CN104558084A - Method for extracting steroid hormone from excrement of forest musk deer - Google Patents
Method for extracting steroid hormone from excrement of forest musk deer Download PDFInfo
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- CN104558084A CN104558084A CN201510031151.8A CN201510031151A CN104558084A CN 104558084 A CN104558084 A CN 104558084A CN 201510031151 A CN201510031151 A CN 201510031151A CN 104558084 A CN104558084 A CN 104558084A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J5/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond
- C07J5/0046—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond substituted in position 17 alfa
- C07J5/0053—Normal steroids containing carbon, hydrogen, halogen or oxygen, substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane and substituted in position 21 by only one singly bound oxygen atom, i.e. only one oxygen bound to position 21 by a single bond substituted in position 17 alfa not substituted in position 16
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J1/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, not substituted in position 17 beta by a carbon atom, e.g. estrane, androstane
- C07J1/0003—Androstane derivatives
- C07J1/0018—Androstane derivatives substituted in position 17 beta, not substituted in position 17 alfa
- C07J1/0022—Androstane derivatives substituted in position 17 beta, not substituted in position 17 alfa the substituent being an OH group free esterified or etherified
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J1/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, not substituted in position 17 beta by a carbon atom, e.g. estrane, androstane
- C07J1/0051—Estrane derivatives
- C07J1/0066—Estrane derivatives substituted in position 17 beta not substituted in position 17 alfa
- C07J1/007—Estrane derivatives substituted in position 17 beta not substituted in position 17 alfa the substituent being an OH group free esterified or etherified
Abstract
The invention discloses a method for extracting steroid hormone from excrement of forest musk deer. The method comprises the following step of leaching animal excrement by virtue of extraction liquid so as to obtain leaching liquid containing the steroid hormone, wherein the extraction liquid is an ethanol water solution, a methanol water solution, ethanol or methanol; the concentration of volume percent of ethanol in the ethanol water solution is more than or equal to 60% and less than 100%; the concentration of volume percent of methanol in the methanol water solution is more than or equal to 60% and less than 100%; the steroid hormone is one or two or all of cortisol, estradiol and testosterone. An experiment proves that the steroid hormone is extracted from the excrement of the forest musk deer by virtue of the extraction method, the optimal extraction liquid is 90E (10% ethanol water solution).
Description
Technical field
The present invention relates to the extracting method of steroid hormone in biological technical field middle forest musk deer ight soil.
Background technology
Woods musk deer is that a kind of small-sized woods dwells and ruminates ungulates, is the precious medicinal animal of Asia special product.Although China starts the woods musk deer stable breeding practice history of existing more than 50 year, stable breeding musk deer class population growth speed is slow, and the phenomenon of negative growth even appears in some year.Analyze its reason to mainly contain: 1. female musk deer of artificial house nonpregnant rate after nursery stage is higher; 2. musk deer class animal natural disposition is timid alert, and quite vigilance, irritability is strong.Hydrocortisone is a kind of steroid hormone, directly can reflect the physiological stress situation of animal; Estradiol and testosterone are steroid hormone, and these two kinds of hormones can well reflect the breeding physiology state of animal, carry out the captive breeding of dynamic monitoring to woods musk deer be of great significance the hormone of the hydrocortisone of woods musk deer, estradiol and testosterone.
Summary of the invention
Technical problem to be solved by this invention is from animal excrement, how to extract more steroid hormone (namely how improving the extracted amount of steroid hormone in animal excrement).
For solving the problems of the technologies described above, the present invention provide firstly the extracting method of steroid hormone in animal excrement.
In animal excrement provided by the present invention, the extracting method (extracting the method for steroid hormone from animal excrement) of steroid hormone, comprises the step obtaining the vat liquor containing described steroid hormone with animal excrement described in extracting solution lixiviate;
Described extracting solution is aqueous ethanolic solution (solute is ethanol, and solvent is water), methanol aqueous solution (solute is methyl alcohol, and solvent is water), ethanol or methyl alcohol;
In described aqueous ethanolic solution, the concentration of volume percent of ethanol is less than 100% for being more than or equal to 60%;
In described methanol aqueous solution, the concentration of volume percent of methyl alcohol is less than 100% for being more than or equal to 60%.
In aforesaid method, described steroid hormone is whole or two kinds or one in hydrocortisone, estradiol and testosterone.
In aforesaid method, described extracting solution is described aqueous ethanolic solution; In described aqueous ethanolic solution, the concentration of volume percent of ethanol is less than 100% for being more than or equal to 80%.
In aforesaid method, described extracting solution is described aqueous ethanolic solution; In described aqueous ethanolic solution, the concentration of volume percent of ethanol is less than 100% for being more than or equal to 90%.
In aforesaid method, described extracting solution is described aqueous ethanolic solution; In described aqueous ethanolic solution, the concentration of volume percent of ethanol is 90%-95%.
In aforesaid method, described extracting solution is described aqueous ethanolic solution; In described aqueous ethanolic solution, the concentration of volume percent of ethanol is 90%.
In aforesaid method, described extracting solution is described methanol aqueous solution; In described methanol aqueous solution, the concentration of volume percent of methyl alcohol is 60%-90%.In described methanol aqueous solution, the concentration of volume percent of methyl alcohol specifically can be 60%-80%.
In aforesaid method, described animal is jenny; Described steroid hormone is hydrocortisone; Described extracting solution is described aqueous ethanolic solution or described methyl alcohol; In described aqueous ethanolic solution, the concentration of volume percent of ethanol can be 90%.
In aforesaid method, described animal is jenny; Described steroid hormone is estradiol; Described extracting solution is described ethanol or described aqueous ethanolic solution; In described aqueous ethanolic solution, the concentration of volume percent of ethanol can be 90%.
In aforesaid method, described animal is buck; Described steroid hormone is hydrocortisone; Described extracting solution is described aqueous ethanolic solution or described ethanol or described methanol aqueous solution; In described aqueous ethanolic solution, the concentration of volume percent of ethanol can be 90%; In described methanol aqueous solution, the concentration of volume percent of methyl alcohol can be 60% or 80%.
In aforesaid method, described animal is buck; Described steroid hormone is testosterone; Described extracting solution is described aqueous ethanolic solution or described ethanol; In described aqueous ethanolic solution, the concentration of volume percent of ethanol can be 90%.
In aforesaid method, described animal is woods musk deer (Moschusberezovskii).
In aforesaid method, described method is carried out at 20-25 DEG C.
In aforesaid method, described method also comprises the step of being preserved in valve bag by the fresh excreta of described animal.The fresh excreta of described animal can be frozen at-20 DEG C.
Experiment proves, in animal excrement of the present invention, the extracting method of steroid hormone can improve the extracted amount of steroid hormone in animal excrement: 90E (concentration of volume percent is the aqueous ethanolic solution of 90%) can from every gram of female woods musk deer argol just extract obtain 425.2373 ± 21.27ng hydrocortisone and 3111.2957 ± 155.70pg estradiol, can from every gram of male woods musk deer argol just extraction obtain 322.3980 ± 32.24ng hydrocortisone and 6.5430 ± 1.21ng testosterone; Ethanol can from every gram of female woods musk deer argol just extract obtain 287.9728 ± 34.96ng hydrocortisone and 3149.2394 ± 203.98pg estradiol, can from every gram of male woods musk deer argol just extraction obtain 319.2465 ± 54.12ng hydrocortisone and 5.8387 ± 1.52ng testosterone; 60M (concentration of volume percent is the methanol aqueous solution of 60%) can from every gram of male woods musk deer argol just extract obtain 329.3007 ± 38.41ng hydrocortisone and 4.8227 ± 0.60ng testosterone, can from every gram of female woods musk deer argol just extraction obtain 368.1832 ± 23.31ng hydrocortisone and 2117.2691 ± 98.89pg estradiol; Methyl alcohol can from every gram of female woods musk deer argol just extract obtain 421.65 ± 50.52ng hydrocortisone and 2168.4580 ± 135.93pg estradiol, can from every gram of male woods musk deer argol just extraction obtain 275.31 ± 10.91ng hydrocortisone and 4.0097 ± 0.62ng testosterone.Experiment proves, the extracting method of steroid hormone in animal excrement of the present invention can be utilized from animal excrement to extract steroid hormone, and optimum extraction liquid is 90E (concentration of volume percent is the aqueous ethanolic solution of 90%).
Accompanying drawing explanation
Fig. 1 extracts with different extracting solution the hydrocortisone extracted amount that female woods musk deer ight soil obtains.
Fig. 2 extracts with different extracting solution the hydrocortisone extracted amount that male woods musk deer ight soil obtains.
Fig. 3 extracts with different extracting solution the estradiol extracted amount that female woods musk deer ight soil obtains.
Fig. 4 extracts with different extracting solution the testosterone extracted amount that male woods musk deer ight soil obtains.
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail, the embodiment provided only in order to illustrate the present invention, instead of in order to limit the scope of the invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
In following embodiment, in woods musk deer ight soil, the leaching process of hydrocortisone all carries out at 20-25 DEG C.
Aqueous ethanolic solution in following embodiment and the solvent of methanol aqueous solution are water, and the solute of aqueous ethanolic solution is ethanol, and the solute of methanol aqueous solution is methyl alcohol.
The extraction of hydrocortisone in embodiment 1, woods musk deer ight soil
1, the extraction of hydrocortisone in female woods musk deer ight soil
Choose 6 female woods musk deers of Sichuan meter Ya Luo Pien Tze Huang Lin She plant, distinguished called after female woods musk deer first, female woods musk deer second, female woods musk deer third, female woods musk deer fourth, female woods musk deer penta and female woods musk deer own.
The collection of 1.1 female woods musk deer ight soil
Within continuous 4 days, gather the fresh excreta of above-mentioned 6 female woods musk deers, every day often only female woods musk deer gather once, a 30g.The fresh excreta at every turn gathered is put into valve bag (often only ight soil valve bag of at every turn gathering of female woods musk deer), excluding air seals, and obtains female woods musk deer first the 1st day ight soil respectively, female woods musk deer first the 2nd day ight soil, female woods musk deer first the 3rd day ight soil, female woods musk deer first the 4th day ight soil, female woods musk deer second the 1st day ight soil, female woods musk deer second the 2nd day ight soil, female woods musk deer second the 3rd day ight soil, female woods musk deer second the 4th day ight soil, female woods musk deer the third the 1st day ight soil, female woods musk deer the third the 2nd day ight soil, female woods musk deer the third the 3rd day ight soil, female woods musk deer the third the 4th day ight soil, female woods musk deer fourth the 1st day ight soil, female woods musk deer fourth the 2nd day ight soil, female woods musk deer fourth the 3rd day ight soil, female woods musk deer fourth the 4th day ight soil, female woods musk deer penta the 1st day ight soil, female woods musk deer penta the 2nd day ight soil, female woods musk deer penta the 3rd day ight soil, female woods musk deer penta the 4th day ight soil, oneself the 1st day ight soil of female woods musk deer, oneself the 2nd day ight soil of female woods musk deer, oneself the 4th day ight soil of oneself the 3rd day ight soil of female woods musk deer and female woods musk deer, above-mentioned ight soil is frozen respectively at-20 DEG C of refrigerators.
The preparation of 1.2 extracting solutions
Configuration volumetric concentration is respectively the methanol aqueous solution (title is respectively 100M, 90M, 80M, 60M) of 100%, 90%, 80%, 60%, volume ratio is as table 1, configuration volumetric concentration is respectively the aqueous ethanolic solution (title is respectively 100E, 90E, 80E, 60E) of 100%, 90%, 80%, 60%, and volume ratio is as table 2.
The formula of table 1, extracting solution
The formula of table 2, extracting solution
The extraction of hydrocortisone in 1.3 female woods musk deer ight soil
1.3.1 the preparation of sample
Take female woods musk deer first the 1st day ight soil getting step 1.1 respectively, female woods musk deer first the 2nd day ight soil, female woods musk deer first the 3rd day ight soil, female woods musk deer first the 4th day ight soil, female woods musk deer second the 1st day ight soil, female woods musk deer second the 2nd day ight soil, female woods musk deer second the 3rd day ight soil, female woods musk deer second the 4th day ight soil, female woods musk deer the third the 1st day ight soil, female woods musk deer the third the 2nd day ight soil, female woods musk deer the third the 3rd day ight soil, female woods musk deer the third the 4th day ight soil, female woods musk deer fourth the 1st day ight soil, female woods musk deer fourth the 2nd day ight soil, female woods musk deer fourth the 3rd day ight soil, female woods musk deer fourth the 4th day ight soil, female woods musk deer penta the 1st day ight soil, female woods musk deer penta the 2nd day ight soil, female woods musk deer penta the 3rd day ight soil, female woods musk deer penta the 4th day ight soil, oneself the 1st day ight soil of female woods musk deer, oneself the 2nd day ight soil of female woods musk deer, oneself the 3rd day ight soil of female woods musk deer, oneself the 4th day each 10g of ight soil of female woods musk deer, 10min mixing is pulverized in pulverizer (23000rpm), obtain female woods musk deer ight soil.
1.3.2 the preparation of vat liquor
In female woods musk deer ight soil, the extracting method of hydrocortisone is as follows, and three repetitions established by each sample:
The female woods musk deer ight soil 0.5g taking step 1.3.1, in 10ml plastic centrifuge tube, adds the extracting solution 100M of 5ml step 1.2 in this centrifuge tube, and after vortex instrument vibration 20min, at 2500rpm, (centrifugal force is 4360g; Whizzer is the low speed autobalancing centrifuge of Beijing Medical Centrifugal Machine Factory, and model is LDZ 5-2) under centrifugal 15min, collect supernatant liquor (called after supernatant liquor I) and precipitation; Add 5ml extracting solution 100M vortex instrument vibration 5min in this precipitation after, at 2500rpm, (centrifugal force is 4360g; Whizzer is the low speed autobalancing centrifuge of Beijing Medical Centrifugal Machine Factory, and model is LDZ 5-2) under centrifugal 15min, collect supernatant liquor (called after supernatant liquor II); Supernatant liquor I and supernatant liquor II are mixed, is female woods musk deer 100M vat liquor.
According to the method described above, respectively 100M is replaced with extracting solution 90M, 80M, 60M, 100E, 90E, 80E and 60E of step 1.2, other steps are all constant, obtain female woods musk deer 90M vat liquor, female woods musk deer 80M vat liquor, female woods musk deer 60M vat liquor, female woods musk deer 100E vat liquor, female woods musk deer 90E vat liquor, female woods musk deer 80E vat liquor, female woods musk deer 60E vat liquor respectively.
The mensuration of the hydrocortisone 1.3.3 extracted
The content of hydrocortisone in the vat liquor of determination step 1.3.2 by the following method:
Adopt radioimmunology utilize iodine [
125i] hydrocortisone radioimmunoassay kit (iodine [
125i] hydrocortisone radioimmunoassay kit is Beijing North Institute of Biological Technology's product, the accurate word S10940097 of traditional Chinese medicines) content of hydrocortisone in the vat liquor of determination step 1.3.2, radioimmunoanalyzer is the XH-6080 type that Xi'an 262 factory produces.
Iodine [
125i] main performance index of hydrocortisone radioimmunoassay kit:
Standard range: 10-500ng/ml
Sensitivity :-2ng/ml
Precision: variation within batch coefficient (CV) <10 ﹪; Interassay coefficient of variation (CV) <15 ﹪
Experiment in triplicate, repeat at every turn test concrete operation step as follows, the reagent in following step be above-mentioned iodine [
125i] reagent in hydrocortisone radioimmunoassay kit:
1) round bottom polystyrene tube is got some, with marking pen numbering NSB, S
0-S
5with testing sample pipe, then press table 3 application of sample with micro sample adding appliance.Before application of sample, all reagent comprises testing sample and shakes up, and equilibrates to room temperature.
Table 3, application of sample programsheet unit (μ L)
Reagent in table 3 except the vat liquor of step 1.3.2, other reagent all from above-mentioned iodine [
125i] hydrocortisone radioimmunoassay kit.
2) data processing
On line processing: automatically processed by computer operation parameter data processing mode and obtain a result.
Knock off and map or counter process:
Percentage combination rate calculates: establish S
0pipe is counted as B
0, each standard pipe or sample hose are counted as B, and NSB pipe is counted as NSB, and percentage combination rate calculation formula is B/B
0=(B-NSB)/(B
0-NSB) × 100%.
Logit calculates: the logit value calculation formula of each standard pipe or sample hose is Logit=ln [(B/B
0)/(1-B/B
0)].
Mapping: take normal concentration as X-coordinate, corresponding B/B
0for ordinate zou makes typical curve.
Counter process: the counter using linear matching function, calculates the content of the hydrocortisone in vat liquor.
Setup Experiments control group measures the water ratio of every only female woods musk deer ight soil, and calculates the hydrocortisone extracted amount (ng/g argol) of every gram of argol:
Hydrocortisone extracted amount (ng/g argol)=(concentration of cortisol × 10ml of kit measurement)/[0.5g × (water ratio of 1-woods musk deer ight soil)] of every gram of argol.
By results averaged identical for extracting solution between different woods musk deer, result is as shown in table 4 and Fig. 1.
Use SPSS16.0 software to carry out one-way analysis of variance (one way-ANOVA) and the multiple comparisons of the Determination of cortisol that different extracting solution extracts in ight soil, significance level is set to P=0.05; Adopt the homogeneity of Levene ' s homogeneity test of variance determination variance of the sample mean.
The hydrocortisone extracted amount of the female woods musk deer ight soil that table 4, different extracting solution obtain
| Extracting solution | Woods musk deer sex | Hydrocortisone extracted amount (ng/g argol) | Hydrocortisone extracted amount rank |
| 100M | ♀ | 421.65±50.52 | 2 |
| 90M | ♀ | 284.9857±8.27 | 8 |
| 80M | ♀ | 377.7693±4.09 | 3 |
| 60M | ♀ | 368.1832±23.31 | 4 |
| 100E | ♀ | 287.9728±34.96 | 7 |
| 90E | ♀ | 425.2373±21.27 | 1 |
| 80E | ♀ | 335.6648±15.87 | 5 |
| 60E | ♀ | 305.3017±48.28 | 6 |
Result shows, the amount extracting the hydrocortisone that female woods musk deer ight soil obtains with 90E (concentration of volume percent is the aqueous ethanolic solution of 90%) is the highest, and respectively higher than with 100M (methyl alcohol), 80M (concentration of volume percent is the methanol aqueous solution of 80%), 60M (concentration of volume percent is the methanol aqueous solution of 60%) and 80E (concentration of volume percent is the aqueous ethanolic solution of 80%) extracts the amount of the hydrocortisone that female woods musk deer ight soil obtains, and be significantly higher than with 100E (ethanol) respectively, 90M (concentration of volume percent is the methanol aqueous solution of 90%) and 60E (concentration of volume percent is the aqueous ethanolic solution of 60%) extracts the amount of the hydrocortisone that female woods musk deer ight soil obtains.Result shows, 90E (concentration of volume percent is the aqueous ethanolic solution of 90%) can be used to the hydrocortisone extracting female woods musk deer ight soil.
2, the extraction of hydrocortisone in male woods musk deer ight soil
Choose 6 male woods musk deers of Sichuan meter Ya Luo Pien Tze Huang Lin She plant, distinguished called after male woods musk deer first, male woods musk deer second, male woods musk deer third, male woods musk deer fourth, male woods musk deer penta and male woods musk deer own.
According to extraction and the measuring method of hydrocortisone in the female woods musk deer ight soil of step 1, by female woods musk deer first, female woods musk deer second, female woods musk deer third, female woods musk deer fourth, female woods musk deer penta and female woods musk deer oneself replace with respectively male woods musk deer first, male woods musk deer second, male woods musk deer third, male woods musk deer fourth, male woods musk deer penta and male woods musk deer oneself, other steps are all constant, obtain male woods musk deer 100M vat liquor, male woods musk deer 90M vat liquor, male woods musk deer 80M vat liquor, male woods musk deer 60M vat liquor respectively, and measure the content of the hydrocortisone in above-mentioned vat liquor.
Setup Experiments control group measures the water ratio of every only male woods musk deer ight soil, and calculates the hydrocortisone extracted amount (ng/g argol) of every gram of argol:
Hydrocortisone extracted amount (ng/g argol)=(concentration of cortisol × 10ml of kit measurement)/[0.5g × (water ratio of 1-woods musk deer ight soil)] of every gram of argol.
By results averaged identical for extracting solution between different woods musk deer, result is as shown in table 5 and Fig. 2.
Use SPSS16.0 software to carry out one-way analysis of variance (one way-ANOVA) and the multiple comparisons of the Determination of cortisol that different extracting solution extracts in ight soil, significance level is set to P=0.05; Adopt the homogeneity of Levene ' s homogeneity test of variance determination variance of the sample mean.
The hydrocortisone extracted amount of the male woods musk deer ight soil that table 5, different extracting solution obtain
| Extracting solution | Woods musk deer sex | Hydrocortisone extracted amount (ng/g argol) | Hydrocortisone extracted amount rank |
| 100M | ♂ | 275.31±10.91 | 6 |
| 90M | ♂ | 258.8379±42.45 | 7 |
| 80M | ♂ | 302.7634±10.11 | 4 |
| 60M | ♂ | 329.3007±38.41 | 1 |
| 100E | ♂ | 319.2465±54.12 | 3 |
| 90E | ♂ | 322.3980±32.24 | 2 |
| 80E | ♂ | 281.4197±9.04 | 5 |
| 60E | ♂ | 257.4543±16.85 | 8 |
Result shows, with 100M (methyl alcohol), 90M (concentration of volume percent is the methanol aqueous solution of 90%), 80M (concentration of volume percent is the methanol aqueous solution of 80%), 60M (concentration of volume percent is the methanol aqueous solution of 60%), 100E (ethanol), 90E (concentration of volume percent is the aqueous ethanolic solution of 90%), 80E (concentration of volume percent is the aqueous ethanolic solution of 80%) and 60E (concentration of volume percent is the aqueous ethanolic solution of 60%) extracts male woods musk deer ight soil and all can obtain hydrocortisone, the hydrocortisone amount that male woods musk deer ight soil obtains is extracted higher with 60M (concentration of volume percent is the methanol aqueous solution of 60%) and 90E (concentration of volume percent is the aqueous ethanolic solution of 90%).Result shows that 60M (concentration of volume percent is the methanol aqueous solution of 60%) and 90E (concentration of volume percent is the aqueous ethanolic solution of 90%) extracts the best results of the hydrocortisone of male woods musk deer ight soil, can be used to the hydrocortisone extracting male woods musk deer ight soil.
The extraction of estradiol in embodiment 2, female woods musk deer ight soil
By the following method, the content of the estradiol in the female woods musk deer 100M vat liquor of embodiment 1, female woods musk deer 90M vat liquor, female woods musk deer 80M vat liquor and female woods musk deer 60M vat liquor vat liquor is measured:
Adopt the content of estradiol in the above-mentioned vat liquor of chemiluminescence determination, analyser uses the Chemiluminescence Apparatus reaching the 200s model of production in sky, Xiamen; (estradiol (E2) immue quantitative detection reagent box (chemoluminescence method) is Beijing North Institute of Biological Technology's product to adopt estradiol (E2) immue quantitative detection reagent box (chemoluminescence method), this test kit main component in table 6, main performance index is as follows:
A. measurement range: 25-1000pg/ml
B. limit of identification :≤13pg/ml
C. precision: CV≤15 ﹪ in crowd, CV≤20 ﹪ between crowd
D. interfering substance: bilirubin 300mg/L, cholesterol 10mg/ml, triglyceride level 7.6mmol/L, oxyphorase 2mg/ml is noiseless to this test kit;
E. cross reaction: with P (100ng/ml), T (100ng/ml) no cross reaction.
Main component in table 6, estradiol (E2) immue quantitative detection reagent box (chemoluminescence method)
In triplicate, the concrete operations at every turn repeating to test are as follows, and the reagent in following step is reagent in above-mentioned estradiol (E2) immue quantitative detection reagent box (chemoluminescence method) in experiment:
Sample and each calibration object all do parallel control experiment, and each analysis design mothod all will do typical curve analysis.Sample and reagent shake up before use and equilibrate to room temperature.
A. poured into by 20mL concentrated cleaning solution in a clean container, obtain the washings diluted after adding the mixing of 380ml distilled water, the washings of dilution is kept at (room temperature condition can preserve 2 weeks) in the container of sealing.
B. coated slab is installed on lath frame.Luminous substrate liquid A and B can by the volume ratio mixing for standby use of 1:1.
C. added 50 μ L calibration objects or a kind of vat liquor in hole to bag, two parallel laboratory tests established by often kind of calibration object or vat liquor.
D. in every hole, add 50 μ L E2 enzyme conjugates.
E. micro oscillator vibrates and solution was mixed in 10 seconds, covers, put 37 DEG C of incubations 1 hour by non-setting adhesive sealer.
F. 5 times are washed with the washings of the dilution of steps A.
G. each 1 each 50 μ L of luminous substrate liquid A and luminous substrate liquid B are added to every hole.
H. micro oscillator vibration 30s, room temperature (22 DEG C) lucifuge is reacted, and in 5-10 minute, measures each hole luminous value with light-emitting appearance.
I. data processing
1) on line processing: use log-logit data processing mode computer automatically to process and obtain a result.
2) be counted as ordinate zou with each standard point, each standard point concentration is X-coordinate, and typical curve drawn by log-log paper, and the counting according to sample checks in concentration of specimens on typical curve.
3) counter process: do log-logit straight-line regression with standard point counting with concentration value, obtain typical curve, calculate the content of estradiol in vat liquor to be measured.
Setup Experiments control group measures the water ratio of every only female woods musk deer ight soil, and calculates the estradiol extracted amount (pg/g argol) of every gram of argol:
Estradiol extracted amount (pg/g argol)=(estradiol concentration × 10ml of kit measurement)/[0.5g × (water ratio of 1-woods musk deer ight soil)] of every gram of argol.
By results averaged identical for extracting solution between different woods musk deer, result is as shown in table 7 and Fig. 3.
Use SPSS16.0 software to carry out one-way analysis of variance (one way-ANOVA) and the multiple comparisons of the estradiol content that different extracting solution extracts in ight soil, significance level is set to P=0.05; Adopt the homogeneity of Levene ' s homogeneity test of variance determination variance of the sample mean.
The estradiol extracted amount of the female woods musk deer ight soil that table 7, different extracting solution obtain
| Extracting solution | Woods musk deer sex | Estradiol extracted amount (pg/g argol) | Estradiol extracted amount rank |
| 100M | ♀ | 2168.4580±135.93 | 4 |
| 90M | ♀ | 1645.3363±169.78 | 8 |
| 80M | ♀ | 2056.6655±31.91 | 6 |
| 60M | ♀ | 2117.2691±98.89 | 5 |
| 100E | ♀ | 3149.2394±203.98 | 1 |
| 90E | ♀ | 3111.2957±155.70 | 2 |
| 80E | ♀ | 2495.6810±226.31 | 3 |
| 60E | ♀ | 1966.1130±111.85 | 7 |
Result shows, the amount extracting the estradiol that female woods musk deer ight soil obtains with 100E (ethanol) and 90E (concentration of volume percent is the aqueous ethanolic solution of 90%) is maximum, all be significantly higher than the amount extracting the estradiol that female woods musk deer ight soil obtains with 80E (concentration of volume percent is the aqueous ethanolic solution of 80%), and all pole is significantly higher than with 100M (methyl alcohol), 90M (concentration of volume percent is the methanol aqueous solution of 90%), 80M (concentration of volume percent is the methanol aqueous solution of 80%), 60M (concentration of volume percent is the methanol aqueous solution of 60%) and 60E (concentration of volume percent is the aqueous ethanolic solution of 60%) extracts the amount of the estradiol that female woods musk deer ight soil obtains.Result shows, 100E (ethanol) and 90E (concentration of volume percent is the aqueous ethanolic solution of 90%) can be used to the estradiol extracting female woods musk deer ight soil.
The extraction of testosterone in embodiment 3, male woods musk deer ight soil
By the following method, the content of the testosterone in the male woods musk deer 100M vat liquor of embodiment 1, male woods musk deer 90M vat liquor, male woods musk deer 80M vat liquor and male woods musk deer 60M vat liquor is measured:
Euzymelinked immunosorbent assay (ELISA) is adopted to measure the content of testosterone in above-mentioned vat liquor.Enzyme micro-plate reader is ThermoMultiscanFC type, adopt testosterone quantitative determination reagent kit (euzymelinked immunosorbent assay (ELISA)) (this test kit is Beijing North Institute of Biological Technology's product), this test kit main component in table 8 and table 9, the main technical details of testosterone quantitative determination reagent kit (euzymelinked immunosorbent assay (ELISA)) is as follows:
A. sensitivity :≤0.05ng/ml
B. precision: variation coefficient CV<15 ﹪
C. specificity: do not have cross reaction with progesterone, estradiol, DHT
The main component of table 8, testosterone quantitative determination reagent kit (euzymelinked immunosorbent assay (ELISA))
| Component/specification | 96T | Main component |
| Pre-coated plate | 12 holes/bar × 8 | Main component is for bag is by goat-anti rabbit |
| Enzyme-labelled antigen | 6ml × 1 bottle | Main component is that HRP marks T |
| Antibody | 6ml × 1 bottle | Main component is the anti-T of rabbit |
| Calibration object is a set of | 0.5ml/ bottle × 6 bottle | Main component is T, removes hormone serum |
| Quality control product is a set of | 0.5ml/ bottle × 2 bottle | Main component is T, positive human serum |
| Developer A | 7ml × 1 bottle | Main component is urea peroxide element |
| Developer B | 7ml × 1 bottle | Main component is TMB |
| Stop buffer | 7ml × 1 bottle | Main component is dilute sulphuric acid |
| Concentrated cleaning solution | 15ml × 1 bottle | Main component is NaCl, phosphoric acid salt, tween 20 |
| Specification sheets | 1 part | |
| Viscose board | 2 | |
| Log-Logit graph paper | 1 part | |
| Sealing bag, siccative | 1 cover |
The concentration of table 9, testosterone quantitative determination reagent kit (euzymelinked immunosorbent assay (ELISA)) alignment product
| Unit | S0 | S1 | S2 | S3 | S4 | S5 |
| ng/ml | 0 | 0.1 | 0.4 | 1.6 | 5 | 20 |
Quality control product concentration in testosterone quantitative determination reagent kit (euzymelinked immunosorbent assay (ELISA)): low value (L) 0.22-0.58ng/ml; High level (H): 1.75-4.13ng/ml.
In triplicate, the concrete operations at every turn repeating to test are as follows, and the reagent in following step is reagent in Immunoglobulin reagents box in experiment:
A, all ingredients in following step moved to room temperature (18-25 DEG C) balance half an hour; Get concentrated cleaning solution, with distilled water diluting 20 times, after mixing, obtain the washings diluted.
B, taken out from sealing bag by pre-wrapper sheet, each setting point and Quality Control once respectively establish holes, and every hole adds respective alignment product and each 50 μ l of Quality Control; All the other each detect aperture directly add a kind of above-mentioned vat liquor 50 μ l.If a blank control wells (this sky does not add any liquid).
C, every hole add enzyme-labelled antigen 50 μ L (blank control wells does not add enzyme-labelled antigen), then add antibody 50 μ L to every hole.Abundant mixing, sticks non-setting adhesive mounting, puts 37 DEG C of incubations 1 hour.
Plate is washed in D, craft: discard liquid in hole, fill each hole with the washings of the dilution of steps A, leaves standstill 10 seconds and dries, pat dry after repeating 3 times.Wash trigger and wash plate: pat dry after selecting washing 3 secondary program.
E, every hole add developer A50 μ L, developer 50 μ L, and after vibration mixing, put 37 DEG C of lucifuges and develop the color 15 minutes, every hole adds stop buffer 50 μ L.
F, use microplate reader reading, measure each hole OD value.
G, data processing
1), percentage combination rate is calculated: by the OD value of calibration object or the sample OD value divided by S0, be percentage combination rate.
2), use linear fit function, select logit line fitting method.First the light absorption value of blank well is set to background.The concentration of calibration object S0 to S5 is as X, corresponding light absorption value subtracts after background as Y, select Logit fit approach, according to fit equation: ln [percentage combination rate/(1-percentage combination rate)]=Blog (concentration)+A, calculate the content of testosterone in vat liquor to be measured from fit line, wherein A is fit equation parameter.
Setup Experiments control group measures the water ratio of every only male woods musk deer ight soil, and calculates the testosterone extracted amount (ng/g argol) of every gram of argol:
Testosterone extracted amount (ng/g argol)=(testosterone concentration × 10ml of kit measurement)/[0.5g × (water ratio of 1-woods musk deer ight soil)] of every gram of argol.
By results averaged identical for extracting solution between different woods musk deer, result is as shown in table 10 and Fig. 4.
Use SPSS16.0 software to carry out one-way analysis of variance (one way-ANOVA) and the multiple comparisons of the testosterone concentration that different extracting solution extracts in ight soil, significance level is set to P=0.05; Adopt the homogeneity of Levene ' s homogeneity test of variance determination variance of the sample mean.
The testosterone extracted amount of the male woods musk deer ight soil that table 10, different extracting solution obtain
| Extracting solution | Woods musk deer sex | Testosterone extracted amount (ng/g argol) | Testosterone extracted amount rank |
| 100M | ♂ | 4.0097±0.62 | 7 |
| 90M | ♂ | 5.4820±1.15 | 4 |
| 80M | ♂ | 4.0790±0.07 | 6 |
| 60M | ♂ | 4.8227±0.60 | 5 |
| 100E | ♂ | 5.8387±1.52 | 2 |
| 90E | ♂ | 6.5430±1.21 | 1 |
| 80E | ♂ | 2.2267±0.37 | 8 |
| 60E | ♂ | 5.8080±1.32 | 3 |
Result shows, the amount extracting the testosterone that male woods musk deer ight soil obtains with 90E (concentration of volume percent is the aqueous ethanolic solution of 90%) is maximum, respectively higher than with 90M (concentration of volume percent is the methanol aqueous solution of 90%), 100E (ethanol) and 60E (concentration of volume percent is the aqueous ethanolic solution of 60%) extracts the amount of the testosterone that male woods musk deer ight soil obtains, and be significantly higher than with 80E (concentration of volume percent is the aqueous ethanolic solution of 80%) respectively, 100M (methyl alcohol), 80M (concentration of volume percent is the methanol aqueous solution of 80%) and 60M (concentration of volume percent is the methanol aqueous solution of 60%) extracts the amount of the testosterone that male woods musk deer ight soil obtains.Result shows, 90E (concentration of volume percent is the aqueous ethanolic solution of 90%) can be used to the testosterone extracting male woods musk deer ight soil.
The result of embodiment 1-3 shows, extract the best results of the steroid hormone in woods musk deer ight soil with 90E (concentration of volume percent is the aqueous ethanolic solution of 90%), 90E (concentration of volume percent is the aqueous ethanolic solution of 90%) can be utilized to extract steroid hormone in woods musk deer ight soil.
Claims (10)
1. the extracting method of steroid hormone in animal excrement, comprises the step obtaining the vat liquor containing described steroid hormone with animal excrement described in extracting solution lixiviate;
Described extracting solution is aqueous ethanolic solution, methanol aqueous solution, ethanol or methyl alcohol;
In described aqueous ethanolic solution, the concentration of volume percent of ethanol is less than 100% for being more than or equal to 60%;
In described methanol aqueous solution, the concentration of volume percent of methyl alcohol is less than 100% for being more than or equal to 60%.
2. method according to claim 1, is characterized in that: described steroid hormone is whole or two kinds or one in hydrocortisone, estradiol and testosterone.
3. method according to claim 1 and 2, is characterized in that: described extracting solution is described aqueous ethanolic solution; In described aqueous ethanolic solution, the concentration of volume percent of ethanol is less than 100% for being more than or equal to 80%.
4. the method according to claim 1 or 2 or 3, is characterized in that: described extracting solution is described aqueous ethanolic solution; In described aqueous ethanolic solution, the concentration of volume percent of ethanol is 90%.
5. method according to claim 1, is characterized in that: described extracting solution is described methanol aqueous solution; In described methanol aqueous solution, the concentration of volume percent of methyl alcohol is 60%-90%.
6., according to the arbitrary described method of claim 1-5, it is characterized in that: described animal is jenny; Described steroid hormone is hydrocortisone; Described extracting solution is described aqueous ethanolic solution or described methyl alcohol; In described aqueous ethanolic solution, the concentration of volume percent of ethanol is 90%.
7., according to the arbitrary described method of claim 1-5, it is characterized in that: described animal is jenny; Described steroid hormone is estradiol; Described extracting solution is described ethanol or described aqueous ethanolic solution; In described aqueous ethanolic solution, the concentration of volume percent of ethanol is 90%.
8., according to the arbitrary described method of claim 1-5, it is characterized in that: described animal is buck; Described steroid hormone is hydrocortisone; Described extracting solution is described aqueous ethanolic solution or described ethanol or described methanol aqueous solution; In described aqueous ethanolic solution, the concentration of volume percent of ethanol is 90%; In described methanol aqueous solution, the concentration of volume percent of methyl alcohol is 60% or 80%.
9., according to the arbitrary described method of claim 1-5, it is characterized in that: described animal is buck; Described steroid hormone is testosterone; Described extracting solution is described aqueous ethanolic solution or described ethanol; In described aqueous ethanolic solution, the concentration of volume percent of ethanol is 90%.
10., according to the arbitrary described method of claim 1-9, it is characterized in that: described animal is woods musk deer (Moschusberezovskii).
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104914098A (en) * | 2015-05-18 | 2015-09-16 | 北京林业大学 | Method for detecting content of substance to be detected in animal manure ethanol extract |
| CN108037216A (en) * | 2018-01-15 | 2018-05-15 | 北京林业大学 | A kind of method that definite woods musk deer takes the fragrant time |
| CN109061199A (en) * | 2018-08-20 | 2018-12-21 | 北京师范大学 | The extraction and detection method of corticosteroid hormone in a kind of Pu goa feaces |
| CN113045615A (en) * | 2021-03-05 | 2021-06-29 | 太原动物园 | Extraction method of steroid hormone in North China leopard feces |
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| 郎冬梅 等: "圈养林麝粪便类固醇激素保存时效性研究", 《四川动物》, vol. 30, no. 3, 31 December 2011 (2011-12-31), pages 357 - 361 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104914098A (en) * | 2015-05-18 | 2015-09-16 | 北京林业大学 | Method for detecting content of substance to be detected in animal manure ethanol extract |
| CN108037216A (en) * | 2018-01-15 | 2018-05-15 | 北京林业大学 | A kind of method that definite woods musk deer takes the fragrant time |
| CN108037216B (en) * | 2018-01-15 | 2021-01-15 | 北京林业大学 | Method for determining aroma taking time of forest musk deer |
| CN109061199A (en) * | 2018-08-20 | 2018-12-21 | 北京师范大学 | The extraction and detection method of corticosteroid hormone in a kind of Pu goa feaces |
| CN113045615A (en) * | 2021-03-05 | 2021-06-29 | 太原动物园 | Extraction method of steroid hormone in North China leopard feces |
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