used for experiments after phenotype observation and sorting operation.
6. The method as claimed in claim 3, wherein in S31, the smell includes BenZ and OCT at a concentration of 100uL/100ml, and the specific steps are as follows:
s311: dissolving BenZ and OCT in Mineral Oil, Heavy (Fisher, #8042-47-5), respectively;
s312: mounting the mineral oil with dissolved odor in a glass bottle corresponding to the diffuser at a rate of 10 ml/bottle;
s313: checking whether the bottle mouth is damaged or not to ensure the air tightness of the diffuser.
7. The method as claimed in claim 6, wherein the step of S32 comprises the following steps:
s321: 50 fruit flies are transferred into a fruit fly tube with wet filter paper to be starved for 24 hours in advance;
s322: putting fruit flies into a training tube which is not soaked with cane sugar, connecting a T-Maze upper fly inlet, aligning an upper airflow port of the middle plate to the fly inlet, connecting an upper air suction port to an air pump, and measuring the airflow in the training tube to be 750 mL/min;
s323: waiting for 1min30s to ensure the fruit fly is adapted to the experimental environment;
s324: 1min30s-3min30s, the OCT (CS) smell was passed through the training tube-)2min, detecting the airflow size to 750 ml/min;
s325: 3min30s-4min, discharging OCT smell, introducing air into the training tube, detecting the size of air flow to 750ml/min, and resting the fruit flies;
s326: 4-6 min, transferring fruit fly into training tube soaked with 2M sucrose saturated solution (US), introducing BenZ odor (CS) into the training tube+) And detecting the size of the airflow to 750 ml/min.
8. The application method of piperine for enhancing learning and memory as claimed in claim 7, wherein the learning ability testing method in S33 comprises the following steps:
s331: after the fruit flies are trained, lifting the lower cavity of the middle plate to a fly loading port, and slightly knocking the fruit flies in the training pipe into the cavity;
s332: collecting pipes are arranged on the left side and the right side of the lower through hole and connected to the corresponding position of the diffuser, and an air pump is connected to an air suction port on the lower side;
s333: the middle plate is pressed to the bottom. At this time, the fruit fly has two kinds of odors (CS)+&CS-) Selecting;
s334: after 2min, the middle plate is lifted to the state that the left and right collecting arms are completely separated, and the fruit flies are collected into the corresponding testing tubes after the smell and the airflow are removed.
9. The method as claimed in claim 8, wherein in S34, the CS obtained in S33 is added+And CS-Counting the total number of the fruit flies respectively according to the formula: PI (proportional integral)1/2＝(#CS+-#CS-)/(#total flies)；PI＝(PIBenZ with US+PIOCT with US) And/2 calculation.
10. The application method of piperine for enhancing learning and memory as claimed in claim 3, wherein in S4, the concentration of the drug is 2mg/mL, the feeding time is 18h, and the hunger time before sugar reward training is 24h, the specific steps are as follows:
s41: collecting eclosion-1-2 d fruit fly, and performing mild CO2Anaesthetizing;
s42: sorting the fruit flies in 30-50 flies/tube;
s43: after sexual maturity of the fruit flies is carried out for 1-2 days, pure hungry is carried out for 4 hours;
s44: feeding piperine (experimental group)/nano microemulsion (control group) for 18 h;
s45: starving wet filter paper (containing water) for 24 h;
s46: carrying out sugar reward training on the fruit flies;
s46: after training, the fruit flies are transferred back to normal solid food for re-feeding for 6 h;
s47: starving the wet filter paper for 18h again;
s48: and (5) performing learning and memory tests on the fruit flies.
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Non-Patent Citations (2)
| Title |
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| MOGHADAMNIA ALI AKBAR等: "Effect of breastfeeding piperine on the learning of offspring mice: interaction with caffeine and diazepam", 《JOURNAL OF EXPERIMENTAL PHARMACOLOGY》 * |
| ROSHANBAKHSH HANNANEH等: "Piperine ameliorated memory impairment and myelin damage in lysolecethin induced hippocampal demyelination", 《LIFE SCIENCES》 * |
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