CN108670961A - The quickly drunk method for establishing model of Caenorhabditis elegans of screening Antialcoholic plant and application - Google Patents
The quickly drunk method for establishing model of Caenorhabditis elegans of screening Antialcoholic plant and application Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
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Abstract
A kind of drunk method for establishing model of Caenorhabditis elegans of quick screening Antialcoholic plant, it is based on Wild-type C. elegans, Liquid Culture is to 2,000 2500 worms/mL, when Caenorhabditis elegans grows to and has just enter into the adult stage, alcoholic solution is added in worm liquid to alcoholic solution volume final concentration of 1% 5%, intervene for 24 hours, at this point, Caenorhabditis elegans shows as state of intoxication.Comparison is detected to the indices of nematode into the drunk model in application, directly the water extract of medical and edible dual purpose plant is added.This method, for evaluating, is conveniently easy to get using model organism Caenorhabditis elegans, can effectively reduce experimentation cost；Caenorhabditis elegans growth cycle is short, and short time interior energy mass propagation obtains thousands of nematodes for testing, and can guarantee enough more of sample size, reduction test error ensures the accuracy of evaluation result；High throughput screening assay can be carried out, efficiently show that test result, test period greatly shorten.
Description
Technical field
The invention belongs to biotechnology, it is related to a kind of plant (medicine-food two-purpose) water extract having sober-up function and quickly sieves
The method of choosing, more particularly to a kind of method for building up of the drunk model of Caenorhabditis elegans of quick screening Antialcoholic plant.
Background technology
China's spirits culture is with a long history, and responsible drinking can improve blood circulation of human body, relieving fatigue, but when intake is more than
After human body limit, the symptoms such as lethargic sleep, gastrorrhagia, hepatic injury, nervous system paralysis are will produce, significant damage is caused to human body, because
This large amount of antialcoholic drug with dispelling effects of alcohol (sober up drink by such as flower of kudzuvine soup, Gan get Le sobering-up beverages, happy sobering-up beverage of waking up, ancient cooking vessel honor
The plants hangover medicines such as material) it comes out.But the drug action of most antialcoholic drug goes to verify by establishing mice drunk model mostly, passes through
Mouse blood index and associated metabolic enzyme are detected, with role of evaluation effect.The method there are processes more complex, test group sample number
Amount limitation, test period are long, and the higher disadvantage of economic cost.
Caenorhabditis elegans (Caenorhabditis elegans, C.elegans) is used as common and non-in scientific research
Often important one mode animal, has the characteristics that and advantage：Individual is smaller, and adult about 1.5mm long is at low cost, convenient for
Mass propgation and operation on laboratory；Life cycle is short, and the average history of life is 3.5 days at 20 DEG C, convenient for quickly carrying out phenotype
Analysis.Therefore a kind of drunk model is established using Caenorhabditis elegans, to solve the shortcoming of mouse test.
Invention content
The object of the present invention is in view of the above shortcomings of the prior art, a kind of the beautiful of quick screening Antialcoholic plant is provided
The drunk method for establishing model of hidden rhabditida and application can eat two by the foundation of the drunk model of Caenorhabditis elegans from a variety of medicines
The significant plant of drug effect of relieving the effect of alcohol quickly is filtered out in plant, has that high-efficiency sieve medicine, to reduce cost, test result reliably special
Point.
In order to achieve the above object, the technical solution adopted in the present invention is：A kind of beautiful hidden bar of quick screening Antialcoholic plant
The drunk method for establishing model of nematode, steps are as follows for this method：
(1) use conventional liq cultivation to Wild-type C. elegans (N2) cultivated, so that worm liquid concentration is controlled
For 2000-2500 worms/mL, Caenorhabditis elegans using bacterial concentration for 100mg/ml concentration OP50 bacterium solutions as food；
The above-mentioned worm liquid concentration referred to refers to the concentration of Caenorhabditis elegans in culture solution；And the worm liquid in text refers to then training
Support the culture solution of a concentration of 2000-2500 worms/mL of Caenorhabditis elegans after Caenorhabditis elegans.
(2) when Caenorhabditis elegans grows to and has just enter into the adult stage, alcoholic solution is added in worm liquid, makes alcoholic solution
The final concentration of 1%-5% of volume intervenes for 24 hours, at this point, Caenorhabditis elegans shows as state of intoxication.
In the drunk model that the above method is established, the head oscillation frequency of Caenorhabditis elegans is 10-40 times/30s, swallows
Movement is 10-40 times/30s, movement locus shows uncoordinated sinusoidal wavelength and has trailing phenomenon, service life 10-12d, line
Total antioxidant capacity is 15-25U/mg in polypide, and it is 80-100A.U. that oxygen radical (ROS) is horizontal, alcohol concentration in nematode body
For 30-50ng/ μ g albumen.
It is above-mentioned to refer to using conventional liq cultivation to Wild-type C. elegans (N2) carry out culture refer to reference to text
Offer (Stiernagle T.Maintenance of C.elegans [J] .Wormbook, 2006:Method 1-11.) is to beautiful
Hidden rhabditida carries out synchronization and is cultivated with basis.
Present invention simultaneously provides application of the above method in quickly screening medicine-food two-purpose Antialcoholic plant.
In application, being to be completely dissolved medical and edible dual purpose plant extract powder with water, and pass through 0.22 micron of filter membrane
Then degerming is added in the drunk model that the above method is established with the volume final concentration of 5-30mg/mL, intervene for 24 hours.Intervene
After complete, Caenorhabditis elegans is detected, the head oscillation frequency of Caenorhabditis elegans is 50-100 times/30s, swallowing movement 50-
90 times/30s, movement locus shows the sinusoidal wavelength relatively coordinated and trailing phenomenon weakens, the service life is 14-20 days, in nematode body
Total antioxidant capacity is 5-12U/mg and oxygen radical (ROS) level is 40-60A.U., and nematode inside alcohol concentration is 0-
30ng/ μ g albumen, Caenorhabditis elegans show a kind of state tending to proper motion, indicate that the medical and edible dual purpose plant has solution
Wine acts on, and Testing index (head oscillation frequency, swallowing movement, movement locus, service life, the total antioxidation of Caenorhabditis elegans
Alcohol concentration in ability, oxygen free radicals and nematode body) be more intended to index when alcohol-free concentration detects in nematode body, then
Show that the medical and edible dual purpose plant antialcoholism action is more notable.
It is above-mentioned to refer to that medical and edible dual purpose plant extract powder refers to being respectively crushed into several medical and edible dual purpose plants of purchase
Powder, respectively with 1g:The solid-liquid ratio of 10ml is extracted repeatedly using water extract method twice with effective component extracting, and filtering merges twice
Filtrate；Then progress filtrate concentration in rotatory vacuum thickener is gone to, concentrate is placed in glass dish and is sealed, it is cold in -80 DEG C
Freeze at least 2h, finally quickly remove, is freeze-dried.
Compared with prior art, the method for the present invention has the beneficial effect that：Using this generally acknowledged pattern of Caenorhabditis elegans
Biology is conveniently easy to get for evaluating, can effectively reduce experimentation cost；Caenorhabditis elegans growth cycle is short, and short time interior energy is big
It measures breeding and obtains thousands of nematodes for testing, it is ensured that enough more, the reduction test errors of sample size, guarantee evaluation
As a result accuracy；High throughput screening assay can be carried out, efficiently show that test result, test period greatly shorten.
Specific implementation mode
The foundation of the drunk model of 1 Caenorhabditis elegans of embodiment
With Wild-type C. elegans (N2) as model foundation basis, synchronization and the basis of Caenorhabditis elegans are trained
Support with reference to bibliography (Stiernagle T.Maintenance of C.elegans [J] .Wormbook, 2006:1-11.)
In liquid culture method carry out.Caenorhabditis elegans using bacterial concentration for 100mg/ml concentration OP50 as food, worm liquid concentration control
It is made as 2000-2500 worms/mL.At this point, detection Caenorhabditis elegans, head oscillation frequency are 50-100 times/30s, swallow fortune
It moves and shows the sinusoidal wavelength that left and right is coordinated, service life 18-25d, total antioxygen in nematode body for 50-90 times/30s, movement locus
Change ability is 5-12U/mg, and horizontal oxygen radical (ROS) is 40-60A.U., and the alcohol of external intake is not detected in nematode body.
When Caenorhabditis elegans grows to and has just enter into the adult stage, alcoholic solution is added in worm liquid, makes alcoholic solution body
The final concentration of 1%-5% of product, intervenes for 24 hours.Detecting Caenorhabditis elegans at this time, the head oscillation frequency of nematode is 10-40 times/
30s, swallowing movement are that 10-40 times/30s, movement locus show uncoordinated sinusoidal wavelength and have trailing phenomenon, and the service life is
10-12d, total antioxidant capacity is 15-25U/mg in nematode body, and it is 80-100A.U. that oxygen radical (ROS) is horizontal, in nematode body
Alcohol concentration is 30-50ng/ μ g albumen, and nematode at this time shows state of intoxication.
The verification of the drunk model of 2 Caenorhabditis elegans of embodiment
The extraction of medical and edible dual purpose plant extract powder：A variety of medical and edible dual purpose plants of purchase are respectively crushed into powder, and
1g is pressed respectively:The solid-liquid ratio of 10ml extracts 2 times with effective component extracting repeatedly using water extract method, and filtering merges 2 filtrates.
Then progress filtrate concentration in rotatory vacuum thickener is gone to, then concentrate is placed in glass dish, is sealed, -80 DEG C of ice are placed
At least 2h is freezed in case.It finally quickly removes the medicine ware after freezing to be placed in freeze drier, is freeze-dried into powder.
Method：Be arranged blank group (worm liquid+water), control group (worm liquid+alcoholic solution), medicine group (worm liquid+alcoholic solution+
Liquid), wherein：
Blank group：The synchronization of Wild-type C. elegans is with basis culture with reference to bibliography (Maintenance
Of C.elegans) in method using liquid culture method carry out.Caenorhabditis elegans is the dense of 100mg/ml with bacterial concentration
Contracting OP50 is food, and the control of worm liquid concentration is 2000-2500 worms/mL.It is cultivated in liquid medium to young adult, at this point,
The alcohol of external intake is not detected in Caenorhabditis elegans body, (head oscillation frequency swallows number, movement rail to each Testing index
Mark, service life, total antioxidant capacity, oxygen free radicals in nematode body) see that paragraph 1 describes in embodiment 1.
Control group：It is on the basis of above-mentioned blank group, when Caenorhabditis elegans, which is grown to, has just enter into the adult stage in worm
Alcoholic solution is added in liquid, makes the final concentration of 1%-5% of alcoholic solution volume, intervenes for 24 hours.At this point, Caenorhabditis elegans is liquor-saturated
Wine state, (head oscillation frequency swallows number, movement locus, service life, total antioxidant capacity, oxygen in nematode body for each Testing index
Free Radical Level, internal alcohol concentration) see the 2nd segment description in embodiment 1.
Medicine group：It is to be removed by 0.22 micron of filter membrane after being completely dissolved medical and edible dual purpose plant extract powder with water
Then bacterium is added to control group with the volume final concentration of 5-30mg/ml and shows as in the nematode worm liquid of state of intoxication, intervene for 24 hours.
It detects the head oscillation frequency of Caenorhabditis elegans, swallow number, movement locus, service life, total antioxidant capacity, oxygen in nematode body
The indices such as Free Radical Level and internal alcohol concentration.
As a result it shows：Detect the nematode head oscillation frequency of medicine group be 50-100 times/30s, swallowing movement 50-90
Secondary/30s, movement locus show the sinusoidal wavelength relatively coordinated and trailing phenomenon weakens, and the service life effectively improves compared with control group, is
14-20 days, nematode inside alcohol concentration was 0-30ng/ μ g albumen, and nematode shows a kind of state and line tending to proper motion
Total antioxidant capacity is 5-12U/mg in polypide and oxygen radical (ROS) level is 40-60A.U..Testing result blank group with
Between control group, show that the plant has sober-up function, then shows that plant alcohol intoxication-alleviating effect is more notable more being intended to blank group state.
Relieve the effect of alcohol application of the 3 pueraria lobata water extract of embodiment to drunk nematode
It is carried out by the group technology of embodiment 2, medical and edible dual purpose plant uses pueraria lobata.
Pueraria lobata is bought from pharmacy, kudzu root extract powder is made by the extracting method in embodiment 2, by kudzu root extract powder
End be completely dissolved with water after by 0.22 micron of filter membrane degerming, control group is then added to the volume final concentration of 5mg/ml
Worm liquid in, intervene for 24 hours.It detects the head oscillation frequency of Caenorhabditis elegans, swallow number, movement locus, service life, nematode body
The indices such as interior total antioxidant capacity, oxygen free radicals and internal alcohol concentration.
As a result it shows：Detect the nematode head oscillation frequency of medicine group be 65 times/30s, swallowing movement be 80 times/30s,
Movement locus shows the sinusoidal wavelength relatively coordinated and trailing phenomenon weakens, service life 20d, and alcohol concentration is inside nematode
10ng/ μ g albumen, it is 9U/mg and oxygen that nematode, which shows total antioxidant capacity in a kind of state tending to proper motion and nematode body,
It is 55A.U. testing results close to blank group that free radical (ROS) is horizontal, shows that pueraria lobata has sober-up function.
Relieve the effect of alcohol application of the 4 flower of kudzuvine water extract of embodiment to drunk nematode
It is carried out by the group technology of embodiment 2, medical and edible dual purpose plant uses flower of kudzuvine.
Flower of kudzuvine is bought from pharmacy, Flos puerariae lobatae extract powder is made by the extracting method in embodiment 2, by Flos puerariae lobatae extract powder
End be completely dissolved with water after by 0.22 micron of filter membrane degerming, control is then added to the volume final concentration of 10mg/ml
In the worm liquid of group, intervene for 24 hours.It detects the head oscillation frequency of Caenorhabditis elegans, swallow number, movement locus, service life, nematode
The indices such as internal total antioxidant capacity, oxygen free radicals and internal alcohol concentration.
As a result it shows：Detect the nematode head oscillation frequency of medicine group be 70 times/30s, swallowing movement be 85 times/30s,
Movement locus shows the sinusoidal wavelength relatively coordinated and trailing phenomenon weakens, service life 18d, and alcohol concentration is inside nematode
10ng/ μ g albumen, nematode show total antioxidant capacity in a kind of state tending to proper motion and nematode body be 12U/mg and
It is 50A.U. testing results close to blank group that oxygen radical (ROS) is horizontal, shows that flower of kudzuvine has sober-up function.
Relieve the effect of alcohol application of the 5 Schisandra chinensis water extract of embodiment to drunk nematode
It is carried out by the group technology of embodiment 2, medical and edible dual purpose plant uses Schisandra chinensis.
Schisandra chinensis is bought from pharmacy, and Schisandra chinens P.E powder is made by the extracting method in embodiment 2, Schisandra chinensis is carried
By 0.22 micron of filter membrane degerming after taking object powder to be completely dissolved with water, then it is added with the volume final concentration of 20mg/ml
Into the worm liquid of control group, intervene for 24 hours.It detects the head oscillation frequency of Caenorhabditis elegans, swallow number, movement locus, longevity
The indices such as total antioxidant capacity, oxygen free radicals and internal alcohol concentration in life, nematode body.As a result it shows：It detects
The nematode head oscillation frequency of medicine group is 60 times/30s, swallowing movement is 75 times/30s, movement locus shows relatively coordination
Sinusoidal wavelength and trailing phenomenon decrease, service life 16d, nematode inside alcohol concentration is 20ng/ μ g albumen, and nematode shows one kind
Tend to total antioxidant capacity in the state of proper motion and nematode body be 8U/mg and oxygen radical (ROS) level be 40A.U. inspection
Result is surveyed close to blank group, shows that Schisandra chinensis has sober-up function.
Relieve the effect of alcohol application of the 6 hoveniae semoveniae semen water extract of embodiment to drunk nematode
It is carried out by the group technology of embodiment 2, medical and edible dual purpose plant uses hoveniae semoveniae semen.
Hoveniae semoveniae semen is bought from pharmacy, and raisin tree seed extract powder is made by the extracting method in embodiment 2, hoveniae semoveniae semen is carried
By 0.22 micron of filter membrane degerming after taking object powder to be completely dissolved with water, then it is added with the volume final concentration of 30mg/ml
Into the worm liquid of control group, intervene for 24 hours.It detects the head oscillation frequency of Caenorhabditis elegans, swallow number, movement locus, longevity
The indices such as total antioxidant capacity, oxygen free radicals and internal alcohol concentration in life, nematode body.
As a result it shows：Detect the nematode head oscillation frequency of medicine group be 55 times/30s, swallowing movement be 70 times/30s,
Movement locus shows the sinusoidal wavelength relatively coordinated and trailing phenomenon weakens, service life 14d, and alcohol concentration is inside nematode
25ng/ μ g albumen, nematode show a kind of state tending to proper motion, and in nematode body total antioxidant capacity be 7U/mg and
It is 40A.U. testing results close to blank group that oxygen radical (ROS) is horizontal, shows that hoveniae semoveniae semen has sober-up function.
The detection method of every Testing index of the Caenorhabditis elegans referred in text：
(1) assay method of nematode movement
The synchronization of Caenorhabditis elegans is with basis culture according to bibliography (Maintenance of C.elegans)
In method carry out.For experiment nematode use Liquid Culture, alcohol intervene when final concentration control 2000 ± 100/
mL.Nematode growth after synchronization is added a concentration of to the L4 phases when completing larval stage last time husking into the adult stage
The FUDR solution of 0.6mM, and make its final concentration of 0.12mM, to inhibit the nematode to lay eggs and ovum is hatched.After 12h, carried out
The alcohol of various concentration is added in grouping and test group, makes the final concentration of 1%-5% of alcohol by volume, (addition has and tries with blank group
Test group the isometric sterile water of the alcohol being added) as a contrast, every group set 3 it is parallel.At 20 DEG C slight shake culture for 24 hours after
Experimental group is drawn to 20 μ l respectively after (nematode population is 40 or so on each plate) on NGM blank plates, 20 DEG C of placement 5min,
The number of oscillations and pharyngeal swallow number on every nematode head in 30s are recorded under stereomicroscope.Observe and record each group nematode
Movement locus on NGM blank plates collects wavelength and amplitude index, the movenent performance of comprehensive analysis each group nematode.
(2) assay method in nematode service life
The statistics in nematode service life, reference literature (Solis G M, Petrascheck M.Measuring
Caenorhabditis elegans Life Span in 96 Well Microtiter Plates[J].Journal of
Visualized Experiments Jove,2011,(49):E2496-e2496. method), when nematode terminates the L4 phases by
Culture is had the culture solution of nematode to be divided into 6 groups by group technology above, is cultivated in 96 orifice plates, per 120 μ l of hole, per hole about 20
Nematode repeats 8 holes.Sealed membrane sealer is placed on 20 DEG C of cultures, is grouped the same day and counts nematode survival number every three days later,
The sealed membrane that sealed membrane ventilation is primary and more renews is opened in an aseptic environment weekly, until nematode is all dead in hole stops note
Record carries out nematode viability durability analysis.
(3) assay method of nematode biochemical indicator
Total antioxidant capacity is measured using commercial reagents box, and sample treatment requires to carry out according to kit.Oxygen radical
(ROS) according to document (Smith J V, Luo Y.Elevation of oxidative free radicals in
Alzheimer's disease models can be attenuated by Ginkgo biloba extract EGb
761.[J].Journal of Alzheimers Disease,2003,5(4):287-300.) use H2DCFDA fluorescence probes
Method measures.
(4) in nematode body alcohol concentration assay method
The measurement of alcohol concentration in nematode body, with reference to bibliography (Alaimo J T, Davis S J, Song S S, et
al.Ethanol Metabolism and Osmolarity Modify Behavioral Responses to Ethanol
in C.elegans[J].Alcoholism Clinical&Experimental Research,2012,36(11):1840-
1850.) gas chromatography is used, and is improved.
Nematode is transferred to after intervention in centrifuge tube, in refrigerated centrifuge under 4 DEG C, 2000 turns, centrifuges 2min,
Remove culture solution.With M9 buffer solution Rapid Cleanings polypide being pre-chilled twice, OP50 bacterium solutions and other impurities on polypide are removed, then
With cold sterile water wash one time.After finally discarding supernatant, each group nematode is settled to 1mL with cold sterile water, sealing is used in combination
Centrifuge tube sealing treatment is prevented gaseous volatilization by film.The centrifuge tube after sealing is placed in supersonic wave cleaning machine again, ice-water bath ring
Ultrasonication is carried out under border, until without complete polypide, is crushed complete.It is broken to be placed in refrigerated centrifuge 4 DEG C, 12000 turns, from
The heart 10 minutes extracts the alcoholic solution in nematode body, and supernatant is extracting solution.
Ethyl alcohol mark song volume final concentration of 0.0005%, 0.001%, 0.002%, 0.003%, 0.004%, in ethyl alcohol mark
N-butanol is added in bent and nematode extracting solution as internal standard, makes internal standard volume final concentration of 0.002%.Mark song and nematode sample warp
The processing of 0.22 Mm filter membrane filtration, waits for machine testing.
Claims (3)
1. a kind of drunk method for establishing model of Caenorhabditis elegans of quick screening Antialcoholic plant, which is characterized in that this method walks
It is rapid as follows：
(1) Wild-type C. elegans are cultivated using conventional liq cultivation, it is 2000- to make the control of worm liquid concentration
2500 worms/mL, Caenorhabditis elegans using bacterial concentration for 100mg/ml concentration OP50 bacterium solutions as food；
(2) when Caenorhabditis elegans grows to and has just enter into the adult stage, alcoholic solution is added in worm liquid, makes alcoholic solution volume
Final concentration of 1%-5% intervenes for 24 hours, at this point, Caenorhabditis elegans shows as state of intoxication.
2. the drunk method for establishing model of Caenorhabditis elegans of quickly screening Antialcoholic plant as described in claim 1, feature
It is, the head oscillation frequency of Caenorhabditis elegans is 10-40 times/30s, swallowing movement in the drunk model that the method is established
Uncoordinated sinusoidal wavelength is showed for 10-40 times/30s, movement locus and has trailing phenomenon, and the service life is 10-12 days, nematode body
Interior total antioxidant capacity is 15-25U/mg, oxygen free radicals 80-100A.U., and alcohol concentration is 30-50ng/ in nematode body
μ g albumen.
3. application of claim 1 the method in quickly screening medicine-food two-purpose Antialcoholic plant.
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| CN112544571A (en) * | 2020-12-03 | 2021-03-26 | 上海应用技术大学 | Establishment and application of caenorhabditis elegans model for evaluating safety and efficacy of sun-screening agent |
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2018
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| DAVID M LOVINGER、JOHN C CRABBE: "Laboratory models of alcoholism: treatment target identification and insight into mechanisms", 《NATURE NEUROSCIENCE》 * |
| JOSEPH T. ALAIMO等: "Ethanol Metabolism and Osmolarity Modify Behavioral Responses to Ethanol in C. elegans", 《ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH》 * |
| THERESA STIERNAGLE: "Maintenance of C. elegans.", 《WORMBOOK》 * |
| 崔淑芳、陈学进主编: "《实验动物学》", 31 January 2013, 第二军医大学出版社 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112544571A (en) * | 2020-12-03 | 2021-03-26 | 上海应用技术大学 | Establishment and application of caenorhabditis elegans model for evaluating safety and efficacy of sun-screening agent |
| CN112544571B (en) * | 2020-12-03 | 2023-03-03 | 上海应用技术大学 | Establishment and application of caenorhabditis elegans model for evaluating safety and efficacy of sun-screening agent |
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