CN104698116B - A kind of method measuring Camp Content in red date - Google Patents
A kind of method measuring Camp Content in red date Download PDFInfo
- Publication number
- CN104698116B CN104698116B CN201510120057.XA CN201510120057A CN104698116B CN 104698116 B CN104698116 B CN 104698116B CN 201510120057 A CN201510120057 A CN 201510120057A CN 104698116 B CN104698116 B CN 104698116B
- Authority
- CN
- China
- Prior art keywords
- red date
- camp
- ultrasonic
- extraction
- camp content
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a kind of method measuring Camp Content in red date, comprise the steps: the extraction of CAMP in (1) red date; (2) high-performance liquid chromatogram determination.The method of the present invention to conventional determining red date CAMP is improved, and improves the extraction ratio of CAMP in red date, and the extraction rate in conventional method 85% is increased to more than 95%; Comprehensive resource utilization rate is high, and due to the significantly minimizing of solvent consumption, in leaching liquor, Camp Content is high, and later stage concentrated cost reduces greatly, environmental protection; The recovery is 109%, and precision is higher, and result is accurate, and method feasibility is high.
Description
Technical field
The present invention relates to extraction and the assay field of effective ingredients in plant, especially a kind of method measuring Camp Content in red date.
Background technology
CAMP (cyclicadenosinemonophosphate is called for short cAMP) molecular formula is: C
10h
12n
5o
6pH
2o, hydrate is off-white color or pale yellow crystals, is slightly soluble in water, is insoluble to ethanol or ether; Fusing point is 219 ~ 220 DEG C, and specific rotation is-51.3 ° (C=0.67); It is to acid, alkali, heat all quite stables.CAMP is a kind of important biomolecule active substance in human body, it is intermediary's factor of intracellular delivery hormone and mediator, and the adjustment of multiple physiological and biochemical procedure is participated in as second messenger, there is significant curative effect to diseases such as miocardial infarction, coronary heart disease, cardiogenic shock, psoriasiss.CAMP is found in nineteen fifty-seven the earliest by Sutherland from mammalian tissues, and Sutherland is because finding that cAMP obtained physiology and the medical science Nobel Prize in 1971 years.According to the literature, in jujube, cAMP content is the highest in animals and plants surveying, and is general vegeto-animal thousands of to tens thousand of times.Thus in jujube, CAMP has much value of exploiting and utilizing.
Supercritical fluid (Supercriticalfluid, SCF) as being in higher than the pure material under the temperature and pressure condition of critical point, be found in 1822, application the earliest then can be traced back to 1970 Nian Mapu research institutes and be used supercritical carbon dioxide from coffee bean, extract caffeine and extract picric acid from hops, from then on the physical property having started the development prelude SCF of SCF special is and density like liquid type, with viscosity like gas, there is no the Modulatory character of interfacial tension and density.The viscosity that SCF is less than liquid, has higher material coefficient of diffusion.Do not have interfacial tension to make to penetrate into better in three-phase system in the middle of the gap of solid particle, improve permeance property, thus improve the efficiency of extraction.In SCF fluid, the condition ratio of supercritical carbon dioxide (supercriticalCarbonDioxide, SCCD) is easier to realize Tc=39.5 DEG C, Pc=72.8atm), thus conventional, be particularly suitable for scale and use.For carbon dioxide, the temperature and pressure of critical point is moderate, easily reaches, and can not destroy the build-in attribute of extracting substance simultaneously.By the control to temperature and pressure, selective extraction can be carried out according to the difference of composition.
In recent years, many scholars have carried out positive exploration and research to the extractive technique of CAMP in red date and assay method.But it is long and incomplete to there is extraction time in the method measuring CAMP in red date at present, and complex operation, the method finding CAMP in a kind of rapid and accurate determination red date is the Focal point and difficult point of the research of this area.Supercritical ultrasonics technology is that (ultrasonic assistant enzyme process extracts the optimization of process conditions of wooden jujube CAMP to a kind of novel effective Chinese herbal medicine extraction process of active component, food science and technology, 2013,7 (38): 220-224), beneficial also widespread use in supercritical carbon dioxide extracting red date, see Chinese patent, denomination of invention: a kind of high-content jujube cyclic adenosine monophosphate extract and preparation method thereof, application number: 201310443550.6, and adopt ultrasonic assistant supercritical carbon dioxide process extract the method for jujube fruit CAMP and measure its content, there is not been reported.
Summary of the invention
Object of the present invention is just to provide a kind of method measuring Camp Content in red date to solve the problem, and it can measure the content of CAMP in red date fast and accurately, and simple to operate, quick, content is accurate.
The present invention is achieved through the following technical solutions above-mentioned purpose:
A kind of method measuring Camp Content in red date of the present invention, is characterized in that, comprise following operation steps:
(1) extraction of CAMP in red date:
(a) pre-service: the red date jujube fruit containing moisture is put into 50 DEG C of dry 72h of air dry oven, then stoning, pulverizing, and crosses 40-60 mesh sieve;
B () ultrasonic overcritical lixiviate: accurately take dry jujube powder 5.0000g, loads in the ultrasonic supercritical fluid extraction still with Vltrasonic device and uses CO
2carry out low temperature supercritical fluid extraction, then the concentration adding powder weight 2-5 times is 5%-10% alcohol extraction liquid, closing kettle gland bonnet, is the CO of 99.8% by purity
2control critical temperature 25-30 DEG C, emergent pressure is 3.5-5.5MPa, CO
2flow 10kg/h, extraction time 1.5-2.5h, ultrasonic time 0.5-1.0h, ultrasonic power: 600w, frequency: 18.0Hz-22.0Hz, ultrasonic 1s, gap 1s;
(c) centrifuging: step (b) is loaded centrifuging in refrigerated centrifuge, rotating speed: 4000r/min, disengaging time: 10min; Temperature: 4 DEG C, get supernatant, washing filter residue, is incorporated to supernatant, is concentrated into 50mL after filtration, for subsequent use.
(2) high-performance liquid chromatogram determination:
(a) CAMP Specification Curve of Increasing:
Accurately take 5.0mgcAMP standard items, dissolve with ultrapure water, then being mixed with concentration is respectively 1 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL, 50 μ g/mL standard solutions, 15 μ L injection liquid chromatographies are drawn with microsyringe, peak area is recorded by after chromatogram integration, with standard concentration (μ g/mL) for ordinate, peak area is horizontal ordinate, drawing standard curve.
The mensuration of (b) sample Camp Content:
By the extract of step (1) through 0.22 μm of membrane filtration, draw 15 μ L with microsyringe and inject high performance liquid chromatograph, record peak area by after chromatogram integration, external standard method calculates, and both obtains the content of sample loop AMP.
Further, choosing water cut in described step (1) at the jujube of 5-15% is raw material.
Further, in described step (1), (b) concentration of alcohol is 6-8%.
Further, (b) time 26-28 DEG C in described step (1).
Further, (b) emergent pressure 4.0-5.0MPa in described step (1).
Further, in described step (1), (b) extraction time is 2.0h.
Further, in described step (1), (b) ultrasonic frequency is 19.5-21.0Hz.
Further, in described step (2), (b) measures according to following chromatographic condition: chromatographic column: ThermoC18 post, 4.6mm × 250mm, 5 μm; Mobile phase: 0.05mol/L potassium dihydrogen phosphate: methyl alcohol volume ratio is 90:10, mobile phase through 0.45 μm of membrane filtration, ultrasonic degas, isocratic elution; Flow velocity: 1ml/min; Sample size: 15 μ L; Column temperature: 30 DEG C; Determined wavelength: 258nm or 268nm or 284nm or 312nm.
Compared with prior art, its useful technique effect is in the present invention:
The method of the present invention to conventional determining red date CAMP is improved, and improves the extraction ratio of CAMP in red date, the extraction rate in conventional method 85% is increased to more than 95%; Comprehensive resource utilization rate is high, and due to the significantly minimizing of solvent consumption, in leaching liquor, Camp Content is high, and later stage concentrated cost reduces greatly, environmental protection; Under chromatographic condition of the present invention, analyze CAMP retention time is 9.113min, and what contrast standard product and sample chromatogram figure retention time can affirm sample determination is exactly CAMP.Noiseless by calculating degree of separation R>>1.5 impurity, and method feasibility is high; In the corresponding range of linearity, various types of linear relationship is good, and the coefficient of determination all reaches more than 0.999, and relative standard deviation (RSD) is 1%, and the recovery is 109%, and precision is higher, and result is accurate, and method feasibility is high.
Accompanying drawing explanation
Fig. 1 is CAMP typical curve.
Embodiment
Below in conjunction with the drawings and specific embodiments, the invention will be further described:
1, raw material and medicine
New fresh date jujube fruit, jujube base, Cangzhou, Hebei; Ultrapure water, self-control; Methyl alcohol, chromatographically pure, Di Ma company; Potassium dihydrogen phosphate, chromatographically pure, Sigma company; CAMP standard items, chromatographically pure, 99.95%, Sigma company.
2, instrument and equipment
SFE-2 supercritical extract instrument, ASI company of the U.S.; JY92-II ultrasonic cell disruptor, NingBo XinZhi Biology Science Co., Ltd; High performance liquid chromatograph: Agilent1260, comprises online degasser, quaternary pump, automatic sampler, column oven, UV-detector, chromatographic work station; Chromatographic column: ThermoC18 chromatographic column (4.6 × 250mm, 5 μm); ML204 analytical balance 0.0001g, plum Teller-Tuo benefit Instrument Ltd.; AP-01P vacuum pump, Tianjin Ao Tesaisi Instrument Ltd.; AKHL-III-24A Ai Ke laboratory ultrapure water instrument, Chengdu Ai Ke Water Management Equipment Ltd.; GL-20G-II type hydro-extractor, Anting Scientific Instrument Factory, Shanghai; Multifunctional crusher, Beijing is bright Medical Instruments factory forever; HDS-D120B-D type Multifunctional dryer, Liaoning Hai Disheng Machinery Co., Ltd..
Embodiment 1
Measure a method for Camp Content in red date, comprise following operation steps:
(1) extraction of CAMP in red date:
(a) pre-service: water cut 5% red date jujube fruit is put into 50 DEG C of dry 72h of air dry oven, then stoning, pulverizing, and crosses 40 mesh sieves;
B () ultrasonic overcritical lixiviate: accurately take dry jujube powder 5.0000g, loads in the ultrasonic supercritical fluid extraction still with Vltrasonic device and uses CO
2carry out low temperature supercritical fluid extraction, then the concentration adding powder weight 2 times is 5% alcohol extraction liquid, closing kettle gland bonnet, is the CO of 99.8% by purity
2control critical temperature 25 DEG C, emergent pressure is 3.5MPa, CO
2flow 10kg/h, extraction time 1.5h, ultrasonic time 0.5, ultrasonic power 600w, frequency 18.0Hz, ultrasonic 1s, gap 1s;
(c) centrifuging: step (b) is loaded centrifuging in refrigerated centrifuge, rotating speed: 4000r/min, disengaging time: 10min; Temperature: 4 DEG C, get supernatant, washing filter residue, is incorporated to supernatant, is concentrated into 50mL after filtration, for subsequent use;
(2) high-performance liquid chromatogram determination:
(a) CAMP Specification Curve of Increasing:
Accurately take 5.0mgcAMP standard items, dissolve with ultrapure water, then being mixed with concentration is respectively 1 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL, 50 μ g/mL standard solutions, 15 μ L injection liquid chromatographies are drawn with microsyringe, peak area is recorded by after chromatogram integration, with standard concentration (μ g/mL) for ordinate, peak area is horizontal ordinate, and drawing standard curve is as accompanying drawing 1.
Know that typical curve is: y=2e-06X-0.6802, coefficient R by upper figure
2=0.9991.Show that cAMP is in 1.0 μ g/mL-50.0 μ g/mL concentration ranges, chromatographic peak area and sample size linear.
This experiment adopts sample mark-on to reclaim, take two increment product of equal in quality, take the jujube powder of 1.0000g drying respectively, portion adds 0.70mg CAMP standard items wherein, according to the sample that disposal methods of the present invention two parts is identical, measure the content of its CAMP.
Result is (sample concentration mg/kg), non-mark-on sample: 396.221; Mark-on sample: 1158.346.Following formula is used to calculate recovery of standard addition:
Recovery of standard addition=(mark-on Specimen Determination value-Specimen Determination value) ÷ adds scalar × 100%;
Recovery of standard addition is: 109%.In the experiment that sample mark-on reclaims according to the rules, the amount general control adding standard items is 0.5-2.0 times of contained material in sample, and this adds 1.7667 times that CAMP mark product amount is Camp Content in dry jujube powder.
The mensuration of (b) sample Camp Content:
By the extract of step (1) through 0.22 μm of membrane filtration, draw 15 μ L with microsyringe and inject high performance liquid chromatograph, peak area is recorded by after chromatogram integration, external standard method calculates, both the content of sample loop AMP had been obtained, chromatographic condition measures: chromatographic column: ThermoC18 post, 4.6mm × 250mm, 5 μm; Mobile phase: 0.05mol/L potassium dihydrogen phosphate: methyl alcohol volume ratio is 90:10, mobile phase through 0.45 μm of membrane filtration, ultrasonic degas, isocratic elution; Flow velocity: 1ml/min; Sample size: 15 μ L; Column temperature: 30 DEG C; Determined wavelength: 258nm.
Embodiment 2
Measure a method for Camp Content in red date, comprise following operation steps:
(1) extraction of CAMP in red date:
(a) pre-service: water cut 15% red date jujube fruit is put into 50 DEG C of dry 72h of air dry oven, then stoning, pulverizing, and crosses 60 mesh sieves;
B () ultrasonic overcritical lixiviate: accurately take dry jujube powder 5.0000g, loads in the ultrasonic supercritical fluid extraction still with Vltrasonic device and uses CO
2carry out low temperature supercritical fluid extraction, then the concentration adding powder weight 5 times is 10% alcohol extraction liquid, closing kettle gland bonnet, is the CO of 99.8% by purity
2control critical temperature 30 DEG C, emergent pressure is 5.5MPa, CO
2flow 10kg/h, extraction time 2.5h, ultrasonic time 1.0h, ultrasonic power: 600w, frequency: 22.0Hz, ultrasonic 1s, gap 1s;
(c) centrifuging: step (b) is loaded centrifuging in refrigerated centrifuge, rotating speed: 4000r/min, disengaging time: 10min; Temperature: 4 DEG C, get supernatant, washing filter residue, is incorporated to supernatant, is concentrated into 50mL after filtration, for subsequent use.
(2) high-performance liquid chromatogram determination:
(a) CAMP Specification Curve of Increasing:
Accurately take 5.0mgcAMP standard items, dissolve with ultrapure water, then being mixed with concentration is respectively 1 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL, 50 μ g/mL standard solutions, 15 μ L injection liquid chromatographies are drawn with microsyringe, peak area is recorded by after chromatogram integration, with standard concentration (μ g/mL) for ordinate, peak area is horizontal ordinate, drawing standard curve.
The mensuration of (b) sample Camp Content:
By the extract of step (1) through 0.22 μm of membrane filtration, draw 15 μ L with microsyringe and inject high performance liquid chromatograph, record peak area by after chromatogram integration, external standard method calculates, and both obtains the content of sample loop AMP.Chromatographic condition measures: chromatographic column: ThermoC18 post, 4.6mm × 250mm, 5 μm; Mobile phase: 0.05mol/L potassium dihydrogen phosphate: methyl alcohol volume ratio is 90:10, mobile phase through 0.45 μm of membrane filtration, ultrasonic degas, isocratic elution; Flow velocity: 1ml/min; Sample size: 15 μ L; Column temperature: 30 DEG C; Determined wavelength: 312nm.
Embodiment 3
Measure a method for Camp Content in red date, it is characterized in that, comprise following operation steps:
(1) extraction of CAMP in red date:
(a) pre-service: water cut 8% red date jujube fruit is put into 50 DEG C of dry 72h of air dry oven, then stoning, pulverizing, and crosses 40-60 mesh sieve;
B () ultrasonic overcritical lixiviate: accurately take dry jujube powder 5.0000g, loads in the ultrasonic supercritical fluid extraction still with Vltrasonic device and uses CO
2carry out low temperature supercritical fluid extraction, then the concentration adding powder weight 2-5 times is 6% alcohol extraction liquid, closing kettle gland bonnet, is the CO of 99.8% by purity
2control critical temperature 26 DEG C, emergent pressure is 4.0MPa, CO
2flow 10kg/h, extraction time 2.0h, ultrasonic time 0.8h, ultrasonic power: 600w, frequency: 19.0Hz, ultrasonic 1s, gap 1s;
(c) centrifuging: step (b) is loaded centrifuging in refrigerated centrifuge, rotating speed: 4000r/min, disengaging time: 10min; Temperature: 4 DEG C, get supernatant, washing filter residue, is incorporated to supernatant, is concentrated into 50mL after filtration, for subsequent use.
(2) high-performance liquid chromatogram determination:
(a) CAMP Specification Curve of Increasing:
Accurately take 5.0mgcAMP standard items, dissolve with ultrapure water, then being mixed with concentration is respectively 1 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL, 50 μ g/mL standard solutions, 15 μ L injection liquid chromatographies are drawn with microsyringe, peak area is recorded by after chromatogram integration, with standard concentration (μ g/mL) for ordinate, peak area is horizontal ordinate, drawing standard curve.
The mensuration of (b) sample Camp Content:
By the extract of step (1) through 0.22 μm of membrane filtration, draw 15 μ L with microsyringe and inject high performance liquid chromatograph, record peak area by after chromatogram integration, external standard method calculates, and both obtains the content of sample loop AMP.Chromatographic condition measures: chromatographic column: ThermoC18 post, 4.6mm × 250mm, 5 μm; Mobile phase: 0.05mol/L potassium dihydrogen phosphate: methyl alcohol volume ratio is 90:10, mobile phase through 0.45 μm of membrane filtration, ultrasonic degas, isocratic elution; Flow velocity: 1ml/min; Sample size: 15 μ L; Column temperature: 30 DEG C; Determined wavelength: 284nm.
Embodiment 4
Measure a method for Camp Content in red date, it is characterized in that, comprise following operation steps:
(1) extraction of CAMP in red date:
(a) pre-service: water cut 12% red date jujube fruit is put into 50 DEG C of dry 72h of air dry oven, then stoning, pulverizing, and crosses 50 mesh sieves;
B () ultrasonic overcritical lixiviate: accurately take dry jujube powder 5.0000g, loads in the ultrasonic supercritical fluid extraction still with Vltrasonic device and uses CO
2carry out low temperature supercritical fluid extraction, then the concentration adding powder weight 3 times is 8% alcohol extraction liquid, closes kettle gland bonnet, the CO2 being 99.8% by purity controls critical temperature 28 DEG C, and emergent pressure is 5.0MPa, CO
2flow 10kg/h, extraction time 1.8h, ultrasonic time 0.8h, ultrasonic power: 600w, frequency 21.0Hz, ultrasonic 1s, gap 1s;
(c) centrifuging: step (b) is loaded centrifuging in refrigerated centrifuge, rotating speed: 4000r/min, disengaging time: 10min; Temperature: 4 DEG C, get supernatant, washing filter residue, is incorporated to supernatant, is concentrated into 50mL after filtration, for subsequent use.
(2) high-performance liquid chromatogram determination:
(a) CAMP Specification Curve of Increasing:
Accurately take 5.0mgcAMP standard items, dissolve with ultrapure water, then being mixed with concentration is respectively 1 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL, 50 μ g/mL standard solutions, 15 μ L injection liquid chromatographies are drawn with microsyringe, peak area is recorded by after chromatogram integration, with standard concentration (μ g/mL) for ordinate, peak area is horizontal ordinate, drawing standard curve.
The mensuration of (b) sample Camp Content:
By the extract of step (1) through 0.22 μm of membrane filtration, draw 15 μ L with microsyringe and inject high performance liquid chromatograph, record peak area by after chromatogram integration, external standard method calculates, and both obtains the content of sample loop AMP.Chromatographic condition measures: chromatographic column: ThermoC18 post, 4.6mm × 250mm, 5 μm; Mobile phase: 0.05mol/L potassium dihydrogen phosphate: methyl alcohol volume ratio is 90:10, mobile phase through 0.45 μm of membrane filtration, ultrasonic degas, isocratic elution; Flow velocity: 1ml/min; Sample size: 15 μ L; Column temperature: 30 DEG C; Determined wavelength: 258nm.
Embodiment 5
Measure a method for Camp Content in red date, comprise following operation steps:
(1) extraction of CAMP in red date:
(a) pre-service: red date jujube fruit is put into 50 DEG C of dry 72h of air dry oven, then stoning, pulverizing, and crosses 55 mesh sieves;
B () ultrasonic overcritical lixiviate: accurately take dry jujube powder 5.0000g, loads in the ultrasonic supercritical fluid extraction still with Vltrasonic device and uses CO
2carry out low temperature supercritical fluid extraction, then the concentration adding powder weight 4 times is 9% alcohol extraction liquid, closing kettle gland bonnet, is the CO of 99.8% by purity
2control critical temperature 27 DEG C, emergent pressure is 4.5MPa, CO
2flow 10kg/h, extraction time 2.2h, ultrasonic time 0.8h, ultrasonic power: 600w, frequency 20.0Hz, ultrasonic 1s, gap 1s;
(c) centrifuging: step (b) is loaded centrifuging in refrigerated centrifuge, rotating speed: 4000r/min, disengaging time: 10min; Temperature: 4 DEG C, get supernatant, washing filter residue, is incorporated to supernatant, is concentrated into 50mL after filtration, for subsequent use.
(2) high-performance liquid chromatogram determination:
(a) CAMP Specification Curve of Increasing:
Accurately take 5.0mgcAMP standard items, dissolve with ultrapure water, then being mixed with concentration is respectively 1 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL, 50 μ g/mL standard solutions, 15 μ L injection liquid chromatographies are drawn with microsyringe, peak area is recorded by after chromatogram integration, with standard concentration (μ g/mL) for ordinate, peak area is horizontal ordinate, drawing standard curve.
The mensuration of (b) sample Camp Content:
By the extract of step (1) through 0.22 μm of membrane filtration, draw 15 μ L with microsyringe and inject high performance liquid chromatograph, record peak area by after chromatogram integration, external standard method calculates, and both obtains the content of sample loop AMP.Chromatographic condition measures: chromatographic column: ThermoC18 post, 4.6mm × 250mm, 5 μm; Mobile phase: 0.05mol/L potassium dihydrogen phosphate: methyl alcohol volume ratio is 90:10, mobile phase through 0.45 μm of membrane filtration, ultrasonic degas, isocratic elution; Flow velocity: 1ml/min; Sample size: 15 μ L; Column temperature: 30 DEG C; Determined wavelength: 258nm.
Embodiment 6
Measure a method for Camp Content in red date, comprise following operation steps:
(1) extraction of CAMP in red date:
(a) pre-service: be that 14% red date jujube fruit puts into 50 DEG C of dry 72h of air dry oven, then stoning, pulverizing by water cut, and cross 53 mesh sieves;
B () ultrasonic overcritical lixiviate: accurately take dry jujube powder 5.0000g, loads in the ultrasonic supercritical fluid extraction still with Vltrasonic device and uses CO
2carry out low temperature supercritical fluid extraction, then the concentration adding powder weight 3.5 times is 6% alcohol extraction liquid, closing kettle gland bonnet, is the CO of 99.8% by purity
2control critical temperature 29 DEG C, emergent pressure is 4.2MPa, CO
2flow 10kg/h, extraction time 1.8h, ultrasonic time 0.6h, ultrasonic power: 600w, frequency 20.5Hz, ultrasonic 1s, gap 1s;
(c) centrifuging: step (b) is loaded centrifuging in refrigerated centrifuge, rotating speed: 4000r/min, disengaging time: 10min; Temperature: 4 DEG C, get supernatant, washing filter residue, is incorporated to supernatant, is concentrated into 50mL after filtration, for subsequent use.
(2) high-performance liquid chromatogram determination:
(a) CAMP Specification Curve of Increasing:
Accurately take 5.0mgcAMP standard items, dissolve with ultrapure water, then being mixed with concentration is respectively 1 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL, 50 μ g/mL standard solutions, 15 μ L injection liquid chromatographies are drawn with microsyringe, peak area is recorded by after chromatogram integration, with standard concentration (μ g/mL) for ordinate, peak area is horizontal ordinate, drawing standard curve.
The mensuration of (b) sample Camp Content:
By the extract of step (1) through 0.22 μm of membrane filtration, draw 15 μ L with microsyringe and inject high performance liquid chromatograph, record peak area by after chromatogram integration, external standard method calculates, and both obtains the content of sample loop AMP.Chromatographic condition measures: chromatographic column: ThermoC18 post, 4.6mm × 250mm, 5 μm; Mobile phase: 0.05mol/L potassium dihydrogen phosphate: methyl alcohol volume ratio is 90:10, mobile phase through 0.45 μm of membrane filtration, ultrasonic degas, isocratic elution; Flow velocity: 1ml/min; Sample size: 15 μ L; Column temperature: 30 DEG C; Determined wavelength: 258nm.
Above-described embodiment is preferred embodiment of the present invention; it is not the restriction to technical solution of the present invention; as long as without the technical scheme that creative work can realize on the basis of above-described embodiment, all should be considered as falling within the scope of the rights protection of patent of the present invention.
Claims (8)
1. measure a method for Camp Content in red date, it is characterized in that, comprise following operation steps:
(1) extraction of CAMP in red date:
(a) pre-service: the red date jujube fruit containing moisture is put into 50 DEG C of dry 72h of air dry oven, then stoning, pulverizing, and crosses 40-60 mesh sieve;
B () ultrasonic overcritical lixiviate: accurately take dry jujube powder 5.0000g, loads in the ultrasonic supercritical fluid extraction still with Vltrasonic device and uses CO
2carry out low temperature supercritical fluid extraction, then the concentration adding powder weight 2-5 times is 5%-10% alcohol extraction liquid, closing kettle gland bonnet, is the CO of 99.8% by purity
2control critical temperature 25-30 DEG C, emergent pressure is 3.5-5.5MPa, CO
2flow 10kg/h, extraction time 1.5-2.5h, ultrasonic time 0.5-1.0h, ultrasonic power: 600w, frequency: 18.0Hz-22.0Hz, ultrasonic 1s, gap 1s;
(c) centrifuging: step (b) is loaded centrifuging in refrigerated centrifuge, rotating speed: 4000r/min, disengaging time: 10mim; Temperature: 4 DEG C, get supernatant, washing filter residue, is incorporated to supernatant, is concentrated into 50mL after filtration, for subsequent use.
(2) high-performance liquid chromatogram determination:
(a) CAMP Specification Curve of Increasing:
Accurately take 5.0mgcAMP standard items, dissolve with ultrapure water, then being mixed with concentration is respectively 1 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL, 50 μ g/mL standard solutions, 15 μ L injection liquid chromatographies are drawn with microsyringe, recording peak area by after chromatogram integration, take standard concentration as ordinate, and the unit of concentration is μ g/mL, peak area is horizontal ordinate, drawing standard curve.
The mensuration of (b) sample Camp Content:
By the extract of step (1) through 0.22 μm of membrane filtration, draw 15 μ L with microsyringe and inject high performance liquid chromatograph, record peak area by after chromatogram integration, external standard method calculates, and both obtains the content of sample loop AMP.
2. the method for Camp Content in mensuration red date according to claim 1, is characterized in that: choosing water cut in described step (1) at the jujube of 5-15% is raw material.
3. the method for Camp Content in mensuration red date according to claim 1, is characterized in that: in described step (1), (b) concentration of alcohol is 6-8%.
4. the method for Camp Content in mensuration red date according to claim 1, is characterized in that: (b) time 26-28 DEG C in described step (1).
5. the method for Camp Content in mensuration red date according to claim 1, is characterized in that: (b) emergent pressure 4.0-5.0MPa in described step (1).
6. the method for Camp Content in mensuration red date according to claim 1, is characterized in that: in described step (1), (b) extraction time is 2.0h.
7. the method for Camp Content in mensuration red date according to claim 1, is characterized in that: in described step (1), (b) ultrasonic frequency is 19.5-21.0Hz.
8. the method for Camp Content in mensuration red date according to claim 1, it is characterized in that: in described step (2), (b) measures according to following chromatographic condition: chromatographic column: ThermoC18 post, 4.6mm × 250mm, 5 μm; Mobile phase: 0.05mol/L potassium dihydrogen phosphate: methyl alcohol volume ratio is 90:10, mobile phase through 0.45 μm of membrane filtration, ultrasonic degas, isocratic elution; Flow velocity: 1ml/min; Sample size: 15 μ L; Column temperature: 30 DEG C; Determined wavelength: 258nm or 268nm or 284nm or 312nm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510120057.XA CN104698116B (en) | 2015-03-18 | 2015-03-18 | A kind of method measuring Camp Content in red date |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510120057.XA CN104698116B (en) | 2015-03-18 | 2015-03-18 | A kind of method measuring Camp Content in red date |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104698116A CN104698116A (en) | 2015-06-10 |
| CN104698116B true CN104698116B (en) | 2015-11-18 |
Family
ID=53345459
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510120057.XA Expired - Fee Related CN104698116B (en) | 2015-03-18 | 2015-03-18 | A kind of method measuring Camp Content in red date |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104698116B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108956527B (en) * | 2018-07-24 | 2020-12-15 | 新疆维吾尔自治区产品质量监督检验研究院 | Method for rapidly detecting cyclic adenosine monophosphate cAMP content in red dates |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109633062B (en) * | 2018-12-21 | 2019-08-02 | 河南农业大学 | The isolation and identification method of cAMP synthetic reaction system ingredient and its application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103623080A (en) * | 2013-09-25 | 2014-03-12 | 中恩（天津）医药科技有限公司 | High-content jujube cyclic adenosine monophosphate extract and preparation method thereof |
-
2015
- 2015-03-18 CN CN201510120057.XA patent/CN104698116B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103623080A (en) * | 2013-09-25 | 2014-03-12 | 中恩（天津）医药科技有限公司 | High-content jujube cyclic adenosine monophosphate extract and preparation method thereof |
Non-Patent Citations (4)
| Title |
|---|
| Content variations of triterpenic acid, nucleoside, nucleobase, and sugar in jujube (Ziziphus jujuba) fruit during ripening;Guo, Sheng; Duan, Jin-ao; Qian, Dawei;《FOOD CHEMISTRY》;20140711;第167卷;468-474 * |
| 不同品种红枣中三萜酸及环核苷酸的测定;高娅， 杨洁， 杨迎春， 苏力坦·阿巴白克力;《中成药》;20121031;第34卷(第10期);1961-1965 * |
| 枣果中环磷酸腺苷(cAMP)的提取工艺及含量测定;张明娟,李薇,庞晓明;《食品与发酵工业》;20120531;第38卷(第5期);228-231 * |
| 超声强化超临界流体萃取机理的研究;胡爱军 等;《高校化学工程学报》;20051031;第19卷(第5期);583-587 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108956527B (en) * | 2018-07-24 | 2020-12-15 | 新疆维吾尔自治区产品质量监督检验研究院 | Method for rapidly detecting cyclic adenosine monophosphate cAMP content in red dates |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104698116A (en) | 2015-06-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100483131C (en) | Method for investigating quality of characterization natural cordyceps sinensis character | |
| CN105111255A (en) | Extraction and separation method for echinacoside and verbascoside in cistanche | |
| CN102928526A (en) | Method for analyzing content of adenosine and cordycepin in cordyceps militaris by virtue of high performance liquid chromatography (HPLC) | |
| CN104698116B (en) | A kind of method measuring Camp Content in red date | |
| CN109503323A (en) | A kind of preprocess method extracting natural d-borneol from Borneolum leaf piece | |
| CN109030678A (en) | The content assaying method of 5 kinds of chemical components in a kind of northeast Chinese Ixeris | |
| CN104730009A (en) | Method for measuring polysaccharide content in tea flower | |
| CN102743598B (en) | var. Denneanum extract and formula particles thereof | |
| CN110261512B (en) | Metabonomics-based quality evaluation method for Uighur coreopsis tinctoria | |
| CN104119433B (en) | A kind of snakegourd anti-tumor active protein and its preparation method and application | |
| CN109134583A (en) | The method of triterpene compound is extracted from schefflera leaf using ionic liquid | |
| CN113267578A (en) | Quality control method of peony and licorice decoction | |
| CN104523820A (en) | Low-temperature dynamic extraction process of sophora alopecuroide alkaloid extract | |
| CN104013703A (en) | Gardenia extract and preparation method thereof | |
| CN103512975A (en) | Method for analyzing contents of effective substances in Cordyceps martialis fruiting body and residue by HPLC | |
| CN103784481B (en) | A kind of method and application thereof extracting antitumor component from Cordyceps militaris (L.) Link. | |
| CN104894206A (en) | Method for enriching huperzine A through huperzia serrata cell and endophytic fungi coculture | |
| Li et al. | Application of fingerprint combined with quantitative analysis and multivariate chemometric methods in quality evaluation of dandelion (Taraxacum mongolicum) | |
| CN110487938B (en) | Quality control method of podocarpus macrophyllus kurz medicinal material | |
| CN102628838B (en) | Fujian herb cusia HPLC fingerprint construction method | |
| CN101732431A (en) | Method for preparing main ester alkaloid of traditional Chinese medicine monkshood by liquid chromatography separation | |
| CN103323551A (en) | Method for detecting content of medlar acid | |
| Chen et al. | Comparative chemical and statistical analysis of cultivated and wild Radix Scutellariae | |
| CN103623240A (en) | Dendrobe processing method and technical screening method of dendrobe | |
| CN114034798A (en) | Red water dendrobium stem flower fingerprint construction and content determination method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Meng Yina Inventor after: Zhang Jian Inventor after: Xu Mingqiang Inventor after: Zhao Xin Inventor after: Zhou Xiaoming Inventor after: Over Limin Inventor after: Miao Haocui Inventor after: Zhang Ping Inventor after: Zhang Qian Inventor after: Ma Yan Inventor after: Zou Shuping Inventor before: Meng Yina Inventor before: Zhao Xin Inventor before: Zhou Xiaoming Inventor before: Zhang Ping Inventor before: Zhang Qian Inventor before: Zou Shuping Inventor before: Ma Yan Inventor before: Zhang Jian Inventor before: Xu Mingqiang |
|
| COR | Change of bibliographic data | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20151118 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |