CN105158041B - A kind of cortex of hair alcohol hormone extracting method - Google Patents
A kind of cortex of hair alcohol hormone extracting method Download PDFInfo
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- CN105158041B CN105158041B CN201510570829.XA CN201510570829A CN105158041B CN 105158041 B CN105158041 B CN 105158041B CN 201510570829 A CN201510570829 A CN 201510570829A CN 105158041 B CN105158041 B CN 105158041B
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Abstract
The invention provides a kind of cortex of hair alcohol hormone extracting method, it the described method comprises the following steps：Step one：Hair sample is placed in isopropanol to the hair carried out after vibration washing after 68 72 DEG C of dry 3.5 4.5h are washed；Step 2：The hair after being ground is ground to the hair after the washing using ball milling instrument；Step 3：Centrifugation obtains supernatant after the hair after grinding is contacted into 15 17h at 75 85 DEG C with methanol.The extracted amount of corticosteroid hormone is improved using method provided by the present invention, sample processing time is more shortened, cortisol extraction efficiency is improved.
Description
Technical field
The present invention relates to herding scientific domain, and in particular to a kind of cortex of hair alcohol hormone extracting method.
Background technology
Corticosteroid hormone belongs to glucocorticoid by adrenal gland synthesis secretion with cortisone hormone.The two has species special
The opposite sex and tissue specificity, but the two is mutually changed in the presence of 11 beta hydroxysteroid hydrogenases in saliva and blood etc. are organized.
Corticosteroid hormone content is more in the primates such as macaque and large mammal, and the skin contained in the rodent such as rat
Matter ketone hormone is more.Corticosteroid hormone is a kind of stress hormone, be often used as reflect animal body stress degree biochemical indicator.
Corticosteroid hormone and body health degree are closely bound up, and there are some researches show the too high or too low meeting of body inner cortex alcohol hormone-content
Cause the generation of the diseases such as Cushing's syndrome, type-II diabetes, addison disease, therefore research corticosteroid hormone
Situation of change in vivo is particularly significant for monitoring body health status in real time.
The serious environmental factor for threatening husbandry sector includes：Temperature, humidity, noise etc., its high temperature or low temperature environment surpass
Animal can be caused heat stress or cold stress reaction occur when crossing animal thermal neutral scope.Therefore research heat stress is to milk cow body
The influence of metabolic mechanism, and then it is very necessary to formulate effective management strategy.Corticosteroid hormone stress degree as reaction
The research of biochemical indicator provide fundamental basis for milk cow thermal stress.The research model of corticosteroid hormone include blood, urine,
Saliva, toe hoof, milk, hair etc., but for long-term temperature stress research be best suitable for model be hair.Hair growth rate
1cm/mon, the hypothesis for entering hair according to Boumba etc. (2006) corticosteroid hormones proposed understands, corticosteroid hormone can be
Accumulated in hair.Therefore cortex of hair alcohol hormone-content can represent the stress situation of a period of time body, such as press close to
At skin in 3cm hairs corticosteroid hormone content can approximately reflect preceding 3 months bodies stress or health status.
As can be seen here, no matter quantitative biochemical index corticosteroid hormone content has to herding scientific research or medical research
It is significant.
Internal 80% corticosteroid hormone is combined presence with corticosteroid hormone receptor protein, and 10% free form is present, remaining
Part then exists otherwise.And there are some researches show in addition to being detected in blood, urine, saliva as free cortisol hormone,
Detected in hair sample to combine corticosteroid hormone, therefore be generally worth based on the total protein content contained in sample to hair
Hair corticosteroid hormone content is quantified.But this area does not develop cortex of hair alcohol detection side rapidly and efficiently also at present
Method.
The content of the invention
It is an object of the invention to overcome defect present in prior art there is provided it is a kind of quick and precisely, it is with low cost/to save
When efficient cortex of hair alcohol hormone extracting method, to promote medical science or herding scientific domain correlative study.
The invention provides a kind of cortex of hair alcohol hormone extracting method, it the described method comprises the following steps：
Step one：Hair sample is placed in isopropanol and carried out after vibration washing in 68-72 DEG C of dry 3.5-4.5h acquisition
Hair after washing；
Step 2：The hair after being ground is ground to the hair after the washing using ball milling instrument；
Step 3：Centrifugation obtains supernatant after the hair after grinding is contacted into 15-17h at 75-85 DEG C with methanol.
It is preferred that, in step one, dry temperature is 70 DEG C, and drying time is 4h.Because with other this areas
Conventional drying temperature compares (such as 50-65 DEG C), and 70 DEG C of dryings can shorten drying time, and the total protein TP extracted at this temperature
Content highest.
It is preferred that, in step 3, the Contact Temperature of hair sample and methanol is 80 DEG C, and the time is 16h.Enter when at 80 DEG C
During row contact incubation compared with 50 DEG C, 60 DEG C and 70 DEG C equitemperatures, its corticosteroid hormone content can increase 1.32-2.21 times,
The efficiency of extraction can be improved.
Optionally, the hair after the grinding relative to 100mg, the consumption of methanol is 1.8-2.2ml.
Optionally, the condition centrifuged in step 3 is to centrifuge 8-14min in 11000-13000rpm/min.
Optionally, in step one, the condition of concussion washing is at 35-40 DEG C, to be washed with 220-260 times/min frequency
Wash 2-3 times, each 2.5-3.5min.
Optionally, the condition of grinding is to grind 4.5-5.5min under 32-38Hz frequency.
Optionally, the hair is derived from mammal.
Optionally, the mammal is milk cow or macaque, preferably milk cow.
Present invention also offers application of the corticosteroid hormone extracting method in the detection of animal hair cortisol.
The present invention chooses 68-72 DEG C of progress hair sample drying, shortens the hair treatment time, and hair is in 75-85
DEG C contact 15-17h reach corticosteroid hormone it is optimal extraction, the extraction efficiency of corticosteroid hormone can be greatly improved, this method can
To be widely used in herding science and clinical medicine disease research field.
Brief description of the drawings
Fig. 1 is hair protein (TP) changes of contents under different incubation temperatures after 70 DEG C of drying.
Fig. 2 changes for cortex of hair alcohol (CORT) hormone-content under different incubation temperatures after 70 DEG C of drying.
Embodiment
The embodiment to the present invention is described in detail below.It should be appreciated that described herein
Embodiment is merely to illustrate and explain the present invention, and is not intended to limit the invention.
Test case 1
Respectively cortex of hair alcohol hormone is extracted according to the drying temperature and incubation temperature provided in table 1.
Utilize shaver collection milk cow belly about 20 × 20cm2The black ox hair sample of area：Weigh 250mg hair samples
Product, are loosely placed in 15ml centrifuge tubes and label, add 10ml isopropanols；Centrifuge tube is positioned on oscillator 37 DEG C with 240
Secondary/min frequency washing 5min, grips hair with tweezers, discards aqueous isopropanol and ttom of pipe impurity；Add again into centrifuge tube
Enter 10ml isopropanols, be placed in 37 DEG C of 240 times/min washing 3min on oscillator, centrifuged with tweezers gripping hair tube to new 15ml
Pipe internal label simultaneously removes lid；By trichoxerosis 4h；Take freezing mixed type ball milling instrument rotor in vacuum flask, add liquid nitrogen frozen
To without sound；Take the peeling of 5ml centrifuge tubes to weigh, take 150mg hair samples add in 5ml centrifuge tubes and respectively at bottom of the tube, in
3 diameter 0.75cm steel balls are placed on portion, top；Rotor after freezing is taken out, the 5ml centrifuge tubes equipped with sample, 35hz grindings is added
5min；Steel ball is poured out, 16h is incubated after adding 3ml methanol；3500rpm/min centrifuges 5min, takes supernatant in 2ml centrifuge tubes
It is interior；- 20 DEG C preserved to detection day, were detected using radio immunoassay RIA (Radio immunoassay) method, this
Secondary detection variation within batch coefficient (CV) is about 5%, CV about 10% between crowd.Testing result is shown in Table 1.
Table 1
It is to combine corticosteroid hormone content due to what is quantitatively detected in hair, therefore based on total protein content in hair
Value, which quantify comparing to be worth based on methanol volume, carries out quantitative more precise and reliable, it is seen that the direct shadow of hair total protein content
Ring final extraction result.Therefore detection discovery is carried out to total protein content in hair in this experiment：In 70 DEG C of optimum condition
Lower drying will not cause total protein content to reduce, and its detection level highest, and cortisol is then carried out under the conditions of 50-80 DEG C swashs
Its TP content of plain extraction experiments does not have significant changes substantially.By relatively more final corticosteroid hormone content it can be found that preferred
80 DEG C be incubated when its CORT content improve 2.21 times compared with 50 DEG C of incubations.Carried it can be seen that the present invention have found optimal hair CORT
Method is taken, the extracted amount of corticosteroid hormone is not only increased, more shortens sample processing time, improve efficiency.
Comparative example 1
According to D.Moya etc. research (Development of a novel method for measuring stress
In beef cattle.) in the method that provides extract Fur of Cow cortisol, take 0.5g Angus beefs head, neck, shoulder
And afterbody, buttocks hair carry out ultrasonic smashing, add methanol and be incubated corticosteroid hormone in 18h extraction hairs in 50 DEG C, using competing
Strive to be immunized and supernatant is tested and analyzed, the detection variation within batch coefficient (CV) is 5.91-9.96%, CV is 5.04- between batch
10.48%, its corticosteroid hormone content finally determined is 0.07-6.62pg/mg.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. a kind of cortex of hair alcohol hormone extracting method, it is characterised in that the described method comprises the following steps：
Step one：Hair sample is placed in after carrying out vibration washing in isopropanol and washed in 68-72 DEG C of dry 3.5-4.5h
Hair afterwards；
Step 2：The hair after being ground is ground to the hair after the washing using ball milling instrument；
Step 3：Centrifugation obtains supernatant after the hair after grinding is contacted into 15-17h at 75-85 DEG C with methanol.
2. extracting method according to claim 1, it is characterised in that in step 3, contact of the hair sample with methanol
Temperature is 80 DEG C, and the time is 16h.
3. extracting method according to claim 1 or 2, it is characterised in that the hair after grinding relative to 100mg, first
The consumption of alcohol is 1.8-2.2ml.
4. extracting method according to claim 3, it is characterised in that the condition centrifuged in step 3 is in 11000-
13000rpm/min centrifuges 8-14min.
5. the extracting method according to any one in claim 1,2 and 4, it is characterised in that in step one, concussion is washed
The condition washed is at 35-40 DEG C, to be washed 2-3 times with 220-260 times/min frequency, each 2.5-3.5min.
6. extracting method according to claim 5, it is characterised in that the condition of grinding is to be ground under 32-38Hz frequency
Grind 4.5-5.5min.
7. the extracting method according to any one in claim 1,2,4 and 6, it is characterised in that the hair is derived from the food in one's mouth
Newborn animal.
8. extracting method according to claim 7, it is characterised in that the mammal is milk cow or macaque.
9. extracting method according to claim 8, it is characterised in that the mammal is milk cow.
10. application of the extracting method in the detection of animal hair cortisol in claim 1-9 described in any one.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107490504A (en) * | 2017-07-19 | 2017-12-19 | 山东大学 | A kind of method of pyrroles's adduct content in measure hair |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105571918B (en) * | 2016-01-18 | 2018-04-10 | 中国农业科学院茶叶研究所 | A kind of sample-pretreating method for the detection of bioactive substance accurate quantification |
| CN105651988B (en) * | 2016-03-05 | 2018-06-26 | 青岛农业大学 | The extracting method of steroid hormone in a kind of penguin excrement sample |
| CN107449644A (en) * | 2017-07-10 | 2017-12-08 | 张红丽 | A kind of method of amphetamine and drugs such as morphine in rapid field examination hair |
| CN110530694A (en) * | 2019-08-30 | 2019-12-03 | 华东师范大学 | A kind of preprocess method of animal hair cortisol detection |
| WO2021054386A1 (en) * | 2019-09-20 | 2021-03-25 | 株式会社あすか製薬メディカル | Method for analyzing steroid hormones contained in animal hair sample, stress evaluation method and method for analyzing cause of hair loss |
| CN111004301B (en) * | 2019-12-11 | 2024-06-11 | 苏州飞康生物医药有限公司 | Extraction and detection method of human hair cortisol |
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| CN101368972A (en) * | 2008-04-02 | 2009-02-18 | 北京科美东雅生物技术有限公司 | Chemical luminescence immune assay determination reagent kit for hydrocortisone and preparation method thereof |
| CN103353535B (en) * | 2013-08-02 | 2015-07-22 | 南通大学 | Pretreatment method for detecting cortisol content in hair |
| CN103776997B (en) * | 2014-02-26 | 2016-03-02 | 南京农业大学 | A kind of method extracted from pig hair and detect cortisol |
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| CN107490504A (en) * | 2017-07-19 | 2017-12-19 | 山东大学 | A kind of method of pyrroles's adduct content in measure hair |
| CN107490504B (en) * | 2017-07-19 | 2019-05-14 | 山东大学 | A kind of method of pyrroles's adduct content in measurement hair |
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