CN104013673A - Salvia extract and preparation method thereof - Google Patents
Salvia extract and preparation method thereof Download PDFInfo
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- CN104013673A CN104013673A CN201410069832.9A CN201410069832A CN104013673A CN 104013673 A CN104013673 A CN 104013673A CN 201410069832 A CN201410069832 A CN 201410069832A CN 104013673 A CN104013673 A CN 104013673A
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Abstract
The invention provides an extracting method of salviae miltiorrhizae. The method comprises the following steps: taking a salviae miltiorrhiza decoction piece; adding water; boiling and extracting at 70 DEG C under decompressing condition; combining an extracting liquid to obtain the decoction piece. The invention further provides a detection method of the salvia extract. According to the detection method of the salvia extract provided by the invention, the content of salvianolic acid B in the extract is far higher than that of a high temperature reflex extract, the content of other components and impurities is higher than that of the high temperature reflex extract, and the purity of the salvia extract prepared by a low-temperature boiling dynamic method is greater than that of the high temperature reflex extract. The detection method provided by the invention can be used for effectively separating multiple components in the salvia extract, is good in spike and stable in base line and provides a reliable base for quality control of the salvia extract.
Description
Technical field
The present invention relates to a kind of Radix Salviae Miltiorrhizae extract and preparation method thereof.The invention still further relates to the detection method of Radix Salviae Miltiorrhizae extract.
Background technology
Radix Salviae Miltiorrhizae is dry root and the rhizome of labiate Radix Salviae Miltiorrhizae Salvia miltiorrhiza Bge., begins to be loaded in Shennong's Herbal, classifies as top gradely, is clinical parts of generic medicinal plants.There is blood circulation promoting and blood stasis dispelling, relieving pain of dysmenorrhea, the relieving restlessness that clears away heart-fire, the effects such as removing heat from blood eliminating carbuncle, the clinical cards such as obstruction of qi in the chest and cardialgia, gastral cavity abdomen hypochondriac pain, lumps in the chest and abdomen, pyretic arthralgia pain, dysphoria and insomnia, menoxenia that are widely used in.
Radix Salviae Miltiorrhizae contains fat-soluble diterpene quinone and water miscible phenolic acid compound.Soluble salvianolic acid is the main active component of Radix Salviae Miltiorrhizae, comprises the compositions such as danshensu, salvianolic acid B, salvianolic acid C and protocatechualdehyde, the highest with content of danshinolic acid B.Salvianolic acid B all has protective effect to cardiovascular system, brain, liver, kidney etc., is effective water soluble ingredient of Radix Salviae Miltiorrhizae.
Radix Salviae Miltiorrhizae extracts the reflux, extract, that adopt more, and extracting temperature is solvent boiling point under atmospheric pressure, and temperature is higher, and salvianolic acid B is to thermally labile, long-time heating, and salvianolic acid B destructions that is easily decomposed, makes its content reduction.Therefore, urgently one can effectively be extracted salvianolic acid B, and impurity is extracted to less Radix Salviae Miltiorrhizae extracting method.
Summary of the invention
Technical scheme of the present invention has been to provide the Radix Salviae Miltiorrhizae extract that a kind of content of danshinolic acid B is higher, and another technical scheme of the present invention has been to provide the preparation method of Radix Salviae Miltiorrhizae extract.
The invention provides the extracting method of Radix Salviae Miltiorrhizae, it comprises following operating procedure: get salvia piece, add water, under reduced pressure, 70 ± 5 DEG C of boilings are extracted, merge extractive liquid,.
Said extracted temperature, is preferably used 70 DEG C.
Boiling described in the present invention is extracted, and, under reduced pressure, makes water be issued to fluidized state in the corresponding temperature of extracting, and under this temperature and pressure condition, extracts, and wherein, vacuum pressure ensures that to meet water seethes with excitement as benchmark under a certain temperature conditions.
Wherein, amount of water is 5～15 times of Radix Salviae Miltiorrhizae dry weight, is preferably 12 times.
Wherein, extract each 0.5～3h 1～3 time; Preferably extract 3 times each 2h.
The present invention also provides a kind of preparation method of Radix Salviae Miltiorrhizae extract, and it comprises following operating procedure:
(1) extract as stated above;
(2) get the extracting solution of step (1), after decompression and solvent recovery, lyophilization, obtain Radix Salviae Miltiorrhizae extract.
Wherein, the condition of described decompression and solvent recovery is :-0.05～-0.095MPa, 40～90 DEG C; Preferably 70 ± 5 DEG C.
The present invention also provides a kind of Radix Salviae Miltiorrhizae extract, and described Radix Salviae Miltiorrhizae extract contains 5～15% salvianolic acid B.
Further, the preparation method of described Radix Salviae Miltiorrhizae extract is described above.
The present invention also provides the detection method of Radix Salviae Miltiorrhizae extract, and it is measured with high performance liquid chromatography, and concrete operation step is as follows:
(1) get Radix Salviae Miltiorrhizae extract to be measured, taking methanol as solvent or diluent, prepare need testing solution;
(2) need testing solution is injected to high performance liquid chromatograph, detect and get final product, its chromatographic condition is as follows:
Chromatographic column: octadecyl silane is immobile phase;
Mobile phase: methanol A-1.0% glacial acetic acid aqueous solution B, gradient elution, elution program A:0min7%, 10min7%, 12min9%, 31min33%, 47min39%, 60min58%, 70min62%;
Detect wavelength: 286 ± 5nm.
Further, in described high performance liquid chromatography, taking one or more in salvianolic acid B, protocatechualdehyde or danshensu sodium as reference substance, preparation reference substance solution.
Further, detecting column temperature is 30 ± 5 DEG C.
Utilize extracting method of the present invention, in the Radix Salviae Miltiorrhizae extract making, content of danshinolic acid B is far above high temperature reflux extract, other compositions and impurity content higher temperatures reflux, extract, are low, and the Radix Salviae Miltiorrhizae extract salvianolic acid B purity that low-temperature boiling dynamic method makes is greater than high temperature reflux extract.
Higher temperatures reflux, extract,, suitable extract at low temperature had both ensured the maximum stripping of liposoluble ingredient, also prevented its destruction that is at high temperature decomposed, and had extracted to greatest extent the liposoluble ingredient in Radix Salviae Miltiorrhizae; Meanwhile, due to low temperature, impurity and other composition stripping quantities reduce, when liposoluble ingredient is separated efficiently from Radix Salviae Miltiorrhizae, in extracting solution, the content of impurity and other compositions also reduces, and is conducive to follow-up separation purifying technique, has improved product quality and added value.
Detection method of the present invention, can effectively effectively separate the multiple compositions in Radix Salviae Miltiorrhizae extract, and peak shape is good, and baseline is steady, for the quality control of Radix Salviae Miltiorrhizae extract provides reliable basis.
Brief description of the drawings
Fig. 1: the different temperature content of danshinolic acid B figure that extract
Fig. 2: Radix Salviae Miltiorrhizae extract surface tension
Fig. 3: Radix Salviae Miltiorrhizae extract Zeta potential
Fig. 4: Radix Salviae Miltiorrhizae extract particle size distribution
Fig. 5: Radix Salviae Miltiorrhizae extract finger printing chromatogram
Fig. 6: 10 crowdes of Radix Salviae Miltiorrhizae extract finger printing stacking charts
Fig. 7: Radix Salviae Miltiorrhizae extract particle size distribution figure
Fig. 8: Radix Salviae Miltiorrhizae extract moisture absorption percentage curves
Fig. 9: 10 crowdes of Radix Salviae Miltiorrhizae extract finger printing stacking charts
Detailed description of the invention
Embodiment 1 Radix Salviae Miltiorrhizae extracting method
Get salvia piece, add 12 times of water gagings of Radix Salviae Miltiorrhizae dry weight, under reduced pressure, 70 DEG C of boilings are extracted 3 times, each 2h, and merge extractive liquid,.
The research of embodiment 2 Radix Salviae Miltiorrhizae extracting method
1 instrument and reagent
Shimadzu LC-10A high performance liquid chromatograph, SCL-6A system controller, N2000 Data Processing in Chromatography Workstation, TC-100 calorstat (Japanese Shimadzu company); Agilent1200 high performance liquid chromatograph series (Agilent company of the U.S.); Mettler AE240 100,000/electronic balance (German Mettler company); FA1104 ten thousand/electronic balance (Shanghai balance equipment factory); KQ3200 type ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); SHZ-D(III) circulating water type vacuum pump (Yuhua Instrument Co., Ltd., Gongyi City); Thermostat water bath (Beijing Zhong Xing great achievement Instrument Ltd.); RE-2000 type Rotary Evaporators (Shanghai Yarong Biochemical Instrument Plant), ALPHA1-4LSC freeze dryer (CHRIST company), PHS-3E type PH counts (Shanghai Precision Scientific Apparatus Co., Ltd); OCAT21 surface/interface tensiometer (German Dataphysics company); Conductivity meter (thunder magnetic DDS ?307A, upper Nereid section) Malvern nano particle size instrument (model Nano-ZS).
Red rooted salvia (the natural pharmaceutcal corporation, Ltd in Cologne, Sichuan); Salvianolic acid B reference substance (lot number: MUST-12020104, Man Site bio tech ltd, Chengdu), danshensu sodium reference substance (lot number: MUST-12020803, Man Site bio tech ltd, Chengdu), protocatechualdehyde reference substance (lot number: 110810-200205, Nat'l Pharmaceutical & Biological Products Control Institute), methanol is chromatographically pure, water is redistilled water (self-control), and other reagent are analytical pure.
The foundation of 2 content of danshinolic acid B mensuration methodologies
Chromatographic condition chromatographic column: octadecylsilane chemically bonded silica post; Agilent TC-C18(250mm × 4.6mm) chromatographic column, 5 μ m; Acetonitrile-0.5% phosphoric acid water (28:72) is mobile phase, detects wavelength 286nm, flow velocity 1.0mL/min; Column temperature: 30 DEG C.
The preparation precision of the preparation reference substance solution of reference substance solution takes salvianolic acid B reference substance 14.08mg in 10mL measuring bottle, and 75% methanol solution standardize solution, shakes up, as stock solution; Precision measures 1mL in 10mL measuring bottle, and 75% methanol solution standardize solution, shakes up, and obtains 0.1408mg/mL salvianolic acid B reference substance solution.
The accurate absorption of the preparation extracting solution of need testing solution is in right amount in 25mL measuring bottle, and 75% methanol solution standardize solution, shakes up, and 0.45 μ m microporous filter membrane filters, and gets subsequent filtrate as need testing solution.
Reference substance solution is got in range of linearity test, and accurate sample introduction 2,4,8,16,20 μ L respectively, measure peak area integrated value according to above-mentioned chromatographic condition, with peak area, sample size are returned, and obtain regression equation: Y=131290X+46157, R=0.9991.Result shows, is that within the scope of 0.2816mg～2.8160mg, linear relationship is good at content of danshinolic acid B.
The accurate reference substance solution 10uL injection liquid chromatography of drawing of precision test, continuous sample introduction 6 times, records peak area, and calculating RSD is 1.16%, shows that instrument precision is good.
The accurate need testing solution of drawing of stability test, respectively at 0h, 2h, 4h, 6h, 8h sample introduction 5uL, records peak area, and RSD is 1.08%, shows that sample is good at 8h internal stability.
Replica test is prepared 6 parts of need testing solutions by " preparation of need testing solution ", and the accurate 5uL injection liquid chromatography of drawing, records peak area respectively, and RSD is 2.14%, shows that this method repeatability is good
Recovery test precision takes the Danshen Root 0.1g of known content, and precision adds salvianolic acid B reference substance 3.8016mg, and 75% methanol constant volume is to 50mL, weighed weight, ultrasonic 1h, takes out, let cool, more weighed weight, supply the weight of less loss with 75% methanol, shake up, filter, get subsequent filtrate, sample introduction 5 μ L measure content, calculate recovery rate, average recovery rate is that 101.77%, RSD is 2.89% (n=6).
In red rooted salvia, content of danshinolic acid B is measured and is got the about 0.2g of Danshen Root (crossing sieve No. three), accurately weighed, puts in tool plug conical flask, precision adds 75% methanol 50mL, weighed weight, ultrasonic 1h, take out, let cool, more weighed weight, supply the weight of less loss with 75% methanol, shake up, filter, get subsequent filtrate, sample introduction 5 μ L measure content, and recording content of danshinolic acid B in red rooted salvia is 3.81%.
3 Orthogonal Experiment and Design
Take salvia piece 30g, taking water as solvent, at 70 DEG C of evacuation boiling extract at temperature to extraction time, solvent load, extraction time three factors carry out L
9(3
4) orthogonal experiment, prepare sample solution, measure content of danshinolic acid B, result is as follows:
Table 1 orthogonal experiment plan is taken into account result
Table 2 variance analysis
Note: P < 0.05 indicates significance, P < 0.01 indicates utmost point significance
Result shows, each factor effect primary and secondary is A>C>B, and A extracts and has the greatest impact salvianolic acid, be secondly C factor, B factor impact minimum.The results of analysis of variance is: A, B, C factor have appreciable impact to extraction effect.The result of comprehensive intuitive analysis and variance analysis, determines that Optimization Technology is A
3b
3c
3, add 12 times of water gagings, extract 3 times each 2h in 70 DEG C of boiling refluxs of low temperature.
The checking of orthogonal test is according to the result of above orthogonal test, to preferred optimum process condition A out
3b
3c
3carry out demonstration test, in triplicate, salvianolic acid B and dry cream yield are respectively 3.11%, 40.36%.
The different difference of extracting temperature takes salvia piece 30g, adopts the process conditions of optimization of orthogonal test, at 50,60,70,80,90,100 DEG C, prepares Radix Salviae Miltiorrhizae extract, measures content of danshinolic acid B and dry cream yield.Result is as table 3, Fig. 1:
The different impacts (n=3) of extracting temperature of table 3
Above data show, along with extracting the rising of temperature, and content of danshinolic acid B first increases and then decreases, it is 2.46% that 70 DEG C of content is up to 3.14%, 100 DEG C of content minimum; Dry cream yield raises and increases gradually with temperature, and 50 DEG C minimum is 38.95%, 100 DEG C and is up to 48.50%.While extracting temperature lower than 70 DEG C, effectively stripping of salvianolic acid B; During higher than 70 DEG C, along with extending heating time, the salvianolic acid B destruction that is decomposed; Therefore 70 DEG C of suitable extraction temperature that are salvianolic acid B.Because dissolubility in material solvent generally increases along with temperature raises, thereby, when low temperature material stripping few, dry cream yield is low, along with temperature raises, material stripping quantity increases, dry cream yield increases.70 DEG C time, effectively stripping of salvianolic acid B, is not destroyed, and in solution, also higher temperatures is low for other compositions and impurity meltage, is conducive to later separation purification.
4 extracting solution physical parameter measurement
(1) the capillary mensuration of Radix Salviae Miltiorrhizae extract
Adopt OCAT21 surface/interface tensiometer to measure the surface tension of Radix Salviae Miltiorrhizae high temperature and extract at low temperature liquid, use SPSS17.0 statistical software to carry out independent sample t inspection relative analysis to test data, measurement result is as table 4, Fig. 2.
The surface tension test (unit: mN) of table 4 Radix Salviae Miltiorrhizae high temperature extraction liquid and extract at low temperature liquid
With high temperature extraction liquor ratio, * P < 0.05
Statistic analysis result shows, two groups of data heterogeneity of variances, two-sided test P=0.027 < 0.05, the surface tension of high temperature extraction liquid and extract at low temperature liquid has significant difference, extract at low temperature liquid surface tension is significantly greater than high temperature extraction liquid surface tension, and this may be relevant with extraction process.
(2) mensuration of Radix Salviae Miltiorrhizae extract electrical conductivity
Adopt conductivity meter to measure the electrical conductivity of Radix Salviae Miltiorrhizae high temperature and extract at low temperature liquid, use SPSS17.0 statistical software to carry out independent sample t inspection relative analysis to test data, measurement result is in table 5.
The conductance measurement (unit: μ s/cm) of table 5 Radix Salviae Miltiorrhizae high temperature extraction liquid and extract at low temperature liquid
Statistical analysis, two groups of data variances are neat, and two-sided test P=0.190 > 0.05 shows that high temperature extraction liquid and extract at low temperature liquid electrical conductivity do not have significant difference.
(3) Radix Salviae Miltiorrhizae extract pH value is measured
Adopt pHS-3C Accurate pH instrumentation to determine the pH of Radix Salviae Miltiorrhizae high temperature and extract at low temperature liquid, use SPSS17.0 statistical software to carry out independent sample t inspection relative analysis to test data, measurement result is in table 6.
The pH of table 6 Radix Salviae Miltiorrhizae high temperature extraction liquid and extract at low temperature liquid measures
With high temperature extraction liquor ratio, * P < 0.05
Statistical result showed, two groups of data variances are neat, two-sided test P=0.000 < 0.05, both have remarkable significant difference, and extract at low temperature liquid pH value is significantly lower than high temperature extraction liquid; Because both extract temperature difference, the stripping difference of material in both, the contained classes of compounds of extracting solution and quantity are variant, therefore the pH value difference of extracting solution.
(4) mensuration of Radix Salviae Miltiorrhizae extract Zeta potential
Adopt zeta potential instrument to measure Radix Salviae Miltiorrhizae high temperature and extract at low temperature liquid Zeta potential, use SPSS17.0 statistical software to carry out independent sample t inspection relative analysis to test data, measurement result is in table 7, Fig. 3.
The conductance measurement (unit: vm) of table 7 Radix Salviae Miltiorrhizae high temperature extraction liquid and extract at low temperature liquid
With high temperature extraction liquor ratio, * P < 0.05
Statistical analysis shows, two groups of data variances are neat, two-sided test P=0.008 < 0.05, and both have significant significant difference, extract at low temperature liquid Zeta potential, significantly lower than high temperature extraction liquid Zeta potential, shows that high temperature extraction liquid stability is better than extract at low temperature liquid.
(5) mensuration of Radix Salviae Miltiorrhizae extract nanometer particle size
Adopt Malvern nano particle size instrument to measure Radix Salviae Miltiorrhizae high temperature and extract at low temperature liquid nanometer particle size, use SPSS17.0 statistical software to carry out independent sample t inspection relative analysis to test data, measurement result is in table 8, Fig. 4.
The conductance measurement (unit: d.nm) of table 8 Radix Salviae Miltiorrhizae high temperature extraction liquid and extract at low temperature liquid
With high temperature extraction liquor ratio, * P < 0.05
Statistical result showed, two groups of data variances are neat, two-sided test P=0.000 < 0.05, both have remarkable significant difference, and extract at low temperature liquid nanometer particle size meansigma methods is significantly greater than high temperature extraction liquid nanometer particle size meansigma methods.From the surface of intensity distribution, high temperature extraction liquid particle size distribution is at 400～800nm, and extract at low temperature liquid particle size distribution, at 2000～6000nm, is coarse dispersion system, and extract at low temperature liquid nanometer particle size is much larger than high temperature extraction liquid.
The finger printing research of embodiment 3 extracting solution
The preparation of need testing solution takes red rooted salvia 30g, adds 12 times of water, in 70 DEG C of reflux, extract, 3 times, and each 2h, filter, merging filtrate, is settled to 1000mL, and the accurate 5mL that draws is in measuring bottle, methanol constant volume, to 25ml, shakes up, and obtains Radix Salviae Miltiorrhizae extract at low temperature liquid need testing solution.
Take red rooted salvia 30g, add 12 times of water, reflux, extract, 3 times, each 2h, filters, and merging filtrate, is settled to 1000mL, and the accurate 5mL that draws is in measuring bottle, and methanol constant volume, to 25ml, shakes up, and obtains Radix Salviae Miltiorrhizae high temperature extraction liquid need testing solution.
The accurately weighed salvianolic acid B of preparation of reference substance solution, protocatechualdehyde, danshensu sodium reference substance are in right amount in 10mL measuring bottle, and methanol constant volume, obtains containing salvianolic acid B 0.1466mgmL
-1, protocatechualdehyde 8.533 μ gmL
-1, danshensu sodium 33.4 μ gmL
-1mixing reference substance solution.
(1) selection of chromatographic condition
Detect the selection of wavelength in order to ensure the maximum information quantification of finger printing, reflect as much as possible sample overall picture, adopt methanol-1.0% glacial acetic acid aqueous solution binary gradient elution, under 30 DEG C of column temperatures, Radix Salviae Miltiorrhizae extract chromatogram under record 254,270,280,286,310nm wavelength, and determine detection wavelength in conjunction with 3D-plot figure.Result shows, under 286nm wavelength, chromatographic peak separating effect is better, and all chromatographic peaks can both embody, and can fully reflect the situation of Radix Salviae Miltiorrhizae sample, therefore, selects 286nm for detecting wavelength.
Being chosen under 30 DEG C of column temperatures of mobile phase kind, taking methanol-0.5% phosphate aqueous solution, methanol-1.0% glacial acetic acid aqueous solution, methanol-1.0% aqueous formic acid, acetonitrile-0.5% phosphate aqueous solution as mobile phase, carries out gradient elution, records chromatogram.Result shows, steady with methanol-1.0% glacial acetic acid aqueous solution gradient elution baseline, separates better, therefore taking methanol-1.0% glacial acetic acid aqueous solution as mobile phase.
The selection of gradient elution program, taking methanol-1.0% glacial acetic acid aqueous solution as mobile phase, under 30 DEG C of column temperatures, adopts different gradient elution programs, measure finger printing, result shows at gradient elution program (A:0min7%, 10min7%, 12min9%, 31min33%, 47min39%, 60min58%, 70min62%) separating degree is better down, characteristic peak is obvious, therefore carry out eluting with this gradient elution program.
The selection of chromatographic column is taking methanol (A)-1.0% glacial acetic acid aqueous solution (B) as mobile phase, under 286nm, adopt respectively Agilent TC-C18, Dikma Kromasil100A C18, Scienhome Kromasil C18 chromatographic column to measure finger printing, result is better with Agilent TC-C18 chromatographic column separating effect, therefore select Agilent TC-C18 chromatographic column to measure.
Result shows, Radix Salviae Miltiorrhizae finger printing chromatographic condition is: chromatographic column is Agilent TC-C18 chromatographic column; Mobile phase is methanol (A)-1.0% glacial acetic acid aqueous solution (B), binary gradient elution, elution program A:0min7%, 10min7%, 12min9%, 31min33%, 47min39%, 60min58%, 70min62%; Column temperature is 30 DEG C; Volume flow is 1.0mLmin
-1, detection wavelength is 286nm, analysis time 70min, test sample, reference substance solution sample size are 15 μ L.
(2) methodological study
Precision test is got No. 1 Radix Salviae Miltiorrhizae normal pressure extracting solution need testing solution continuous sample introduction 5 times, records finger printing, calculates total peak relative retention time, relative peak area and RSD value, is all less than < 3%, shows that instrument precision is good.
Stability test is got precision test need testing solution, measure finger printing respectively at 0h, 2h, 4h, 8h, 16h, 36h, calculate total peak relative retention time, relative peak area and RSD value, be all less than < 3%, show that sample 36h internal stability is good.
Replica test is got same batch of 6 parts of red rooted salvias, accurately weighed, prepares need testing solution by " preparation of need testing solution " method, measures finger printing, calculates total peak relative retention time, relative peak area and RSD value.Result shows, total fingerprint peaks relative retention time, relative peak area RSD < 3%, and this assay method repeatability is good.
(3) foundation of Radix Salviae Miltiorrhizae finger printing
10 batches of red rooted salvias are got respectively in the detection of Radix Salviae Miltiorrhizae finger printing, accurately weighed, prepare need testing solution by " preparation of need testing solution " method, detect by the preparation of test chromatographic condition, record chromatogram in 70min, the total peak of selecting each collection of illustrative plates to produce by the retention time at more each peak, the total peak-to-peak area of record, in table 9, table 10, Fig. 5, Fig. 6.
Table 9 Radix Salviae Miltiorrhizae extract at low temperature liquid peak area
Table 10 Radix Salviae Miltiorrhizae high temperature extraction liquid peak area
Fingerprint similarity calculates the chromatographic fingerprints of Chinese materia medica similarity evaluation software (2004A version) that adopts Chinese Pharmacopoeia committee to publish and carries out similarity calculating, in table 11.
Table 11 Radix Salviae Miltiorrhizae extract similarity result of calculation
From extracting solution finger printing, both differ greatly; Radix Salviae Miltiorrhizae high temperature extraction liquid finger printing chromatographic peak quantity is more, and Radix Salviae Miltiorrhizae extract at low temperature liquid finger printing chromatographic peak negligible amounts shows that the contained classes of compounds lower temperature of Radix Salviae Miltiorrhizae high temperature extraction liquid extracting solution is many, and the stripping in the time of extract at low temperature of some compositions reduces.High temperature extraction liquid and extract at low temperature liquid have 6 total peaks, and 13min left and right chromatographic peak is danshensu sodium, and 21min left and right chromatographic peak is protocatechualdehyde, and 56min left and right chromatographic peak is salvianolic acid B.From total peak-to-peak area, both are also variant; Extract at low temperature liquid danshensu sodium, protocatechualdehyde peak area are much smaller than high temperature extraction liquid, and salvianolic acid B chromatographic peak peak area is much larger than high temperature extraction liquid.More than test, Radix Salviae Miltiorrhizae extract at low temperature liquid content of danshinolic acid B is high, and impurity and other compositions are few, and in extracting solution, salvianolic acid B purity is higher.
The parametric measurement of embodiment 4 Radix Salviae Miltiorrhizae extracts
Get low temperature (embodiment 1), high temperature extraction liquid and be concentrated into 200mL at 70 DEG C of Rotary Evaporators, concentrated solution cold preservation 12 hours at-5 DEG C, form ice cube, in the dry 24h of freeze dryer, take out, weigh, in exsiccator, keep in Dark Place, measure the content of salvianolic acid B, extract at low temperature thing is 7.38%, high temperature extraction thing is 5.62%, and extract at low temperature thing content of danshinolic acid B is significantly higher than high temperature extraction thing.
The mensuration of 1 extract physical parameter
(1) mensuration of Radix Salviae Miltiorrhizae extract specific surface area
Get extract powder appropriate, adopt specific surface area measuring instrument to measure the specific surface area of Radix Salviae Miltiorrhizae high temperature and extract at low temperature thing, use SPSS17.0 statistical software to carry out independent sample t inspection relative analysis to test data, measurement result is in table 12
Specific area measuring (the unit: m of table 12 Radix Salviae Miltiorrhizae high temperature extraction thing and extract at low temperature thing
2/ g)
With the comparison of high temperature extraction thing, * P < 0.05
Statistical analysis, two groups of data variances are neat, the P < 0.05 of two-sided test, can not think that the specific surface area of two kinds of Radix Salviae Miltiorrhizae extracts is identical, because the specific surface area average of extract at low temperature thing is greater than high temperature extraction thing, therefore the specific surface area of extract at low temperature thing is significantly greater than high temperature extraction thing specific surface area, the surface adsorption ability of prompting extract at low temperature thing may be stronger than high temperature extraction thing.
(2) Radix Salviae Miltiorrhizae extract porosity measurement
Get extract powder appropriate, adopt specific surface area measuring instrument to measure the specific surface area of Radix Salviae Miltiorrhizae high temperature and extract at low temperature thing, use SPSS17.0 statistical software to carry out independent sample t inspection relative analysis to test data, measurement result is in table 13.
Porosity measurement (the unit: cm of table 13 Radix Salviae Miltiorrhizae high temperature extraction thing and extract at low temperature thing
2/ g)
With the comparison of high temperature extraction thing, * P < 0.05
Statistical analysis, two groups of data variances are neat, the P < 0.05 of two-sided test, both have remarkable significant difference, and the porosity of extract at low temperature thing is significantly greater than high temperature extraction thing porosity.
(3) particle size determination of Radix Salviae Miltiorrhizae extract
Adopt powder body dry method to measure Radix Salviae Miltiorrhizae extract particle diameter, measurement result is in table 14, Fig. 7.
Particle size determination (the unit: μ m) of table 14 Radix Salviae Miltiorrhizae high temperature extraction thing and extract at low temperature thing
Result shows, extract at low temperature thing d (0.1), d (0.9) particle diameter are less than high temperature extraction thing, extract at low temperature thing d (0.5) particle diameter is greater than high temperature extraction thing, Radix Salviae Miltiorrhizae high temperature extraction thing particle size distribution is at 0.8～2000nm, Radix Salviae Miltiorrhizae extract at low temperature thing particle size distribution is at 0.8～700nm, and extract at low temperature thing higher temperatures extract particle size distribution is more concentrated.
(4) mensuration of Radix Salviae Miltiorrhizae extract angle of repose
Adopt fixed funnel method to measure Radix Salviae Miltiorrhizae extract angle of repose, funnel is fixed on to the top certain distance of the diagram paper of horizontal positioned, carefully the extract powder body preparing is poured in funnel, on graph paper, be piled into cone, measure diameter 2R and the height H of cone, according to tan θ=H/R, calculate θ angle of repose, in table 15.
Measure (unit: degree) angle of repose of table 15 Radix Salviae Miltiorrhizae high temperature extraction thing and extract at low temperature thing
Use SPSS17.0 statistical software to carry out independent sample t inspection relative analysis to above data, two master data variances are neat, two-sided test P=0.067 > 0.05, both are without remarkable significant difference, high temperature and extract at low temperature thing are less than 40 ° angle of repose, mobility is better, can meet need of production, and extract at low temperature thing mobility is better than high temperature extraction thing.
(5) Radix Salviae Miltiorrhizae extract bulk density and tap density are measured
Take Radix Salviae Miltiorrhizae extract powder body 10g, be placed in 25mL graduated cylinder, after striking off gently, read volume V
0, vibration graduated cylinder not changing, reads volume V to powder body volume
f, calculating bulk density, tap density, the results are shown in Table 16.
The bulk density of table 16 Radix Salviae Miltiorrhizae high temperature extraction thing and extract at low temperature thing and tap density are measured (unit: g/mL)
With the comparison of high temperature extraction thing, * P < 0.05
Use SPSS17.0 statistical software to carry out independent sample t inspection relative analysis to above data, variance is neat, two-sided test bulk density and tap density P value are 0.000 < 0.05, have remarkable significant difference, extract at low temperature thing bulk density and tap density are significantly less than high temperature extraction thing.
(6) Radix Salviae Miltiorrhizae extract moisture absorption percentage rate is measured
Getting clean weighing botle constant weight weighs, get Radix Salviae Miltiorrhizae extract tiling (thickness 1～2mm) in weighing botle, in 60 DEG C of dry 6h, take out, be put in the 12h that dewaters in the exsiccator that fills discolour silica gel, weigh, weighing botle is put in the exsiccator that fills supersaturation sodium chloride solution (relative humidity is 75%), close drying device, be placed under room temperature, respectively at 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168h takes out and weighs, press hydroscopicity (%)=(the front extract weight of extract weight-moisture absorption after moisture absorption) front extract weight × 100% formula of/moisture absorption, calculate moisture absorption percentage rate, with moisture absorption percentage rate, the time is mapped, obtain moisture equilibrium at dry side curve, see Fig. 8:
Data show, when 0～120h, along with time lengthening, Radix Salviae Miltiorrhizae extract moisture absorption percentage rate enlarges markedly, and exceedes after 120h when the time, and along with time lengthening, moisture absorption percentage rate slowly increases, and shows that Radix Salviae Miltiorrhizae extract reaches moisture equilibrium at dry side at 120h.After Radix Salviae Miltiorrhizae extract moisture absorption, darken, and liquefaction.Extract balance Moisture absorption percentage rate is all greater than 15%, illustrate Radix Salviae Miltiorrhizae extract have draw moist.Extract at low temperature thing balance Moisture absorption percentage rate is greater than high temperature extraction thing balance Moisture absorption percentage rate, and the easier moisture absorption of extract at low temperature thing higher temperatures extract, may be the difference of two kinds of extract ingredients, causes its hygroscopicity to change.
Embodiment 5 Radix Salviae Miltiorrhizae extract finger printing researchs
The preparation precision of need testing solution takes the Radix Salviae Miltiorrhizae extract that is equivalent to crude drug amount 0.15g in 25mL measuring bottle, methanol constant volume, and ultrasonic 30min dissolves, and filters, and removes subsequent filtrate, obtains Radix Salviae Miltiorrhizae extract need testing solution.
Reference substance solution is by the preparation method preparation of " research of extracting solution finger printing " lower reference substance solution.
(1) foundation of finger printing
Get need testing solution and reference substance solution, press the preparation of extracting solution finger printing test chromatographic condition and detect, record chromatogram in 70min, the total peak of selecting each collection of illustrative plates to produce by the retention time at more each peak, calculate the peak area at total peak, in table 17, table 18, Fig. 9.
Table 17 Radix Salviae Miltiorrhizae extract at low temperature thing peak area
Table 18 Radix Salviae Miltiorrhizae high temperature extraction thing peak area
(2) fingerprint similarity calculates
The chromatographic fingerprints of Chinese materia medica similarity evaluation software (2004A version) that adopts Chinese Pharmacopoeia committee to publish carries out similarity calculating, in table 19.
Table 19 Radix Salviae Miltiorrhizae extract similarity result of calculation
As from the foregoing, Radix Salviae Miltiorrhizae extract finger printing differs greatly.High temperature extraction thing finger printing chromatographic peak is more, and extract at low temperature thing chromatographic peak is less, and high temperature extraction thing ingredient kind lower temperature extract is many; High temperature extraction thing danshensu and protocatechualdehyde chromatographic peak peak area are greater than extract at low temperature thing, and salvianolic acid B peak area is less than extract at low temperature thing.Show, in Radix Salviae Miltiorrhizae extract at low temperature thing, content of danshinolic acid B is greater than high temperature extraction thing, and impurity and other component content higher temperatures extracts are few, and extract at low temperature thing salvianolic acid B purity is higher than high temperature extraction thing.
9 conclusions
By above test, Radix Salviae Miltiorrhizae low-temperature boiling dynamic extraction, salvianolic acid B is stripping to greatest extent, prevents from being destroyed by pyrolytic, and impurity and other composition strippings reduce; Higher temperatures is extracted, and extract at low temperature content of danshinolic acid B is high, and purity is large.Owing to extracting temperature difference, classes of compounds is different from quantity, and extracting solution physical parameter and extract powder property are also variant.
Detection method of the present invention, can effectively effectively separate the multiple compositions in Radix Salviae Miltiorrhizae extract, and peak shape is good, and baseline is steady, for the quality control of Radix Salviae Miltiorrhizae extract provides reliable basis.
Claims (10)
1. the extracting method of Radix Salviae Miltiorrhizae, is characterized in that: it comprises following operating procedure: get salvia piece, add water, under reduced pressure, 70 ± 5 DEG C of boilings are extracted, merge extractive liquid,.
2. extracting method according to claim 1, is characterized in that: amount of water is 5～15 times of Radix Salviae Miltiorrhizae dry weight, is preferably 12 times.
3. extracting method according to claim 1, is characterized in that: extract each 0.5～3h 1～3 time; Preferably extract 3 times each 2h.
4. a preparation method for Radix Salviae Miltiorrhizae extract, is characterized in that: it comprises following operating procedure:
(1) extract by method described in claim 1-3 any one;
(2) get the extracting solution of step (1), after decompression and solvent recovery, lyophilization, obtain Radix Salviae Miltiorrhizae extract.
5. the preparation method of Radix Salviae Miltiorrhizae extract according to claim 4, is characterized in that: the condition of described decompression and solvent recovery is :-0.05～-0.095MPa, 40～90 DEG C; Preferably 70 ± 5 DEG C.
6. the Radix Salviae Miltiorrhizae extract that described in claim 4 or 5 prepared by method, is characterized in that: described Radix Salviae Miltiorrhizae extract contains 5～15% salvianolic acid B.
7. the detection method of Radix Salviae Miltiorrhizae extract, is characterized in that: it is measured with high performance liquid chromatography, and concrete operation step is as follows:
(1) get Radix Salviae Miltiorrhizae extract to be measured, prepare need testing solution;
(2) need testing solution is injected to high performance liquid chromatograph, detect and get final product, its chromatographic condition is as follows:
Chromatographic column: octadecyl silane is immobile phase;
Mobile phase: methanol A-1.0% glacial acetic acid aqueous solution B, gradient elution, elution program A:0min7%, 10min7%, 12min9%, 31min33%, 47min39%, 60min58%, 70min62%;
Detect wavelength: 286 ± 5nm.
8. detection method according to claim 7, is characterized in that: in step (1), taking methanol as solvent or diluent, prepare need testing solution.
9. detection method according to claim 7, is characterized in that: in described high performance liquid chromatography, and taking one or more in salvianolic acid B, protocatechualdehyde or danshensu sodium as reference substance, preparation reference substance solution.
10. detection method according to claim 7, is characterized in that: detecting column temperature is 30 ± 5 DEG C.
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| CN104458957A (en) * | 2014-12-12 | 2015-03-25 | 广西科技大学 | Online sweeping-micellar electrokinetic chromatography method for separating and measuring protocatechualdehyde in acute bronchitis syrup |
| CN105461548A (en) * | 2015-12-22 | 2016-04-06 | 贵州景峰注射剂有限公司 | Method converting danshinolic acid B into tanshinol |
| CN105628696A (en) * | 2015-12-28 | 2016-06-01 | 上海景峰制药有限公司 | Method for determining content of total phenolic acid in salivae miltiorrhizae liguspyragine hydrochloride and glucose injection |
| CN116832124A (en) * | 2023-02-23 | 2023-10-03 | 华润三九现代中药制药有限公司 | Method for preparing glabrous greenbrier rhizome extract |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104458957A (en) * | 2014-12-12 | 2015-03-25 | 广西科技大学 | Online sweeping-micellar electrokinetic chromatography method for separating and measuring protocatechualdehyde in acute bronchitis syrup |
| CN105461548A (en) * | 2015-12-22 | 2016-04-06 | 贵州景峰注射剂有限公司 | Method converting danshinolic acid B into tanshinol |
| CN105628696A (en) * | 2015-12-28 | 2016-06-01 | 上海景峰制药有限公司 | Method for determining content of total phenolic acid in salivae miltiorrhizae liguspyragine hydrochloride and glucose injection |
| CN116832124A (en) * | 2023-02-23 | 2023-10-03 | 华润三九现代中药制药有限公司 | Method for preparing glabrous greenbrier rhizome extract |
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