CN104483424B - Rubusoside quality control method - Google Patents
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Abstract
The invention discloses a preparation method and a quality control method of high-purity rubusoside by use of rubusoside rosaceae rubus plants as raw materials. The method is characterized in that the method comprises the steps of extracting rubusoside plants, adsorbing with a macroporous resin column, detecting through the HPLC (High Performance Liquid Chromatography), collecting the flow ingredient containing rubusoside, separating by a reverse-phase preparative high-performance liquid phase method, detecting each part of the collected eluant by use of analytical type HPLC, mixing and keeping the high-purity rubusoside component, and carrying out quality control by the thin-layer chromatography and the liquid chromatogram. According to the method, the purity of the rubusoside can be improved and the production cost can be reduced.
Description
Technical field
The present invention relates to a kind of method of quality control field of medicinal material and in particular to a kind of Sweet tea element quality control side
Method.
Background technology
Sweet tea element is a kind of Diterpene glucoside class chemical composition, is one of active components of plants, is also many plants and medicine
The index composition that standard quality controls.Not yet corresponding national drug standards material at present, both at home and abroad to Sweet tea element reference substance
System research have no report, with reference to the technical requirements of traditional Chinese chemical contrast (for assay), right to Sweet tea element chemistry
Studied according to product, set up the analysis of the batch extracting technique, purity and content and determination of foreign matter of Sweet tea element chemical reference substance
Assay method, thus setting up the technical standard of Sweet tea element chemical reference substance, for its as traditional Chinese chemical contrast and medicinal material and
The quality standard research of preparation provides scientific basic and guarantee.
Highly purified Sweet tea element reference substance, is the key problem in technology controlling plant Sweet tea and the quality containing Sweet tea, numerous enterprises,
Scientific research and testing department are required for highly purified Sweet tea element reference substance, and its market demand is very big, because Sweet tea element is in medicinal material
Content is low, and extraction and separation technology requirement is very high, difficulty is very big.The present invention carries out Sweet tea element Chemistry for Chinese Traditional Medicine standard items preparation and its matter
Amount control technology research, solves the problems, such as high-purity Sweet tea element chemical reference substance, the effect to the modernization of Chinese medicine is apparent
, there is great practical significance and learning value.
Sweet tea element is to separate, from sweet tea plant, the active material obtaining, and has the extracting method of document report Sweet tea element,
As: 1.[autograph] application in purifying Rubusoside technique for the preparative chromatography: have studied the system purifying Rubusoside from sweet tea
Standby technique.Extract Rubusoside with boiling water from sweet tea first, extract 3 times, each 1h, recycle hp-20 macroporous absorbent resin
Prepurification is carried out to Rubusoside crude extract, with 90% ethanol as eluent, elutes 6 times of column volumes；Concentrate after removing ethanol, use d-
941 anion exchange resin decolourings, wash and are dried to obtain Rubusoside crude product, and purity is 68.6%.Last Rubusoside slightly produces
Product through preparative chromatography after purification, obtain the Rubusoside that purity is 97.9%, and chromatographic condition is mobile phase vMethyl alcohol:vWater=70:30, stream
Fast 10ml/min, applied sample amount 200mg.2.[inscribes one's name] the extraction process screening of Sweet tea element in the Yao Shan Sweet tea of Guangxi: preferably Guangxi precious jade
The optimum extraction process of Sweet tea element in the Sweet tea of mountain.With orthogonal experiment contrived experiment, it is measured using high-efficient liquid phase technique, with
In extract, the content of Sweet tea element is screened for index.3.[inscribes one's name] research of Guangxi Folium hydrangeae strigosae glycosides post purifying process: with containing 70%
Guangxi Folium hydrangeae strigosae CE be raw material, by post purify with recrystallize Rubusoside is purified, thus preparing highly purified
Rubusoside monomer.4.[inscribe one's name] macroporous absorbent resin extract Rubusoside research: screened suitable proposing from 6 kinds of macroreticular resins
Take the adsorbent of separating sweet tea glycosides, have studied the technique that macroporous absorbent resin extracts Rubusoside, including the quality of Rubusoside solution
Concentration, the impact to adsorption process of ph value and flow velocity, desorb the impact to desorption process of chaste tree and desorption temperature.5.[inscribes one's name] utilize
" Guangxi Folium hydrangeae strigosae " produces the new technology of 98% Rubusoside: and Guangxi Folium hydrangeae strigosae leaf → pulverizing → two part solvent set carries → and vacuum decompression is dense
Contracting → water soluble chitosan flocculation clarification → efficient centrifugal separation → Macroporous Adsorption Resin → ion-exchange resin decolorization, carry
Pure → desalination resin desalination, de- bitter and purification → composite decoloration → vacuum-concentrcted → be spray-dried → produce >=98%
Rubusoside.6.[inscribes one's name] the plain development of Sweet tea: the process → extraction → concentration → adsorbing separation → concentration → spraying of Sweet tea raw material
→ packaging is dried.The preparation of 7.[glycosides in high purity] with sweet tea as raw material, through means such as water extraction, column chromatography, recrystallizations, make
Standby go out monomer, and with fusing point, tlc, liquid chromatogram, purity detecting is carried out to it, infrared spectrum, nuclear magnetic resonance carry out structural characterization,
Show that this change house thing is glycosides.
The extraction that said method discusses Sweet tea element from different perspectives separates, and separation purity is higher, but purity is all not up to
The requirement of traditional Chinese chemical contrast, that is, more than 98%, separation process is loaded down with trivial details for purity, and does not have Sweet tea element purity and quality control side
Method is it is impossible to meet the needs of high-purity Sweet tea element chemical reference substance.
Content of the invention
The purpose of the present invention in order to overcome the shortcomings of that existing Sweet tea element purity is low, yield is low or production cost is high, and
No system Sweet tea element extraction purification and method of quality control, provides a kind of preparation of high-purity Sweet tea element and its quality control side
Method, the Sweet tea element purity that the method prepares is high, quality is good, can be used as chemical reference substance or standard items, quality control side
Method is effective, stable, homogeneous.
Crude drug source of the present invention:
Sweet tea: the leaf of rose rose family rubus plant Sweet tea rubus suavissirnus s.lee.
Sweet tea element: from Sweet tea leaf extracting and developing, refined, purify and be obtained, English name: rubusoside.
Molecular formula: c32h50o13；
Structural formula:
The method of quality control of Sweet tea element of the present invention is realized by below scheme:
The method of quality control of Sweet tea element adopts high effective liquid chromatography for measuring, and its chromatographic condition is as follows:
Chromatographic column is octadecyl silane filler；
Respectively with acetonitrile-water volume ratio as 10-90:90-10, methanol-water volume ratio for 20-80:90-10 as mobile phase；
Detection wavelength 205-215nm；
Flow velocity is 0.5～5 ml/min；
When preferably, using high effective liquid chromatography for measuring, chromatographic condition is as follows:
Respectively with acetonitrile-water volume ratio as 50:50, methanol-water volume ratio for 40:60 as mobile phase；
Detection wavelength 205,210nm；
Flow velocity is 0.8～1.2 ml/min；
Content and purity testing: respectively with 2 mobile phase solvent systems and 2 Detection wavelengths, record chromatogram is to main one-tenth
More than 2.5 times of the appearance retention time divided, calculate content with area normalization method, result system measurement sample size is equal
More than 98%；Determination of foreign matter, respectively in the chromatogram of different system record, in addition to solvent peak, impurity peak area summation result
It is respectively less than 2.0%.
Preferably, the method for quality control of Sweet tea element also includes thin-layered chromatography detection method, can be in Sweet tea element coarse crystallization
Carry out rapid previewing survey when preparation, improve the quality of Sweet tea element coarse crystallization it is also possible to after refined acquisition crystallization, carry out matter
Amount monitoring, the method method of Sweet tea element reference substance reference, its chromatographic condition is, with silica gel g as chromatoplate, with acetic acid second
Ester-acetone-acetic acid-water weight ratio example 8-10:3-5:1-2:1-2 is solvent, chloroform-methanol-water weight ratio example 15-25:6-
10:0.5 is solvent, and acetate-methanol-water weight ratio example 12-20:2-5:0.5 is solvent；Then iodine colour developing, is placed in
Inspect under white light, in thin-layer chromatography, under three development systems, be the single fluorescence spot of yellow, then represent purity and meet
Require, otherwise undesirable.
Preferably, in the method for quality control of Sweet tea element, thin-layered chromatography detection method is:
Take the solution of Sweet tea element sample methyl alcohol 1mg/ml, on same silica gel g, by different point sample amount gradient point
Sample, point sample amount is respectively 20 μ g, 40 μ g, 60 μ g, 80 μ g, 100 μ g；Respectively with ethyl acetate-acetone-acetic acid-water weight ratio example
8:3:1:1；Chloroform-methanol-water weight ratio example 20:6:0.5；Acetate-methanol-water weight ratio example 16:3:0.5 is to launch
Agent；Put expansion cylinder to launch respectively, away from for 15cm, then iodine colour developing, is placed in and inspects under white light, in the gradient of 5 variable concentrations for exhibition
In point sample and three development system thin-layer chromatographys, it is the single fluorescence spot of yellow.
The method of quality control of Sweet tea element of the present invention is it is characterised in that described Sweet tea element sample is by with lower section
Method is prepared from:
1) extract: take Sweet tea that leaf, extracting in water are dried, concentrate, filter, through macroporous resin column chromatography, molten with alcohol-water
Liquid system carries out gradient elution, collects the eluent that ethanol volumn concentration is 40%-80%, merges, concentrates, obtain final product Sweet tea
Plain coarse crystallization；
2) refine: isolate and purify Sweet tea element coarse crystallization with preparative high performance liquid chromatography, use c-18 chromatographic column, coordinate first
Alcohol-water system carries out gradient elution, with methanol-water solution by after Sweet tea element coarse crystallization dissolving, injects preparative high-efficient liquid phase color
Spectrometer, collects Sweet tea element solution more than 90% for the purity, reduced pressure concentration, obtains final product.
Preferably, described step 1) in, the ethanol eluate of variable concentrations, different eluents are collected in packet during gradient elution
Using thin-layered chromatography detection, with the method for Sweet tea element reference substance reference, its chromatographic condition is, with silica gel g as chromatoplate, with second
Acetoacetic ester-acetone-acetic acid-water weight ratio example 8-10:3-5:1-2:1-2 is solvent, or chloroform-methanol-water weight ratio example
15-25:6-10:0.5 be solvent, or acetate-methanol-water weight ratio example 12-20:2-5:0.5 be solvent, exhibition away from
For 15cm；Iodine develops the color, and is placed in and inspects under white light, selects in thin-layer chromatography, the high wash-out of yellow single fluorescence spot brightness
Liquid, merges, and concentrates, and obtains final product Sweet tea element coarse crystallization.
Preferably, described step 2) refine in, respectively collect purity between 90%-98% and purity be more than 98% with
On eluent, reduced pressure concentration, obtain purity between 90%-98% and more than more than 98% Sweet tea element.
Preferably, described step 2) refine in, collect purity more than 90% Sweet tea element solution after, using efficient liquid
Phase chromatography measures its purity again, and its chromatographic condition is:
Chromatographic column is octadecyl silane filler；
Respectively with acetonitrile-water volume ratio as 10-90:90-10, methanol-water volume ratio is mobile phase for 20-80:90-10；
Detection wavelength 205-215nm；
Flow velocity is 0.5～5 ml/min.
Compared with prior art, the substantive distinguishing features that the present invention projects and significantly progress are:
1st, the Sweet tea element purity that the present invention extracts is very high, can reach more than 98%, purity meets chemical reference substance requirement,
Solve the supply problem of Sweet tea element chemical reference substance, the quality control for Sweet tea medicinal material provides offer scientific basic and guarantor
Card.
2nd, the present invention is reasonable in design, process is simple, is extracted using aqueous solvent, ties after a macroporous resin column chromatography again
Crystalline substance can get purity higher Sweet tea element, after through preparative high performance liquid chromatography prepare purity reach more than 98% sweet
Theine chemical reference substance, method is simple.
3rd, separating rate of the present invention is fast, and with short production cycle, suitable industrialized production has good application prospect.
4th, the present invention carries out purity test, assay and quality control using thin-layer chromatography and high performance liquid chromatography, really
Protect the quality of product.
By studying to Sweet tea element chemical reference substance, set up the batch extracting technique, pure of Sweet tea element chemical reference substance
Degree and the analysis determining method of content and determination of foreign matter, thus setting up the technical standard of Sweet tea element chemical reference substance, make for it
Quality standard research for traditional Chinese chemical contrast and medicinal material and preparation provides scientific basic and guarantee.Result of study can be sweet
Theine chemical reference substance provides more complete Essential Chemistry foundation, grasps its chemical information and analysis and testing technology, is conducive to phase
Close the exploitation further of product, and for the peculiar product of exploitation China, exploitation has the product of high-tech, high added value
Product, improve the market competitiveness, it will produce potential and immeasurable social benefit and economic benefit.
Specific embodiment
Embodiment 1: the preparation of high-purity Sweet tea element
Take Sweet tea dried leaf, add water to cook extraction 3 times, each amount of water is 8 times of medicinal material weight, each extraction time is 2
Hour, merge extract, filter, filtrate concentrates, and obtains medicinal extract, through macroporous resin column absorption, with alcohol-water (0:100～60:40)
Gradient elution, collects alcohol-water (60:40) elution fraction, concentrates, and obtains Sweet tea element coarse crystallization.
Coarse crystallization preparative rp-hplc is prepared (chromatographic column: c-18 post；Mobile phase: methyl alcohol: water volume ratio is
65:35；Detection wavelength is 205nm；Flow velocity is 5ml/min), collect purity respectively and be more than with purity between 90%-98%
More than 98% eluent, reduced pressure concentration, obtain the Sweet tea element between 90%-98% and more than more than 98% for the purity.
With hplc analysis method, all preparation solutions are detected: chromatographic column c-18 post；Mobile phase is acetonitrile: water volume ratio
For 30:70；Detection wavelength is 205nm；Flow velocity 1ml/min；Merging retention time is identical and purity is in weight 90%～98%
Between and purity be more than weight more than 98% Sweet tea element preparation solution, reduced pressure concentration, obtain purity in weight 90%～98%
Between and the purity Sweet tea element white powder that is more than more than 98%, finished product hplc testing result is shown in Table 1.
Table 1, the hplc detection chromatogram of Sweet tea element quality control
| Sequence number | Retention time | Peak area | % area | Highly | Separating degree | Theoretical cam curve |
| 1 | 3.283 | 178642 | 0.71 | 15989 | 3193 | |
| 2 | 10.225 | 2902 | 0.01 | 228 | 24.32 | 13657 |
| 3 | 11.466 | 25025568 | 99.26 | 1093439 | 2.55 | 4631 |
| 4 | 15.439 | 3843 | 0.02 | 193 | 6.76 | 12164 |
Embodiment 2: the preparation of high-purity Sweet tea element
Take Sweet tea dried leaf, add water to cook extraction 2 times, each amount of water is 10 times of medicinal material weight, each extraction time is 3
Hour, filtration, merging filtrate, concentrate, obtain medicinal extract, extract adsorbs through macroporous resin column, with alcohol-water (0:100～70:30)
Gradient elution, collects alcohol-water (40:70) elution fraction, concentrates, and obtains Sweet tea element coarse crystallization.
Coarse crystallization preparative rp-hplc is prepared (chromatographic column: c-18 post；Mobile phase methanol: water volume ratio is
60:40；Detection wavelength is 205nm；Flow velocity is 5ml/min), collect purity respectively and be more than with purity between 90%-98%
More than 98% eluent, reduced pressure concentration, obtain the Sweet tea element between 90%-98% and more than more than 98% for the purity.
With hplc analysis method, all preparation solutions are detected: chromatographic column: c-18 post；Mobile phase acetonitrile: water volume ratio
For 25:75；Detection wavelength is 205nm；Flow velocity is 1ml/min)；Merge that retention time is identical and purity weight 90%～
Between 98% and purity be more than weight more than 98% Sweet tea element, reduced pressure concentration, obtain purity weight 90%～98% it
Between and purity be more than more than 98% Sweet tea element white powder.
Embodiment 3: the preparation of high-purity Sweet tea element
Take Sweet tea dried leaf, add water to cook extraction 8 times, each amount of water is 2 times of medicinal material weight, each extraction time is 1
Hour, filtration, merging filtrate, concentrate, obtain medicinal extract, extract adsorbs through macroporous resin column, with alcohol-water (0:100～50:50)
Gradient elution, collects alcohol-water (80:20) elution fraction, concentrates, and obtains Sweet tea element coarse crystallization.
With hplc detection, collect the flow point containing Sweet tea element, merge, concentrate, obtain Sweet tea element coarse crystallization.Coarse crystallization is used
Preparative rp-hplc is prepared (chromatographic column: c-18 post；Mobile phase methanol: water volume ratio is 50:50；Detection wavelength is
205nm；Flow velocity is 5ml/min), collect the eluent that purity is between 90%-98% and purity is more than more than 98% respectively,
Reduced pressure concentration, obtains the Sweet tea element between 90%-98% and more than more than 98% for the purity.
With hplc analysis method, all preparation solutions are detected: chromatographic column: c-18 post；Mobile phase: acetonitrile: water volume ratio
For 30:70；Detection wavelength is 205nm；Flow velocity is 1ml/min)；Merge that retention time is identical and purity weight 90%～
Between 98% and purity be more than weight more than 98% Sweet tea element, reduced pressure concentration, obtain purity weight 90%～98% it
Between and purity be more than more than 98% Sweet tea element white powder.
Embodiment 4: the preparation of high-purity Sweet tea element
1) take Sweet tea dried leaf, add water to cook extraction 3 times, each amount of water is 8 times of medicinal material weight, and each extraction time is
2 hours, merge extract, filter, filtrate concentrate, obtain medicinal extract, through macroporous resin column absorption, with alcohol-water (0:100～60:
40) gradient elution, is grouped by different concentration ethanol and collects eluent part；
2) each group eluent adopts thin-layered chromatography to detect, with the method for Sweet tea element reference substance reference, its chromatographic condition is,
With silica gel g as chromatoplate, launched using following solvent system:
Solvent system: ethyl acetate-acetone-acetic acid-water weight ratio example 8:3:1:1；
Exhibition is away from for 15cm；Iodine develops the color, and is placed in and inspects under white light, selects in thin-layer chromatography, the single fluorescent spot of yellow is lighted
Spend high eluent, merge, concentrate, obtain final product Sweet tea element coarse crystallization；
3) coarse crystallization preparative rp-hplc is prepared (chromatographic column: c-18 post；Mobile phase methanol: water volume ratio is
50:50；Detection wavelength is 205nm；Flow velocity is 5ml/min), collect purity respectively and be more than with purity between 90%-98%
More than 98% eluent, reduced pressure concentration, obtain the Sweet tea element between 90%-98% and more than more than 98% for the purity.
Embodiment 5: the detection of Sweet tea element
Sweet tea element more than 98% for the content that Example 1-4 is obtained, is detected by following method of quality control:
1st, thin-layered chromatography detection method:
Respectively the final sample of Example 1-4 add methyl alcohol make every 1ml contain 1mg solution, in same polyamide film
On, respectively by the concentration gradient point sample that 20 μ g, 40 μ g, 60 μ g, 80 μ g, 100 μ g are different；It is respectively adopted following solvent system to put
Expansion cylinder launches respectively, and exhibition is away from for 15cm:
Solvent system 1: with ethyl acetate-acetone-acetic acid-water weight ratio example 8:3:1:1；
Solvent system 2: with ethyl acetate-acetone-acetic acid-water weight ratio example 10:5:2:2；
Solvent system 3: chloroform-methanol-water weight ratio example 15:6:0.5；
Solvent system 4: chloroform-methanol-water weight ratio example 25:10:0.5；
Solvent system 5: acetate-methanol-water weight ratio example 12:2:0.5；
Solvent system 6: acetate-methanol-water weight ratio example 20:5:0.5；
Take out after expansion, dry, iodine develops the color, and puts and inspects under white light；Result in thin-layer chromatography it is seen that yellow single
Fluorescence spot, 6 kinds of solvent systems, the gradient point sample of 5 variable concentrations of 4 samples, it is single spot, have no impurity spot
Point.
2nd, high performance liquid chromatography:
Chromatographic condition and system suitability octadecylsilane chemically bonded silica are filler；
It is respectively adopted following flow phase system, flow velocity is 1.2 ml/min:
Flow phase system 1: acetonitrile-aqueous solution volume ratio is 50:50；
Flow phase system 2: acetonitrile-aqueous solution volume ratio is 10:90；
Flow phase system 3: acetonitrile-aqueous solution volume ratio is 90:10；
Flow phase system 4: methanol-water volume ratio is 40:60；
Flow phase system 5: methanol-water volume ratio is 20:80；
Flow phase system 6: methanol-water volume ratio is 90:10；
Detection wavelength is respectively 205nm, 2015nm, and number of theoretical plate is calculated by Sweet tea element peak and should be not less than 3000.
It is appropriate to this product of constant weight that determination method is taken at 105 DEG C of dryings, accurately weighed, goes up six flow phase system respectively
Solution makes the solution that every 1ml contains 3mg, shakes up, and precision measures 10 μ g injection liquid chromatographs, and record chromatogram is to principal component
More than 2.5 times of appearance retention time, calculate content with area normalization method, and results sample measures Sweet tea cellulose content equal 98%
More than.Determination of foreign matter, respectively in the chromatogram of different system record, in addition to solvent peak, impurity peak area summation result is all little
In 2.0%.
Peak purity detects: takes reference substance appropriate, presses two above flow phase system respectively, on high performance liquid chromatograph,
Carry out Peak homogeneity, the hplc chromatographic peak of Sweet tea element with diode array dad detector > 98%, its chromatographic peak UV absorption
Spectrogram, three-dimensional collection of illustrative plates and 5 points of spectrograms are completely superposed, and are shown to be single pure material peak.
[structural identification]
uvλh 2 oMax nm: end absorbs.
irνkbr max cm-1: 3387 (- oh), 2931 (phenyl ring), 1723 (carbonyls), 1638 (double bonds), 1460,1076,
887.
1hnmr(c5d5N, 500mhz, tms) δ ppm:1.23 (3h, s, h-18), 1.30 (3h, s, h-20), 4.98 (1h,
Brs, h-17a), 5.51 (1h, brs, h-17b), 6.11 (1h, d, j=8.4, glc-h1 '), 5.10 (1h, d, j=8.4, glc-
h1").
13cnmr(c5d5N, 125mhz, tms) δ ppm:40.8 (c-1), 19.5 (c-2), 38.4 (c-3), 44.1 (c-4),
57.4 (c-5), 22.2 (c-6), 41.7 (c-7), 42.5 (c-8), 54.0 (c-9), 39.9 (c-10), 20.7 (c-11), 37.3
(c-12), 86.0 (c-13), 44.6 (c-14), 47.8 (c-15), 154.6 (c-16), 104.5 (c-17), 28.4 (c-18),
177.0 (c-19), 15.7 (c-20), 99.8 (c-1'), 75.5 (c-2'), 78.9 (c-3'), 72.4 (c-4'), 78.1 (c-
5'), 63.1 (c-6'), 95.9 (c-1 "), 74.1 (c-2 "), 79.1 (c-3 "), 71.2 (c-4 "), 79.4 (c-5 "), 62.2 (c-
6").
Ms (esi-ms) m/z:665 [m+na]+、162、134(100)、133、117.
Test result indicate that: the Sweet tea element chemical reference substance that the present invention separates, purifies, through infrared spectrum, ultraviolet spectra, core
Magnetic resonance, mass spectrum and Physico-chemical tests confirm chemical constitution.Through 6 development system tlc detection disclosed by the invention, 6 flowings
The hplc detection of phase system and 2 different wave lengths, does purity test to chromatographic peak with dad simultaneously, shows that meeting Chinese medicine contains measurement
Surely use the requirement of chemical reference substance, content is more than 98%.
Claims (7)
1. a kind of detection method of Sweet tea element is it is characterised in that Sweet tea element sample adopts high performance liquid chromatography and thin-layer chromatography inspection
Survey method measures, and described high-efficient liquid phase chromatogram condition is:
Chromatographic column is octadecyl silane filler；
Respectively with acetonitrile-water volume ratio as 10-90:90-10, as 20:80 or 90:10 is as mobile phase for methanol-water volume ratio；
Detection wavelength is 205nm or 215nm；
Flow velocity is 0.5～5 ml/min；
Described thin-layered chromatography detection method, with the method for Sweet tea element reference substance reference, its chromatographic condition is, with silica gel g for chromatography
Plate, respectively with ethyl acetate-acetone-acetic acid-water weight ratio example 8-10:3-5:1-2:1-2 as solvent, chloroform-methanol-water weight
Amount ratio 15-25:6-10:0.5 is solvent, and acetate-methanol-water weight ratio example 12-20:2-5:0.5 is solvent；So
Iodine colour developing, is placed in and inspects under white light, be the single fluorescence spot of yellow, then under three development systems in thin-layer chromatography afterwards
Represent purity to meet the requirements, otherwise undesirable.
2. detection method as claimed in claim 1 it is characterised in that:
Respectively with acetonitrile-water volume ratio as 50:50, as 20:80 or 90:10 is as mobile phase for methanol-water volume ratio；
Detection wavelength is 205nm or 215nm；
Flow velocity is 0.8～1.2 ml/min；
Content and purity testing: respectively with 3 mobile phase solvent systems and 2 Detection wavelengths, record chromatogram is to principal component
More than 2.5 times of appearance retention time, calculate content with area normalization method, result system measurement sample size all 98% with
On；Determination of foreign matter, respectively in the chromatogram of different system record, in addition to solvent peak, impurity peak area summation result is respectively less than
2.0%.
3. detection method as claimed in claim 1 it is characterised in that:
Take the solution of Sweet tea element sample methyl alcohol 1mg/ml, on same silica gel g, by different point sample amount gradient point samples, point
Sample amount is respectively 20 μ g, 40 μ g, 60 μ g, 80 μ g, 100 μ g；Respectively with ethyl acetate-acetone-acetic acid-water weight ratio example 8:3:1:
1；Chloroform-methanol-water weight ratio example 20:6:0.5；Acetate-methanol-water weight ratio example 16:3:0.5 is solvent；Put exhibition
Open cylinder to launch respectively, away from for 15cm, then iodine colour developing, is placed in and inspects under white light, in the gradient point sample and three of 5 variable concentrations for exhibition
In individual development system thin-layer chromatography, it is the single fluorescence spot of yellow.
4. the detection method as described in claim 1-3 any one is it is characterised in that described Sweet tea element sample is by with lower section
Method is prepared from:
1) extract: take Sweet tea that leaf, extracting in water are dried, concentrate, filter, through macroporous resin column chromatography, use ethanol-water solution body
System carries out gradient elution, collects the eluent for 40%-80% for the ethanol volumn concentration, merges, concentrates, and obtains final product the thick knot of Sweet tea element
Brilliant；
2) refine: isolate and purify Sweet tea element coarse crystallization with preparative high performance liquid chromatography, use c-18 chromatographic column, cooperation methyl alcohol-
Aqueous systems carry out gradient elution, with methanol-water solution by after Sweet tea element coarse crystallization dissolving, inject preparative high performance liquid chromatography
Instrument, collects Sweet tea element solution more than 90% for the purity, reduced pressure concentration, obtains final product.
5. detection method as claimed in claim 4 it is characterised in that:
Described step 1) in, the ethanol eluate of variable concentrations is collected in packet during gradient elution, and different eluents adopt thin layer color
Spectrometry detects, with the method for Sweet tea element reference substance reference, its chromatographic condition is: with silica gel g as chromatoplate, with ethyl acetate-the third
Ketone-acetic acid-water weight ratio example 8-10:3-5:1-2:1-2 is solvent, or chloroform-methanol-water weight ratio example 15-25:6-
10:0.5 is solvent, or acetate-methanol-water weight ratio example 12-20:2-5:0.5 is solvent, and exhibition is away from for 15cm；
Iodine develops the color, and is placed in and inspects under white light, selects in thin-layer chromatography, and the high eluent of yellow single fluorescence spot brightness merges, dense
Contracting, obtains final product Sweet tea element coarse crystallization.
6. detection method as claimed in claim 4 it is characterised in that:
Described step 2) refine in, respectively collect purity between 90%-98% and purity more than 98% eluent, decompression dense
Contracting, obtain purity between 90%-98% and more than 98% Sweet tea element.
7. detection method as claimed in claim 4 it is characterised in that:
Described step 2) refine in, collect purity more than 90% Sweet tea element solution after, using high performance liquid chromatography again
Measure its purity, its chromatographic condition is:
Chromatographic column is octadecyl silane filler；
Respectively with acetonitrile-water volume ratio as 10-90:90-10, methanol-water volume ratio is 20:80 or 90:10 is mobile phase；
Detection wavelength is 205nm or 215nm；
Flow velocity is 0.5～5 ml/min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410789690.3A CN104483424B (en) | 2014-12-18 | 2014-12-18 | Rubusoside quality control method |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101003552A (en) * | 2006-01-18 | 2007-07-25 | 广西师范大学 | Method for preparing glycosides in high purity |
| EP2236043A1 (en) * | 2009-04-03 | 2010-10-06 | Plantextrakt GmbH&Co. Kg | Method for producing an extraction substance from leaves of the Chinese blackberry and use of same |
| CN102239983A (en) * | 2011-05-16 | 2011-11-16 | 向华 | Method for extracting rubusoside from sweet tea leaves |
| CN104049047A (en) * | 2014-06-25 | 2014-09-17 | 曲阜圣香远生物科技有限公司 | Method for evaluating quality of stevioside |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101003552A (en) * | 2006-01-18 | 2007-07-25 | 广西师范大学 | Method for preparing glycosides in high purity |
| EP2236043A1 (en) * | 2009-04-03 | 2010-10-06 | Plantextrakt GmbH&Co. Kg | Method for producing an extraction substance from leaves of the Chinese blackberry and use of same |
| CN102239983A (en) * | 2011-05-16 | 2011-11-16 | 向华 | Method for extracting rubusoside from sweet tea leaves |
| CN104049047A (en) * | 2014-06-25 | 2014-09-17 | 曲阜圣香远生物科技有限公司 | Method for evaluating quality of stevioside |
Non-Patent Citations (4)
| Title |
|---|
| Analysis of Rubusoside and Related Compounds in Tenryocha Extract Sweetener;Naoki Sugimoto et al;《J. Food Hyg. Soc. Japan》;20020825;第43卷(第4期);250-253 * |
| Production of Ru·busoside Derivatives by Transgalactosylation of Various α-Galactosidases;Sumio KITAHATA et al;《Agricultural and Biological Chemistry》;19891130;第53卷(第11期);2929-2934 * |
| 制备色谱法在纯化甜茶苷工艺中的应用;吕鑫华 等;《北京化工大学学报(自然科学版)》;20121031;第39卷(第5期);92-97 * |
| 广西甜茶质量控制方法的研究进展;吴柳春;《中国民族民间医药》;20120815;第21卷(第8（上）期);53-55 * |
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