CN106198777A - Whether a kind of functional Monascus adds the discrimination method of lovastatin - Google Patents
Whether a kind of functional Monascus adds the discrimination method of lovastatin Download PDFInfo
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- CN106198777A CN106198777A CN201610474007.6A CN201610474007A CN106198777A CN 106198777 A CN106198777 A CN 106198777A CN 201610474007 A CN201610474007 A CN 201610474007A CN 106198777 A CN106198777 A CN 106198777A
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G01N30/72—Mass spectrometers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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Abstract
The present invention relates to Monas cuspurpureus Went Components identification field, disclose the discrimination method whether adding lovastatin in a kind of functional Monascus, including: the formulation of (1) standard curve: with Monacolin K, lovastatin as raw material, prepare n detection sample, measure δ in detection sample13C value；With the mass percent of lovastatin as abscissa, with δ13C value is vertical coordinate, each δ that will record13C value, prepares δ13The standard curve A of the mass percent relation of C value and lovastatin；(2) functional Monascus treats that test sample differentiates: functional Monascus being treated, the Monacolin K in test sample and lovastatin extract, and measures the δ of extract13C value；(3) try to achieve functional Monascus and treat the mass percent of lovastatin in the extract of test sample.The discrimination method of the present invention is the most accurate, it is possible to carries out quantitative identification to whether adding lovastatin in functional Monascus, has filled up the blank in this field current.
Description
Technical field
The present invention relates to Monas cuspurpureus Went Components identification field, particularly relate to whether a kind of functional Monascus adds lovastatin
Discrimination method.
Background technology
Monas cuspurpureus Went, is the Chinese medicine health product of Dietotherapy double-purpose.Beginning for the Tang Dynasty, existing more than 1,000 year history, during Ming Dynasty Lee
Precious the most on the books in<<Compendium of Materia Medica>>: Monas cuspurpureus Went cures mainly to help digestion and invigorates blood circulation, spleen and stomach dry, traumatic injury trauma, and women's vim and vigour pain
With stagnant blood in puerperal not to the utmost.
Monas cuspurpureus Went is the fermentation product that monascus colonizes on rice, and modern science further discloses Monas cuspurpureus Went, and also to have other only
Special effect.Containing lipid regulating agent HMG-COA reductase inhibitor in Japanese scholars remote rattan Zhang Faxian Monas cuspurpureus Went in 1979
(Monacolin K), and it is thought that the main composition of Monas cuspurpureus Went.Monacolin K contestable ground blocks total cholesterol of liver
Synthesis, make serum total cholesterol (TC) reduce, and triglyceride (TG) and low density lipoprotein, LDL (LDL-C) can be reduced, raise
High density lipoprotein (HDL-C).Therefore Monas cuspurpureus Went can reduce cholesterol, prevention cardiovascular disease etc..
Owing to complexity prepared by the Monas cuspurpureus Went of pure natural, and in Monas cuspurpureus Went, the content of Monacolin K is relatively low, expensive.At present, city
Chang Shangyou producer adds to lovastatin in functional Monascus, improves the total content of functional component Monacolin K, palms off sky
So functional Monascus.
Lovastatin is to obtain through purification after aspergillus terreus ferments, and Merck company lovastatin makes fall hyperlipemia
Product " Teroltrat ".The structure of lovastatin is identical with the structure of closed loop Monacolin K, itself be do not have activated, only warp
The Carboxylesterase hydrolysis crossing human body generation is effect that activated open loop Monacolin K just has regulation blood fat, but this
Process needs to consume the Carboxylesterase of human body, can increase hepatic and/or renal burden.The residual being additionally used for extracting lovastatin is organic
Human body can be had side effects by solvent the most unavoidably.And Monas cuspurpureus Went, it is a kind of natural Fermentation Condition of Monascus spp product, it is not necessary to carry through any
Pure, prove through modern science experiment: owing to Monas cuspurpureus Went is natural fermentation products, more pacify than prescription drugs lovastatin in terms of safety
Complete reliable.Simultaneously possibly together with multiple essential amino acids, unsaturated fatty acid, various organized enzymes and sugar etc..Owing to its composition is many
Sample, determines its multisystem, the synergistic feature of Mutiple Targets: except the liver benefiting stomach function regulating, outside the effect helped digestion, moreover it is possible to regulation
Blood fat, reduce blood viscosity, blood pressure lowering, improve diabetes, pre-preventing bone rarefaction, antitumor, anti-Alzheimer disease and prevention
The application of the aspects such as atherosclerosis.Above-mentioned fully show, the effect of Monas cuspurpureus Went considerably beyond the effect of single lovastatin,
Its future is more wide, thus is favored by countries in the world.
But at present, a kind of simple efficiently and accurately method whether can have in identification function monascus product
Artificially add lovastatin.Therefore, the most effectively differentiate natural function monascus product the most manually adds lovastatin one
Be directly this area want solve technical barrier, for ensure the people healthy and Monas cuspurpureus Went industry normal development all have
Critically important meaning.
Summary of the invention
In order to solve above-mentioned technical problem, the invention provides the mirror whether adding lovastatin in a kind of functional Monascus
Other method.The discrimination method of the present invention is the most accurate, it is possible to whether adding lovastatin in functional Monascus quantitatively reflect
, the blank in this field current has not been filled up.
The concrete technical scheme of the present invention is: whether add the discrimination method of lovastatin in a kind of functional Monascus, bag
Include following steps:
(1) formulation of standard curve: with Monacolin K, lovastatin as raw material, respectively n different detection sample of preparation, wherein
In detection sample, lovastatin mass percent is 0-100%；Detection sample injects the detached dowel of HPLC pretreatment with methanol after dissolving
In；Use isotope ratio mass spectrometer, elemental analyser, measure δ in detection sample13C value；With the mass percent of lovastatin it is
Abscissa, with δ13C value is vertical coordinate, each δ that will record13C value, prepares δ13C value and the mass percent relation of lovastatin
Standard curve A；
(2) functional Monascus treats that test sample differentiates: functional Monascus being treated, the Monacolin K in test sample and lovastatin carry
Take, obtain extract；Extract injects after dissolving with methanol in the detached dowel of HPLC pretreatment；Use isotope ratio mass spectrum
Instrument, elemental analyser, measure the δ of extract13C value；
(3) by the δ of extract13C value is updated in standard curve A, tries to achieve functional Monascus and treats that in the extract of test sample, him is cut down in Lip river
The mass percent in spit of fland.
As preferably, the n described in step (1) is the natural number more than or equal to 3.
As preferably, the n described in step (1) is 10, respectively the mass percent difference of lovastatin in detection sample
It is 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%.
As preferably, the n described in step (1) is 6, and respectively in detection sample, the mass percent of lovastatin is respectively
0%, 26%, 40%, 57.5%, 73%, 100%.
As preferably, in step (1), measure δ in detection sample simultaneously2H-number；With the mass percent of lovastatin as horizontal stroke
Coordinate, with δ2H-number is vertical coordinate, each δ that will record2H-number, prepares δ2The mark of the mass percent relation of H-number and lovastatin
Directrix curve B；In step (2), measure the δ of extract simultaneously2H-number；In step (3), by the δ of extract2H-number is updated to mark
In directrix curve B, try to achieve functional Monascus and treat the mass percent of lovastatin in the extract of test sample.
The present inventor, can especially for Mo Na by Monacolin K and lovastatin are carried out long-term research
The research of both woods K and lovastatin training method, finds following difference: lovastatin comes from liquid submerged fermentation, mesh
Before the culture medium of lovastatin on the market contain glucose, lactose, Semen Maydis pulp or soybean derivatives etc..Thing in these culture medium
Matter, it is that carbon source, hydrogen source are all synthesized by C4 approach (photosynthesis), the therefore δ of lovastatin13C (meansigma methods is-
About 16 ‰), δ2H (meansigma methods is about-278 ‰) is consistent with the C4 source in culture medium dispensing.Monacolin K is from rice
Fermented product, rice is synthesized by C3 approach (photosynthesis), its δ13C value relative isotope standard V-PDB is characterized in-
Between 25 ‰ and-27 ‰, Monacolin K13C (meansigma methods is about-30 ‰), δ2H (meansigma methods is about-310 ‰) and base
The botanical source of matter is consistent.
After finding above-mentioned distinguishing characteristics, the present inventor uses the difference of substrate route of synthesis, and Monacolin K and Lip river are cut down him
Spit of fland carries out stable isotope ratio and differentiates, and can carry out quantitative analysis, and method is simple, and stability is preferable.The present invention
Being tried to achieve by two standard curves and treat the mass percent of lovastatin in test sample extract, double verification, reliability is higher.
As preferably, described in step (2), the extracting method of extract is:
Functional Monascus is treated, and test sample is crushed to 40 mesh and is sufficiently mixed uniformly, weighs 400.0-600.0mg in 50mL volumetric flask
In, add 75vol% ethanol 30mL, shake up, ultrasonic 40-60min under room temperature, add 75vol% ethanol to close to volumetric flask scale,
The most ultrasonic 8-12min after shaking up, is cooled to room temperature afterwards, is settled to 50mL with 75vol% ethanol, with the rotation of 3000-4000rpm
Rotary speed is centrifuged 8-12min, removes insoluble residue, takes the filtering with microporous membrane that supernatant is through 0.45 micron, and filtrate is made after drying
Obtain extract.
Use said method that functional Monascus being treated, the Monacolin K in test sample and lovastatin extract, method letter
Single, extraction ratio is high.
As preferably, by injecting the separation of 2mL to HPLC pretreatment after described detection sample or the dissolving of extract methanol
In post, described detached dowel specification is: kromaton, Annonay F 21*250mm, 5 μm.
It is compared with the prior art, the invention has the beneficial effects as follows: the discrimination method of the present invention is the most accurate, it is possible to function
Whether property Monas cuspurpureus Went adds lovastatin and carries out quantitative identification, fill up the blank in this field current.
Accompanying drawing explanation
Fig. 1 is the standard curve A in embodiment 1；
Fig. 2 is the standard curve B in embodiment 1.
Detailed description of the invention
Below in conjunction with embodiment, the invention will be further described.
Embodiment 1
Whether a kind of functional Monascus adds the discrimination method of lovastatin, comprises the following steps:
(1) formulation of standard curve: with Monacolin K, lovastatin as raw material, preparation 6 detection sample, respectively detects respectively
In sample, the mass percent of lovastatin is respectively 0%, and 26%, 40%, 57.5%, 73%, 100%.Detection sample methanol is molten
Inject after solution in the detached dowel (kromaton, Annonay F 21*250mm, 5 μm) of 2mL to HPLC pretreatment；Use isotope
Ratio mass spectrometer, elemental analyser, measure δ in detection sample2H-number and δ13C value；Each δ recorded2H-number, δ13C value, such as following table institute
Show:
| Lovastatin mass percent (%) | δ13C(‰) | δ2H(‰) |
| 0 | -29.7 | -310 |
| 26 | -25.74 | -302 |
| 40 | -23.88 | -298 |
| 57.5 | -21.85 | -292 |
| 73 | -19.63 | -286 |
| 100 | -15.92 | -278 |
According to data in upper table, with the mass percent of lovastatin as abscissa, with δ13C value, δ2H-number is respectively vertical coordinate,
Prepare δ respectively13Standard curve A and δ of the mass percent relation of C value and lovastatin2H-number and the quality hundred of lovastatin
The standard curve B of proportion by subtraction relation, the most as shown in Figure 1 and Figure 2.
(2) functional Monascus treats that test sample differentiates: functional Monascus being treated, test sample is crushed to 40 mesh and is sufficiently mixed uniformly, claims
Take 500.0mg in 50mL volumetric flask, add 75vol% ethanol 30mL, shake up, ultrasonic 50min under room temperature, add 75vol% second
Alcohol to close to volumetric flask scale, the most ultrasonic 10min after shaking up, be cooled to room temperature afterwards, be settled to 50mL with 75vol% ethanol,
It is centrifuged 10min with the rotary speed of 3500rpm, removes insoluble residue, take the filtering with microporous membrane that supernatant is through 0.45 micron, filter
Liquid prepares extract after drying.Extract with methanol dissolve after by inject 2mL to HPLC pretreatment detached dowel
In (kromaton, Annonay F 21*250mm, 5 μm)；Use isotope ratio mass spectrometer, elemental analyser, measure and extract
The δ of thing2H-number and δ13C value；Wherein δ13C is-25.55 ‰, δ2H-number is-300.44 ‰
(3) by the δ of extract2H-number and δ13C value is updated in standard curve B and standard curve A respectively, tries to achieve function respectively
Property Monas cuspurpureus Went treats the mass percent of lovastatin, δ in the extract of test sample13Numerical value a corresponding for C is 29.26%, δ2H is corresponding
Numerical value b is 30.43%.Therefore during functional Monascus treats the extract of test sample, the mass percent of lovastatin is at 29.26%-
30.43% is interval.
Embodiment 2
Whether a kind of functional Monascus adds the discrimination method of lovastatin, comprises the following steps:
(1) formulation of standard curve: with Monacolin K, lovastatin as raw material, preparation 10 detection sample, respectively examines respectively
In test sample, the mass percent of lovastatin is respectively 0%, and 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 100%.Detection sample injects detached dowel (kromaton, the Annonay of 2mL to HPLC pretreatment with methanol after dissolving
F21*250mm, 5 μm) in；Use isotope ratio mass spectrometer, elemental analyser, measure δ in detection sample2H-number and δ13C value；With
The mass percent of lovastatin is abscissa, with δ2H-number, δ13C value is respectively vertical coordinate, each δ that will record2H-number, δ13C
Value, prepares δ respectively2Standard curve B and δ of the mass percent relation of H-number and lovastatin13C value and the matter of lovastatin
The standard curve A of amount percentage relation.
(2) functional Monascus treats that test sample differentiates: functional Monascus being treated, test sample is crushed to 40 mesh and is sufficiently mixed uniformly, claims
Take 400.0mg in 50mL volumetric flask, add 75vol% ethanol 30mL, shake up, ultrasonic 40min under room temperature, add 75vol% second
Alcohol to close to volumetric flask scale, the most ultrasonic 8min after shaking up, be cooled to room temperature afterwards, be settled to 50mL with 75vol% ethanol, with
The rotary speed of 3000rpm is centrifuged 12min, removes insoluble residue, takes the filtering with microporous membrane that supernatant is through 0.45 micron, filtrate
Dried prepared extract.Extract with methanol dissolve after by inject 2mL to HPLC pretreatment detached dowel (kromaton,
Annonay F 21*250mm, 5 μm) in；Use isotope ratio mass spectrometer, elemental analyser, measure the δ of extract2H-number and
δ13C value；
(3) by the δ of extract2H-number and δ13C value is updated in standard curve B and standard curve A respectively, tries to achieve function respectively
The mass percent of lovastatin in the extract of test sample treated by property Monas cuspurpureus Went.
Embodiment 3
Whether a kind of functional Monascus adds the discrimination method of lovastatin, comprises the following steps:
(1) formulation of standard curve: with Monacolin K, lovastatin as raw material, preparation 10 detection sample, respectively examines respectively
In test sample, the mass percent of lovastatin is respectively 0%, and 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 100%.Detection sample injects detached dowel (kromaton, the Annonay of 2mL to HPLC pretreatment with methanol after dissolving
F21*250mm, 5 μm) in；Use isotope ratio mass spectrometer, elemental analyser, measure δ in detection sample2H-number and δ13C value；With
The mass percent of lovastatin is abscissa, with δ2H-number, δ13C value is respectively vertical coordinate, each δ that will record2H-number, δ13C
Value, prepares δ respectively2Standard curve B and δ of the mass percent relation of H-number and lovastatin13C value and the matter of lovastatin
The standard curve A of amount percentage relation.
(2) functional Monascus treats that test sample differentiates: functional Monascus being treated, test sample is crushed to 40 mesh and is sufficiently mixed uniformly, claims
Take 600.0mg in 50mL volumetric flask, add 75vol% ethanol 30mL, shake up, ultrasonic 60min under room temperature, add 75vol% second
Alcohol to close to volumetric flask scale, the most ultrasonic 12min after shaking up, be cooled to room temperature afterwards, be settled to 50mL with 75vol% ethanol,
It is centrifuged 8min with the rotary speed of 4000rpm, removes insoluble residue, take the filtering with microporous membrane that supernatant is through 0.45 micron, filter
Liquid prepares extract after drying.Extract with methanol dissolve after by inject 2mL to HPLC pretreatment detached dowel
In (kromaton, Annonay F 21*250mm, 5 μm)；Use isotope ratio mass spectrometer, elemental analyser, measure and extract
The δ of thing2H-number and δ13C value；
(3) by the δ of extract2H-number and δ13C value is updated in standard curve B and standard curve A respectively, tries to achieve function respectively
The mass percent of lovastatin in the extract of test sample treated by property Monas cuspurpureus Went.
Raw materials used, equipment in the present invention, unless otherwise noted, is the conventional raw material of this area, equipment；In the present invention
Method therefor, unless otherwise noted, is the conventional method of this area.
The above, be only presently preferred embodiments of the present invention, not impose any restrictions the present invention, every according to the present invention
Any simple modification, change and the equivalent transformation that above example is made by technical spirit, all still falls within the technology of the present invention side
The protection domain of case.
Claims (7)
1. whether a functional Monascus adds the discrimination method of lovastatin, it is characterised in that comprise the following steps:
(1) formulation of standard curve: with Monacolin K, lovastatin as raw material, respectively n different detection sample of preparation, wherein
In detection sample, lovastatin mass percent is 0-100%；Detection sample injects the detached dowel of HPLC pretreatment with methanol after dissolving
In；Use isotope ratio mass spectrometer, elemental analyser, measure δ in detection sample13C value；With the mass percent of lovastatin it is
Abscissa, with δ13C value is vertical coordinate, each δ that will record13C value, prepares δ13C value and the mass percent relation of lovastatin
Standard curve A；
(2) functional Monascus treats that test sample differentiates: functional Monascus being treated, the Monacolin K in test sample and lovastatin carry
Take, obtain extract；Extract injects after dissolving with methanol in the detached dowel of HPLC pretreatment；Use isotope ratio mass spectrum
Instrument, elemental analyser, measure the δ of extract13C value；
(3) by the δ of extract13C value is updated in standard curve A, tries to achieve functional Monascus and treats that in the extract of test sample, him is cut down in Lip river
The mass percent in spit of fland.
Whether a kind of functional Monascus the most as claimed in claim 1 adds the discrimination method of lovastatin, it is characterised in that
N described in step (1) is the natural number more than or equal to 3.
Whether a kind of functional Monascus the most as claimed in claim 2 adds the discrimination method of lovastatin, it is characterised in that
N described in step (1) is 10, and respectively in detection sample, the mass percent of lovastatin is respectively 0%, 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 100%.
Whether a kind of functional Monascus the most as claimed in claim 2 adds the discrimination method of lovastatin, it is characterised in that
N described in step (1) is 6, and respectively in detection sample, the mass percent of lovastatin is respectively 0%, 26%, 40%, 57.5%,
73%, 100%.
5. whether adding the discrimination method of lovastatin in a kind of functional Monascus as described in one of claim 1-4, it is special
Levy and be, in step (1), measure δ in detection sample simultaneously2H-number；With the mass percent of lovastatin as abscissa, with δ2H
Value is vertical coordinate, each δ that will record2H-number, prepares δ2The standard curve B of the mass percent relation of H-number and lovastatin；?
In step (2), measure the δ of extract simultaneously2H-number；In step (3), by the δ of extract2H-number is updated in standard curve B,
Try to achieve functional Monascus and treat the mass percent of lovastatin in the extract of test sample.
6. whether adding the discrimination method of lovastatin in a kind of functional Monascus as described in one of claim 1-4, it is special
Levying and be, described in step (2), the extracting method of extract is:
Functional Monascus is treated, and test sample is crushed to 40 mesh and is sufficiently mixed uniformly, weighs 400.0-600.0 mg in 50 mL capacity
In Ping, add 75vol% ethanol 30mL, shake up, ultrasonic 40-60 min under room temperature, add 75 vol % ethanol and carve to close to volumetric flask
Degree, after shaking up, the most ultrasonic 8-12 min, is cooled to room temperature afterwards, is settled to 50mL with 75vol% ethanol, with 3000-4000rpm
Rotary speed be centrifuged 8-12 min, remove insoluble residue, take the filtering with microporous membrane that supernatant is through 0.45 micron, filtrate is dried
Rear prepared extract.
7. whether adding the discrimination method of lovastatin in a kind of functional Monascus as described in one of claim 1-4, it is special
Levy and be, described detection sample or extract methanol dissolve after by injecting in the detached dowel of 2mL to HPLC pretreatment, described point
From post specification it is: kromaton, Annonay F 21*250mm, 5 μm.
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Cited By (1)
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| CN108169381A (en) * | 2018-01-26 | 2018-06-15 | 昆明理工大学 | The detection method of drug is illegally added in a kind of reducing blood lipid class Chinese patent drug and health food |
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| CN1985858A (en) * | 2005-12-19 | 2007-06-27 | 北京维信学知科技发展有限公司 | Quality detection method for red yeast medicine |
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