CN108037216A - A kind of method that definite woods musk deer takes the fragrant time - Google Patents
A kind of method that definite woods musk deer takes the fragrant time Download PDFInfo
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- CN108037216A CN108037216A CN201810036152.5A CN201810036152A CN108037216A CN 108037216 A CN108037216 A CN 108037216A CN 201810036152 A CN201810036152 A CN 201810036152A CN 108037216 A CN108037216 A CN 108037216A
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Abstract
The present invention relates to woods musk deer to cultivate field, more particularly to a kind of method that definite woods musk deer takes the fragrant time.The methods of observation of the method for the present invention synthesis microscopic morphology, seasoning analysis change of moisture content, low-polarity components and elisa assay excrement sample Hormone change, by the way that by the mode of appearance color of Moschus sample, composition transfer and with taking fragrant woods musk deer excrement sample hormone correlation dynamic change to carry out system contrast, the multi-level woods musk deer of multi-angle for having explored set of system takes the fragrant time to determine method.Never ipsilateral to Moschus quality judging and it can determine optimal to take the fragrant time to provide scientific basis using the method for the present invention, available for situation of change in grasp woods musk deer Moschus after secretion maturing process, so as to take the fragrant time for definite artificial breeding woods musk deer science, collection Moschus quality is ensured.
Description
Technical field
The present invention relates to woods musk deer to cultivate field, more particularly to a kind of method that definite woods musk deer takes the fragrant time.
Background technology
Woods musk deer (Moschusberezovskii) is also referred to as musk deer, woods river deer, is one kind of build minimum in musk deer animals, mainly
China is originated in, is country-level wild protection animal.It is China's tradition rare traditional Chinese medicine and internationally recognized that male musk deer, which produces Moschus,
Fine perfumery, the treasure being considered as in medicinal material, the musk deer in traditional Chinese and western medicine clinicing aspect extensive application, many important Chinese medicine formulas
Perfume (or spice) is essential component, such as Angong Niuhuang Wan, Pien Tze Huang, Xihuang pills, Moschus brain refreshing liquid.However, due to throughout the year by
Hunt movable influence unlawfully, the quantity of domestic wild woods musk deer is seldom at present, and Moschus resource is very in short supply, manually supports musk deer industry in recent years
Development is very fast, and the quantity of Shaanxi Province woods musk deer cultivation at present is about more than 10,000, and Moschus is obtained also by relying primarily on wild woods musk deer in the past
Resource, which is gradually transitions, manually cultivates the acquisition of woods musk deer, due to the expansion of woods musk deer cultivation scale, establishes a set of Moschus quality judging
System simultaneously determines suitable to take the fragrant time very necessary.
Moschus quality is generally traditionally judged using mode of appearance, color and luster and smell, but due to a lack of quantized data, identification knot
Fruit varies with each individual, and can not provide the judgement conclusion of science.Have some research reports of Moschus composition measurement in recent years, but it is restricted
In the complexity of Moschus component and the limitation of study condition, these Moschus Study on composition change all lack systematicness, so far for
The standards system that Moschus quality component judges is still not perfect, this also directly affects the judgement of Moschus maturity period.Sichuan is artificial at present
Cultivation woods musk deer takes the fragrant time to concentrate on annual November, and Shaanxi woods musk deer takes the fragrant time to concentrate on annual 2 month, this when taking perfume (or spice)
Between define and be built upon on the basis of Conventional wisdom, lack experimental data and support, therefore still suffer from certain blindness.
The present invention provides a kind of method that definite woods musk deer takes the fragrant time, comprehensive microscopic morphology observation, seasoning analysis contain
The methods of water change, gas chromatography mass spectrometry (GC-MS) technology and elisa assay excrement sample Hormone change, by by outside Moschus sample
See form color, composition transfer and with taking fragrant woods musk deer excrement sample hormone correlation dynamic change to carry out system contrast, explore one
The set multi-level woods musk deer of multi-angle takes the fragrant time to determine method.Using the method for the present invention can never ipsilateral to Moschus quality judging
And determine it is optimal take the fragrant time to provide scientific basis, woods musk deer most preferably takes determining for fragrant time, can ensure that pharmacy corporation obtains
Moschus material quality, so as to bring more preferable Social benefit and economic benefit.Further, since the method for the present invention possesses definite behaviour
Make specification and operating condition, the personnel's subjective factor that is put to the test influences small, the accuracy and reliability significantly larger than tradition of its result
Experience criterion, and the method for the present invention is simple and easy to do, of less demanding to the professional standards and practical experience of operating personnel, it is very suitable
It is suitable for and is promoted the use of in drug tests.
The content of the invention
The present invention is intended to provide a kind of method that easy-to-use definite woods musk deer takes the fragrant time, the method for the present invention includes following
Step：
(1) Moschus sample appearance morphological analysis
By stereoscopic sem observation Moschus mode of appearance, describe and record Moschus mode of appearance；
(2) Moschus sample determination of moisture
A. prepare 200 μ l specification centrifuge tubes, add Moschus sample after correct amount in pipe, liquid measures 10 with pipettor
μ l, solid weigh 5-10mg；
B. the centrifuge tube equipped with sample is weighed, takes the centrifuge tube of a same size, 10 μ l distilled water are added after weighing,
Together dried with Moschus sample as control sample after weighing again；
C. all samples to be tested and control sample are placed on centrifugation tube sheet, open the centrifuge tube mouth of pipe, be placed in equipped with foot
In the closing drier for measuring calcium sulfate, magnesium sulfate and sodium sulphate, standing and drying 48h；
D. represent dry thorough when the centrifuge tube weight equipped with control sample is identical with blank pipe weight, to all centrifugations
Pipe is weighed, and calculates the moisture in Moschus sample；
(3) Moschus sample principal component gas chromatography mass spectrometry GC-MS chromatography
A. sample pretreatment：Moschus sample to be detected is weighed, is freeze-dried with the freeze drier for being pre-chilled to -50 DEG C
12-24h, obtains Moschus freeze-dried powder, weighs above-mentioned Moschus freeze-dried powder 0.05g, with anhydrous CaSO4Absorbent drying 72h；
B. add 1ml dichloromethane extraction 15min into above-mentioned dried Moschus freeze-dried powder sample, GC- is obtained after filtering
MS test samples；
C. gas chromatograph test condition is as follows：
Input mode：Splitless injecting-Sample；
Sample size：1μl；
Injector temperature：250-252℃；
Tubing string temperature：80 DEG C, kept for 1 minute,
5 DEG C of increase per minute is kept for 1 minute up to 245 DEG C afterwards,
10 DEG C of increase per minute is kept for 5 minutes up to 280 DEG C afterwards；
Carrier gas：Helium；
Flow rate of carrier gas：1-1.05ml/min；
The bulk analysis time：60min；
D. mass spectrograph test condition is as follows：
Converting interface temperature：280-282℃；
Mass spectrum room temperature：230-232℃；
Quadrupole rod analyzer temperature：150℃；
(4) woods musk deer fecal hormone assay
A. sample pretreatment：Woods musk deer fecal specimens are fully ground crushing, 0.50g is weighed and is put into 10ml centrifuge tubes, add
Enter 90% ethanol of 5ml, after oscillation extraction 20min, 2500rpm centrifugation 20min, take supernatant, lower sediment adds 5ml
90% ethanol oscillation extraction 20min, 2500rpm centrifuges 20min again, merges supernatant twice and in 60 DEG C of water bath methods, adds
Enter 1ml PBS vibration recycling, be placed in freezing in -20 DEG C of refrigerators to be measured；
B. radioimmunoassay method woods musk deer fecal specimens corticosteroid hormone, the parameter of cortisol kit used are used
For：Sensitivity≤2.0ng/ml, variation within batch coefficient<10%, interassay coefficient of variation<15%；
C. using electrochemiluminescent immunoassay method measure woods musk deer fecal specimens testosterone and estradiol hormone, testosterone kit used and female two
The parameter of alcohol kit is：Rate of recovery 90-110%, precision in analysis<15.0%, betweenrun precision<20.0%；
D. data processing：By collection of each month Moschus sample corresponding woods musk deer individual excrement testosterone, estradiol and cortisol
Determination data is arranged, and is manufactured box traction substation and is calculated the significance of difference of adjacent two months hormonal readiness.
After completing said determination step, the method for the present invention can determine that woods musk deer takes the fragrant time by following several aspects：
1. take the fragrant time from the change judgement of Moschus mode of appearance
When Moschus sample is light water oily liquids, the particulate Moschus containing light brown in rear liquid is staticly settled
When, this stage starts to secrete the first fragrant form in fragrant stage for woods musk deer.As the time elapses, Moschus becomes brown color solid-state, can observe
Larger to this period Moschus humidity, this stage is Moschus from liquid to the solid state transformed stage, and Moschus appearance color does not have afterwards
There is significant change, but can intuitively observe that Moschus appearance humidity is decreased obviously, then in ensuing about 6 months, musk deer
Fragrant mode of appearance color change is little, and humidity is gradually reduced, but changes unobvious.After testing it was found that when Moschus appearance is wet
Degree starts when being decreased obviously, you can is determined as suitable taking the fragrant time.
2. judge to take the fragrant time from Moschus moisture content change scope
Moschus water content is the results show that the Moschus water content of Early insulin secretion is very high, this stage Moschus is liquid, then musk deer
Perfume (or spice) is gradually converted into solid-state, and Moschus color is gradually deepened, and slowly becomes brown color, and moisture is remarkably decreased, this stage Moschus
Solid-state is become by liquid, Moschus water content declines by a big margin.After certain phase is reached, Moschus water content continues to decline, but
Downward trend slows down, and tends towards stability substantially.After testing it was found that starting when Moschus water content is basicly stable, you can determine
The fragrant time is taken for suitable.
3. take the fragrant time from the change judgement of Moschus chromatogram
Early stage Moschus chromatogram shows that secreting fragrant Moschus at initial stage has begun to fractions Fingerprints occur, but material
Component species is less, Moschus chromatogram composition characteristics peak negligible amounts.As the time elapses, there is obvious component in Moschus chromatogram
Fingerprints, composition characteristics peak number amount significantly increase, and Moschus main component characteristic peak occurs substantially, afterwards close to 8
In month, the above-mentioned Moschus composition characteristics peak number amount in Moschus chromatogram does not have significant changes, kept stable.Therefore,
When significant changes do not occur for the above-mentioned Moschus composition characteristics peak number amount in Moschus chromatogram, you can it is suitable when taking perfume (or spice) to be determined as
Between.
4. judge to take the fragrant time from Moschus principal component changes of contents
By carrying out GC-MS analyses to specially treated Moschus sample, suitable GC-MS testing conditions are determined, and it is right
The higher principal component of wherein 10 kinds of contents carries out area normalization method semi-quantitative analysis, it can be seen that a variety of principal component December in Moschus
Part content reaches highest, such as：Cyclopentadecanone, cholesterol, cholestanol, 3a- hydroxyl -5b- androstane -17- ketone and cis-1,
2-Cyclododecanediol is in content highest in December, and Cholest-2-ene, Cholest-3-ene, and (5.beta.)-,
Cholesta-3,5-diene contents were 2 months next years or reached highest April.A variety of principal components start ripple occur during December
Dynamic, part principal component has downward trend, this shows various principal component variation tendencies, and there are difference.It is considered that by the end of December after analysis
Stage woods musk deer mating ending phase for this, mentioned component may in Musk Secretion and during curing latter portions into distribution
Biochemical reaction, changes into other organic matters, as the content of cis-9-Hexadecenal is gradually reduced since October, but
The content of the ring ketone compounds such as cis-1,2-Cyclododecanediol is begun to ramp up, and may be deposited between these organic components
Chemically reacting or mutually converting, but related conclusions are still needed and further to be verified.It is therefore believed that consider it is various it is main into
Point changes of contents trend, December for artificial breeding woods musk deer it is suitable take the fragrant time.
5. judge to take the fragrant time from woods musk deer excrement sample Hormone change scope
Hormone plays an important role in the various physiology courses of animal, and Hormone change can reflect that animal physiological becomes
Change, therefore will be seen that the health status of animal by Hormone change.This research employs non-damaging method to woods musk deer excrement sample
Hormone has carried out dynamic detection, and woods musk deer excrement sample physiological hormone level is the results show that breeding physiology associated hormone testosterone and estradiol
It is horizontal June content it is higher, fluctuation is larger, and August part is relatively low to second year content in April, and fluctuation is smaller, this research will secrete perfume (or spice)
Initial stage and secrete the time of fragrant phase and separate, analyze hormonal readiness and the correlation of Moschus principal component, the results showed that secrete fragrant two kinds of initial stage
Sex hormone level and ten kinds of component contents are significantly correlated, remaining period only a few Moschus principal component and hormonal readiness phase
Close.June is that woods musk deer secretes fragrant initial stage, is shown by hormone and Moschus component correlation analysis, and testosterone and estradiol hormone are in perfume (or spice)
Important function has been played during capsule gland primary starting secretion Moschus, and the correlation unobvious in later stage maturing process.
Woods musk deer excrement sample corticosteroid hormone analysis result be shown in June, 10 to December content it is higher, other month contents
It is relatively low, June woods musk deer be in secrete fragrant beginning period, 10 to December be in woods musk deer breeding the breeding season, show that woods musk deer is secreting
Fragrant phase and breeding period stress level is higher, remaining period cortisol levels is relatively stablized.Originally corticosteroid hormone is researched and analysed
The horizontal correlation with Moschus principal component, the results showed that corticosteroid hormone is not horizontal notable with ten kinds of component content correlations.Cause
This, woods musk deer excrement sample Hormone change analysis result shows, considers that woods musk deer secretes fragrant and mating season corticosteroid hormone change and and musk deer
Fragrant component correlation, woods musk deer most preferably take the fragrant time, if taking fragrant woods musk deer to be not involved in mating, the fragrant time should be taken to be advanced in January
December.
6. integrated judgment most preferably takes the fragrant time
Comprehensive items woods musk deer Moschus analysis result：1. Moschus mode of appearance seasonal variations；2. Moschus moisture season becomes
Change；3. Moschus chromatogram and principal component content seasonal variations；4. excrement physiological hormone seasonal variations and component correlation analysis；Woods
Musk deer takes the fragrant time to determine to take the fragrant time according to breeding musk deer and non-breeding musk deer, participates in breeding woods musk deer and most preferably takes the fragrant time should be in January
Carry out taking perfume (or spice) after part mating, non-breeding woods musk deer takes the fragrant time at the beginning of being advanced to 12 months to start.
Preferably, present invention determine that the method that woods musk deer takes the fragrant time, wherein (3) step carries out by the following method：
(3) Moschus sample principal component gas chromatography mass spectrometry GC-MS chromatography
A. sample pretreatment：Moschus sample to be detected is weighed, is freeze-dried with the freeze drier for being pre-chilled to -50 DEG C
24h, obtains Moschus freeze-dried powder, weighs above-mentioned Moschus freeze-dried powder 0.05g, with anhydrous CaSO4Absorbent drying 72h；
B. add 1ml dichloromethane extraction 15min into above-mentioned dried Moschus freeze-dried powder sample, GC- is obtained after filtering
MS test samples；
C. gas chromatograph test condition is as follows：
Input mode：Splitless injecting-Sample；
Sample size：1μl；
Injector temperature：250℃；
Tubing string temperature：80 DEG C, kept for 1 minute,
5 DEG C of increase per minute is kept for 1 minute up to 245 DEG C afterwards,
10 DEG C of increase per minute is kept for 5 minutes up to 280 DEG C afterwards；
Carrier gas：Helium；
Flow rate of carrier gas：1ml/min；
The bulk analysis time：60min；
D. mass spectrograph test condition is as follows：
Converting interface temperature：280℃；
Mass spectrum room temperature：230℃；
Quadrupole rod analyzer temperature：150℃.
Moreover, it relates to a kind of definite woods musk deer takes application of the method for fragrant time in drug test.
Since Moschus is expensive, Sparklet testing method has more practicality, and the present invention is by improving pretreatment of raw material
Technique, introduces freeze-drying step in raw material extraction process, inhibits while drying efficiency is improved and volatilizees in Moschus
Property component scatter and disappear, greatly improve the extraction efficiency of the significant component of sample, the possibility for possessing trace detection.
The method of the present invention is available for situation of change in grasp woods musk deer Moschus after secretion maturing process, so that for determining people
Work cultivation woods musk deer science takes the fragrant time, ensures collection Moschus quality.
Present invention determine that the method that woods musk deer takes the fragrant time, comprehensive microscopic morphology observation, seasoning analysis change of moisture content, gas
Matter is combined (GC-MS) technology and the methods of elisa assay excrement sample Hormone change, by by the mode of appearance color of Moschus sample,
Composition transfer and with taking fragrant woods musk deer excrement sample hormone correlation dynamic change to carry out system contrast, has explored the polygonal of set of system
Spending multi-level woods musk deer takes the fragrant time to determine method.Can never ipsilateral to Moschus quality judging and it be determined using the method for the present invention
Optimal takes the fragrant time to provide scientific basis, and woods musk deer most preferably takes determining for fragrant time, and it is former can to ensure that pharmacy corporation obtains Moschus
Quality is expected, so as to bring more preferable Social benefit and economic benefit.Further, since the method for the present invention possesses definite working specification
And operating condition, the personnel's subjective factor that is put to the test influence is small, and the accuracy and reliability of its result is significantly larger than Conventional wisdom and sentences
Determine method, overcome experience and judge that there is the defects of blindness, and the method for the present invention is simple and easy to do, professional standards to operating personnel
It is of less demanding with practical experience, it is extremely suitable for promoting the use of in drug tests.
Brief description of the drawings
Fig. 1 is Moschus sample appearance metamorphosis figure (A：In June, 2016, B：In August, 2016, C：In October, 2016, D：
In December, 2016, E：2 months 2017, F：In April, 2017)；
Fig. 2 takes fragrant time Moschus sample moisture content data variation figure for difference；
Fig. 3 is the chromatogram trend chart that difference takes fragrant time Moschus sample；
Fig. 4 be difference take fragrant time Moschus sample muskone changes of contents tendency chart (muskone content June to December by
Edge up height, is gradually reduced afterwards)；
Fig. 5 is the 3a- hydroxyl -5b- androstane -17- ketone changes of contents tendency charts (3a- that difference takes fragrant time Moschus sample
Hydroxyl -5b- androstane -17- ketone gradually rises content June to December, is gradually reduced afterwards)；
Fig. 6 be it is different take fragrant time Moschus sample cholesterol level trend chart (cholesterol level is 0 in June,
August to December gradually rises, and the content decline of 2 months, April, content was returned as 0)；
Fig. 7 is that (cholestanol content is June the different cholestanol changes of contents tendency charts for taking fragrant time Moschus sample
0, August to December gradually rises, and 2 months and content in April are returned as 0)；
Fig. 8 is the Cholesta-3,5-diene changes of contents tendency charts (Cholesta- that difference takes fragrant time Moschus sample
3,5-diene contents are 0 in June, and August is more stable to December, and the content rise of 2 months, declines April)；
Fig. 9 takes the Cholest-3-ene of fragrant time Moschus sample, (5.beta.)-changes of contents tendency chart for difference
(Cholest-3-ene, (5.beta.)-June to December, content was 0 or less, was gradually risen afterwards)；
Figure 10 is the Cholest-2-ene changes of contents tendency charts (Cholest-2-ene that difference takes fragrant time Moschus sample
Content June gradually rises to October by 0, is reduced to December 0,2 months and is persistently raised to April)；
Figure 11 is the cis-9-Hexadecenal changes of contents tendency charts (cis-9- that difference takes fragrant time Moschus sample
Hexadecenal June content be 0, August part rises to highest, starts to be gradually reduced afterwards, is reduced to 2 months and April
0)；
Figure 12 is the cis-1,2-Cyclododecanediol changes of contents tendency charts that difference takes fragrant time Moschus sample
(cis-1,2-Cyclododecanediol June content be 0, August and October are more stable, and December, content rose to highest, 2
0) moon and April drop to；
Figure 13 is that difference takes the cyclopentadecanone changes of contents tendency chart of fragrant time Moschus sample (cyclopentadecanone is in June
Part content is 0, and the rise of August part content, October and December tends towards stability, be gradually reduced afterwards)；
Figure 14 is that (testosterone concentration is most June, other moons for testosterone concentration trend chart in different time sections woods musk deer excrement
Part content is very low and tends towards stability)；
Figure 15 be different time sections woods musk deer excrement in estradiol content trend chart (estradiol content is most June, its
His month content is very low and tends towards stability)；
Figure 16 is that (Determination of cortisol is more June, 8- for cortisol levels tendency chart in different time sections woods musk deer excrement
December, hormone-content was gradually increasing, and was begun to decline to the 2-4 months).
Embodiment
Illustrate embodiments of the present invention below by way of specific instantiation, those skilled in the art can be by this specification
Disclosed content understands other advantages and effect of the present invention easily.The present invention can also pass through in addition different specific realities
The mode of applying is embodied or practiced, the various details in this specification can also be based on different viewpoints with application, without departing from
Various modifications or alterations are carried out under the spirit of the present invention.
Before the specific embodiment of the invention is further described, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, the protection domain being not intended to be limiting of the invention.
Unless otherwise defined, all technical and scientific terms and those skilled in the art of the present technique used in the present invention are usual
The meaning of understanding is identical.Except used in embodiment specific method, equipment, in addition to material, according to those skilled in the art
The record of grasp and the present invention to the prior art, can also use and the method described in the embodiment of the present invention, equipment, material
Any method, equipment and the material of the similar or equivalent prior art realizes the present invention.
Embodiment
1. Moschus sample collection
28 constitution ages of Fengxian County, Shaanxi, China Pien Tze Huang woods musk deer cultivation base it is consistent woods musk deer individual, as seminar.According to
Planning of experiments, sampling work are divided into 6 stages, on May 1st, 2017, and all stage sampling works are completed：First stage (2016
On June 18, on May 12, to 2016), second stage (on August 1st, 2016), the phase III (on October 25th, 2016), the
Four stages (on December 25th, 2016), the 5th stage (on 2 23rd, 2017), the 6th stage (on April 26th, 2017).Foundation takes
Fragrant scheme, in May, 2017, smoothly completes the Moschus sampling work all planned, and collects qualified Moschus sample, and sample is carried out
Freezen protective is used for subsequent analysis.It has recorded the information such as sampling woods musk deer individual age, weight and genetic background in sampling process,
And it tentatively have recorded each stage Moschus appearance, color and take the information such as fragrant situation.2. Moschus mode of appearance changes (2016.5-
2017.5)
Analysis method：By stereoscopic sem observation Moschus mode of appearance, describe and record Moschus mode of appearance.
In June, 2016, Moschus was light water oily liquids, and pale brown color substance contain in inside, to August part after color burn, change
For yellow oily liquid or semisolid, April October to next year Moschus for the granular ripe Moschus of yellow-brown solid (see Fig. 1, its
Middle A：In June, 2016, B：In August, 2016, C：In October, 2016, D：In December, 2016, E：2 months 2017, F：In April, 2017).
3. water content data variation analyzes (2016.5-2017.5)
Moschus sample moisture content assaying method：
A. prepare 200 μ l specification centrifuge tubes, add Moschus sample after correct amount in pipe, liquid measures 10 with pipettor
μ l, solid weigh 5-10mg；
B. the centrifuge tube equipped with sample is weighed, takes the centrifuge tube of a same size, 10 μ l distilled water are added after weighing,
Together dried with Moschus sample as control sample after weighing again；
C. all samples to be tested and control sample are placed on centrifugation tube sheet, open the centrifuge tube mouth of pipe, be placed in equipped with foot
In the closing drier for measuring calcium sulfate, magnesium sulfate and sodium sulphate, standing and drying 48h；
D. represent dry thorough when the centrifuge tube weight equipped with control sample is identical with blank pipe weight, to all centrifugations
Pipe is weighed, and calculates the moisture in Moschus sample.
As the time elapses, the water content of Moschus sample, which is presented, is gradually reduced trend (see Fig. 2), and in June, 2016 is aqueous
Amount excursion (84.99%-87.66%), part change of moisture content scope of in August, 2016 (45.34%-69.44%), 2016
Change of moisture content scope in October (44.39%-58.95%), in December, 2016 change of moisture content scope (37.99%-
49.31%), 2 month change of moisture content scopes (34.90%-49.54%) in 2017, in April, 2017 change of moisture content scope
(34.44%-49.46%).Fall part of in August, 2016 is more maximum than fall in June, behind fall gradually become
It is slow.In July, 2016, moisture content range fluctuation was larger, and analysis is due to 6-8 month Moschus by light light water oily liquids shape
State is converted into brown yellow granule solid state process, changes more violent, the difference in change procedure existence time, water after October
Point changes of contents scope diminishes, and December, content continued to decline compared to October.2017 2 month Moschus water content compare 2016
Year December, content continued to decline, but declined unobvious, April Moschus water content compared to 2 month content continue to decline, but trend
Unobvious, it is believed that water content is basicly stable after in December, 2016.Pass through the water content to above-mentioned each period Moschus sample
Analysis, we draw the following conclusions：Ripe Moschus water content ranges standard should be 35%-50% in Moschus berezovskii Herov capsule gland, when being adopted
When the water content of the Moschus sample of collection is within above range, prompt arrive suitably take the fragrant time.
4. chromatogram changes (2016.5-2017.5)
Moschus sample principal component gas chromatography mass spectrometry GC-MS chromatogram analysis methods：
A. sample pretreatment：Moschus sample to be detected is weighed, is freeze-dried with the freeze drier for being pre-chilled to -50 DEG C
24h, obtains Moschus freeze-dried powder, weighs above-mentioned Moschus freeze-dried powder 0.05g, with anhydrous CaSO4Absorbent drying 72h；
B. add 1ml dichloromethane extraction 15min into above-mentioned dried Moschus freeze-dried powder sample, GC- is obtained after filtering
MS test samples；
C. gas chromatograph test condition is as follows：
Gas chromatograph-mass spectrometer (GC-MS)：GC MS-QP2010；
Input mode：Auto injection, Splitless injecting-Sample；
Sample size：1μl；
Injector temperature：250℃；
Tubing string temperature：80 DEG C, kept for 1 minute,
5 DEG C of increase per minute is kept for 1 minute up to 245 DEG C afterwards,
10 DEG C of increase per minute is kept for 5 minutes up to 280 DEG C afterwards；
Carrier gas：Helium；
Flow rate of carrier gas：1ml/min；
The bulk analysis time：60min；
D. mass spectrograph test condition is as follows：
Converting interface temperature：280℃；
Mass spectrum room temperature：230℃；
Quadrupole rod analyzer temperature：150℃.
The chromatogram variation tendency of Moschus sample (see Fig. 3).
In June, 2016 total ion current (TIC) collection of illustrative plates shows that Moschus starts Fingerprints occur, but various material kinds
Class and peak value are relatively low, and many components of analysis shows are impurity peaks, and Moschus chromatogram does not have obvious composition characteristics peak substantially.
The part of in August, 2016 total ion current (TIC) collection of illustrative plates shows that Moschus starts obvious composition characteristics peak occur, various effective
Component species and content substantially rise compared to June, mass spectral analysis display component species kind about more than 100.Analyze this stage Moschus
Moisture is largely absorbed, and water content is reduced, and Moschus has begun to concentration and forms brown color solid-state.
In October, 2016 total ion current (TIC) collection of illustrative plates shows that Moschus has a Moschus component TuPu method, but it is various effectively into
Point species and the content difference with insignificance with August part compared with, show component species kind about more than 100.
In December, 2016 total ion current (TIC) collection of illustrative plates shows that Moschus has a Moschus component TuPu method, but it is various effectively into
Point species and content show component species kind about more than 100 with August part and difference with insignificance October compared with.
Month total ion current (TIC) collection of illustrative plates shows that Moschus has Moschus component TuPu method, but various active ingredients within 2017 2
Species and the content difference with insignificance compared with August part, October and December, display component species kind about more than 100.
In April, 2017 total ion current (TIC) collection of illustrative plates shows that Moschus has Moschus component TuPu method, but various active ingredients
Species and the content difference with insignificance compared with August part, October, December and 2 months in 2017, display component species is about
Kind more than 100.Analysis, Moschus is in brown color solid-state, and Moschus active ingredient characteristic peak is notable, and Moschus chromatogram is included substantially into dtex
Peak is levied, but characteristic component peak part occurs and changes.
Substantially occur part of in August, 2016 in April, 2017 the characteristic peak positions time respectively 26.8 points, 34.6 points,
35.5 points, 38.7 points, 44.6 points, 45 points, 48.2 points, 48.6 points.Therefore it can be seen that Moschus chromatogram master from Moschus chromatogram
Want characteristic peak to occur substantially in August part, therefore, can carry out taking perfume (or spice) after the bright August part of Moschus makings coupled HPLC chart.
5. area normalization method Moschus sample principal component analysis (2016.5-2017.5)
The principal component changes of contents trend of Moschus sample (see Fig. 4-13).
In June, 2016, Moschus chromatogram showed that Moschus Contents of Main Components is relatively low, substantially not substantially into dtex
Levy peak.It is notable that August starts Moschus active ingredient characteristic peak, and obvious composition characteristics peak occurs in chromatogram, and component species is about more than 100
Kind.August-December characteristic peak peak area difference is little, and 2 months 2017 and characteristic peak in April part occur and change.This research is screened
Go out muskone, cyclopentadecanone, cis-9-Hexadecenal, cholesterol, 3a- hydroxyl -5b- androstane -17- ketone, Cholest-
3-ene, (5.beta.)-, Cholesta-3,5-diene, Cholest-2-ene, cholestanol, cis-1,2-
Cyclododecanediol totally ten kinds of active ingredients.Detect data and show 6 Monthly changes scope (0.35%- of muskone
2.04%), August part excursion (8.57%-13.52%), 10 Monthly changes scopes (7.34%-12.72%), December become
Change scope (6.39%-14.28%), 2 Monthly changes scopes (3.20%-8.73%), 4 Monthly changes scope (1.53%-
5.11%)；Cyclopentadecanone June is 0, August part excursion (0.16%-0.51%), 10 Monthly changes scopes
(0.14%-0.48%), 12 Monthly changes scopes (0.11%-0.49%), 2 Monthly changes scopes (0.94%-3.32%), 4
Monthly changes scope (0.05%-0.23%)；Cis-9-Hexadecenal June is 0, August part excursion (4.10%-
9.85%), 10 Monthly changes scopes (0.78%-3.54%), 12 Monthly changes scopes (0.46%-1.64%), 2 Monthly changes
Scope (0.01%-0.08%), 4 Monthly changes scopes (0.04%-0.14%)；6 Monthly changes scope (1.73%- of cholesterol
5.37%), August part excursion (5.19%-7.37%), 10 Monthly changes scopes (6.34%-10.16%), December become
Change scope (9.05%-12.05%), 2 Monthly changes scopes (0.16%-4.96%), April 0；3a- hydroxyl -5b- androstanes
6 Monthly changes scope (0.37%-2.55%) of alkane -17- ketone, August part excursion (4.90%-7.08%), 10 Monthly changes
Scope (4.07%-7.91%), 12 Monthly changes scopes (4.45%-11.71%), 2 Monthly changes scope (0.21%-
2.67%), 4 Monthly changes scope (0.15%-0.38%)；Cholest-3-ene, (5.beta.)-June are that 0, August part becomes
Change scope (0.43%-0.80%), 10 Monthly changes scopes (0.90%-1.80%), 12 Monthly changes scope (0.42%-
1.08%), 2 Monthly changes scopes (0.24%-2.82%), 4 Monthly changes scopes (1.56%-3.43%)；Cholesta-3,
6 Monthly changes scopes (0.95%-2.46%) of 5-diene, August part excursion (2.98%-4.41%), 10 Monthly changes
Scope (2.42%-6.29%), 12 Monthly changes scopes (3.35%-6.15%), 2 Monthly changes scope (2.63%-
13.21%), 4 Monthly changes scope (1.14%-6.93%)；6 Monthly changes scope (0.33%- of Cholest-2-ene
0.85%), August part excursion (1.03%-1.84%), 10 Monthly changes scopes (2.24%-3.62%), 12 Monthly changes
Scope (0.20%-0.94%), 2 Monthly changes scopes (1.32%-7.40%), 4 Monthly changes scope (4.94%-
8.29%)；Cholestanol June is 0, August part excursion (1.69%-4.22%), 10 Monthly changes scope (3.09%-
4.93%), 12 Monthly changes scopes (2.71%-7.09%), 2 Monthly changes scopes (0.16%-0.79%), April 0；
Cis-1,2-Cyclododecanediol June is 0, August part excursion (0.14%-0.80%), 10 Monthly changes models
Enclose (0.25%-2.09%), 12 Monthly changes scopes (1.10%-3.66%), 2 Monthly changes scopes (0.04%-0.30%),
April is 0.
6. woods musk deer fecal hormone changes of contents trend (2016.5-2017.5)
Woods musk deer fecal hormone content assaying method：
A. sample pretreatment：Woods musk deer fecal specimens are fully ground crushing, 0.50g is weighed and is put into 10ml centrifuge tubes, add
Enter 90% ethanol of 5ml, after oscillation extraction 20min, 2500rpm centrifugation 20min, take supernatant, lower sediment adds 5ml
90% ethanol oscillation extraction 20min, 2500rpm centrifuges 20min again, merges supernatant twice and in 60 DEG C of water bath methods, adds
Enter 1ml PBS vibration recycling, be placed in freezing in -20 DEG C of refrigerators to be measured；
B. radioimmunoassay method woods musk deer fecal specimens corticosteroid hormone is used, puts and exempts from instrument model GC-2016 (in Anhui
Ke Zhongjia scientific instrument Co., Ltd, Anhui, China), the parameter of cortisol kit (Beijing North biotech company) is：
Sensitivity≤2.0ng/ml, variation within batch coefficient<10%, interassay coefficient of variation<15%；
C. using electrochemiluminescent immunoassay method measure woods musk deer fecal specimens testosterone and estradiol hormone, luminescence immunoassay instrument model
TZD-CL-200S (reaches bio tech ltd, Fujian, China) in Xiamen day, testosterone kit (Beijing North biotechnology
Company) and the parameter of estradiol kit (Beijing North biotech company) be：Rate of recovery 90-110%, precision in analysis
<15.0%；Betweenrun precision<20.0%；
D. data processing：By collection of each month Moschus sample corresponding woods musk deer individual excrement testosterone, estradiol and cortisol
Determination data is arranged, and is manufactured box traction substation and is calculated the significance of difference of adjacent two months hormonal readiness.
Different time sections woods musk deer fecal hormone changes of contents trend (see Figure 14-16).
Each month variance analysis is the results show that the 6-8 months and 12-2 month woods musk deer individual testosterone levels variations are notable；Female two
Alcohol is in addition to the 6-8 months and the 12-2 months, and also there were significant differences for the hormonal readiness change of the 10-12 months；Each month difference of cortisol levels is equal
Not significantly, in relatively stable state.
7. take fragrant woods musk deer fecal hormone and Moschus component correlation analysis table
(1) testosterone
A
| Component | Related coefficient | Sig. (bilateral) | N |
| Muskone | -.743** | 0 | 22 |
| Cyclopentadecanone | -.749** | 0 | 22 |
| cis-9-Hexadecenal | -.721** | 0 | 22 |
| Cholesterol | -.750** | 0 | 22 |
| 3a- hydroxyl -5b- androstane -17- ketone | -.789** | 0 | 22 |
| Cholest-3-ene,(5.beta.)- | -.738** | 0 | 22 |
| Cholesta-3,5-diene | -.832** | 0 | 22 |
| Cholest-2-ene | -.776** | 0 | 22 |
| Cholestanol | -.783** | 0 | 22 |
| cis-1,2-Cyclododecanediol | -.789** | 0 | 22 |
B
| Component | Related coefficient | Sig. (bilateral) | N |
| Muskone | -.015 | 0.91 | 57 |
| Cyclopentadecanone | -.146 | 0.28 | 57 |
| cis-9-Hexadecenal | .048 | 0.722 | 57 |
| Cholesterol | .295* | 0.026 | 57 |
| 3a- hydroxyl -5b- androstane -17- ketone | .156 | 0.247 | 57 |
| Cholest-3-ene,(5.beta.)- | .019 | 0.887 | 57 |
| Cholesta-3,5-diene | -.174 | 0.195 | 57 |
| Cholest-2-ene | -.13 | 0.335 | 57 |
| Cholestanol | .316* | 0.017 | 57 |
| cis-1,2-Cyclododecanediol | .062 | 0.648 | 57 |
* for when confidence level (double surveys) is 0.05, correlation is significant；
For * when confidence level (double surveys) is 0.01, correlation is significant.
(2) estradiol
A
B
| Component | Related coefficient | Sig. (bilateral) | N |
| Muskone | -.061 | 0.651 | 57 |
| Cyclopentadecanone | -.181 | 0.178 | 57 |
| cis-9-Hexadecenal | -.274* | 0.039 | 57 |
| Cholesterol | .278* | 0.036 | 57 |
| 3a- hydroxyl -5b- androstane -17- ketone | -.045 | 0.739 | 57 |
| Cholest-3-ene,(5.beta.)- | -.016 | 0.904 | 57 |
| Cholesta-3,5-diene | .044 | 0.743 | 57 |
| Cholest-2-ene | -.305* | 0.021 | 57 |
| Cholestanol | .143 | 0.288 | 57 |
| cis-1,2-Cyclododecanediol | .039 | 0.773 | 57 |
* for when confidence level (double surveys) is 0.05, correlation is significant；* are when confidence level (double surveys) is 0.01, phase
Closing property is significant.
(3) cortisol
A
| Component | Related coefficient | Sig. (bilateral) | N |
| Muskone | -.337 | 0.171 | 18 |
| Cyclopentadecanone | -.255 | 0.307 | 18 |
| cis-9-Hexadecenal | -.268 | 0.282 | 18 |
| Cholesterol | -.287 | 0.249 | 18 |
| 3a- hydroxyl -5b- androstane -17- ketone | -.208 | 0.408 | 18 |
| Cholest-3-ene,(5.beta.)- | -.480* | 0.044 | 18 |
| Cholesta-3,5-diene | -.323 | 0.191 | 18 |
| Cholest-2-ene | -.316 | 0.201 | 18 |
| Cholestanol | -.438 | 0.069 | 18 |
| cis-1,2-Cyclododecanediol | -.302 | 0.223 | 18 |
B
* for when confidence level (double surveys) is 0.05, correlation is significant；
For * when confidence level (double surveys) is 0.01, correlation is significant.
The preferred embodiments of the disclosure and embodiment are explained in detail above, but the present invention is not limited to
The above-described embodiment and examples, in the knowledge that those skilled in the art possess, can also not depart from the present invention
Various changes can be made on the premise of design.
Claims (3)
1. a kind of method that definite woods musk deer takes the fragrant time, it is characterised in that comprise the following steps：
(1) Moschus sample appearance morphological analysis
By stereoscopic sem observation Moschus mode of appearance, describe and record Moschus mode of appearance；
(2) Moschus sample determination of moisture
A. preparing 200 μ l specification centrifuge tubes, add Moschus sample after correct amount in pipe, liquid measures 10 μ l with pipettor,
Solid weighs 5-10mg；
B. the centrifuge tube equipped with sample is weighed, takes the centrifuge tube of a same size, 10 μ l distilled water are added after weighing, again
Together dried with Moschus sample as control sample after weighing；
C. all samples to be tested and control sample are placed on centrifugation tube sheet, open the centrifuge tube mouth of pipe, be placed in equipped with enough sulphur
In the closing drier of sour calcium, magnesium sulfate and sodium sulphate, standing and drying 48h；
D. represent dry thorough when the centrifuge tube weight equipped with control sample is identical with blank pipe weight, all centrifuge tubes are claimed
Weight, calculates the moisture in Moschus sample；
(3) Moschus sample principal component gas chromatography mass spectrometry GC-MS chromatography
A. sample pretreatment：Moschus sample to be detected is weighed, 12- is freeze-dried with the freeze drier for being pre-chilled to -50 DEG C
24h, obtains Moschus freeze-dried powder, weighs above-mentioned Moschus freeze-dried powder 0.05g, with anhydrous CaSO4Absorbent drying 72h；
B. add 1ml dichloromethane extraction 15min into above-mentioned dried Moschus freeze-dried powder sample, GC-MS surveys are obtained after filtering
Test agent；
C. gas chromatograph test condition is as follows：
Input mode：Splitless injecting-Sample；
Sample size：1μl；
Injector temperature：250-252℃；
Tubing string temperature：80 DEG C, kept for 1 minute,
5 DEG C of increase per minute is kept for 1 minute up to 245 DEG C afterwards,
10 DEG C of increase per minute is kept for 5 minutes up to 280 DEG C afterwards；
Carrier gas：Helium；
Flow rate of carrier gas：1-1.05ml/min；
The bulk analysis time：60min；
D. mass spectrograph test condition is as follows：
Converting interface temperature：280-282℃；
Mass spectrum room temperature：230-232℃；
Quadrupole rod analyzer temperature：150℃；
(4) woods musk deer fecal hormone assay
A. sample pretreatment：Woods musk deer fecal specimens are fully ground crushing, 0.50g is weighed and is put into 10ml centrifuge tubes, add 5ml
90% ethanol, after oscillation extraction 20min, 2500rpm centrifugation 20min, take supernatant, lower sediment adds 90% ethanol of 5ml
Oscillation extraction 20min, 2500rpm centrifuge 20min again, merge supernatant twice and in 60 DEG C of water bath methods, add 1ml PBS
Vibration recycling, is placed in freezing in -20 DEG C of refrigerators to be measured；
B. radioimmunoassay method woods musk deer fecal specimens corticosteroid hormone is used, the parameter of cortisol kit used is：Spirit
Sensitivity≤2.0ng/ml, variation within batch coefficient<10%, interassay coefficient of variation<15%；
C. using electrochemiluminescent immunoassay method measure woods musk deer fecal specimens testosterone and estradiol hormone, testosterone kit and estradiol examination used
The parameter of agent box is：Rate of recovery 90-110%, precision in analysis<15.0%, betweenrun precision<20.0%；
D. data processing：By the corresponding woods musk deer individual excrement testosterone of collection of each month Moschus sample, estradiol and cortisol measure
Data are arranged, and are manufactured box traction substation and are calculated the significance of difference of adjacent two months hormonal readiness.
2. the method that definite woods musk deer takes the fragrant time as claimed in claim 1, wherein (3) step carries out by the following method：
(3) Moschus sample principal component gas chromatography mass spectrometry GC-MS chromatography
A. sample pretreatment：Moschus sample to be detected is weighed, 24h is freeze-dried with the freeze drier for being pre-chilled to -50 DEG C, obtains
To Moschus freeze-dried powder, above-mentioned Moschus freeze-dried powder 0.05g is weighed, with anhydrous CaSO4Absorbent drying 72h；
B. add 1ml dichloromethane extraction 15min into above-mentioned dried Moschus freeze-dried powder sample, GC-MS surveys are obtained after filtering
Test agent；
C. gas chromatograph test condition is as follows：
Input mode：Splitless injecting-Sample；
Sample size：1μl；
Injector temperature：250℃；
Tubing string temperature：80 DEG C, kept for 1 minute,
5 DEG C of increase per minute is kept for 1 minute up to 245 DEG C afterwards,
10 DEG C of increase per minute is kept for 5 minutes up to 280 DEG C afterwards；
Carrier gas：Helium；
Flow rate of carrier gas：1ml/min；
The bulk analysis time：60min；
D. mass spectrograph test condition is as follows：
Converting interface temperature：280℃；
Mass spectrum room temperature：230℃；
Quadrupole rod analyzer temperature：150℃.
3. definite woods musk deer takes application of the method for fragrant time in drug test as claimed in claim 1 or 2.
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