CN104820026A - Litchi seed dripping pill quality detection method - Google Patents
Litchi seed dripping pill quality detection method Download PDFInfo
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- CN104820026A CN104820026A CN201410195930.7A CN201410195930A CN104820026A CN 104820026 A CN104820026 A CN 104820026A CN 201410195930 A CN201410195930 A CN 201410195930A CN 104820026 A CN104820026 A CN 104820026A
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- procyanidin
- dripping pill
- reference substance
- acetone
- semen litchi
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Abstract
The present invention provides a litchi seed dripping pill quality detection method, which comprises: taking a procyanidin reference substance, and adding methanol to dissolve so as to prepare a reference substance solution; taking litchi seed dripping pills, grinding, precisely weighing 0.5 g of the litchi seed dripping pills, adding to 40 mL of acetone with a volume concentration of 70% to carry out ultrasonic extraction, filtering, washing the residue with acetone, combining the filtrates, carrying out water bath heating so as to completely evaporate the acetone, dissolving the residue with methanol and transferring into a measuring flask, diluting with methanol to achieve the scale, shaking to achieve a uniform state, filtering with a 0.2 [mu]m micro-pore filtration membrane, and adopting the subsequent filtrate as a test solution; precisely sucking the reference substance solution to load, and measuring the peak area to obtain a standard curve equation; and precisely sucking the test solution to load, measuring the peak area, and substituting into the standard curve equation to calculate the procyanidin content. According to the present invention, the specificity of the method is good, and the procyanidin content in the litchi seed dripping pill can be well, efficiently, stably, quickly and sensitively determined so as to effectively detect the litchi seed dripping pill quality and ensure the medication safety.
Description
Technical field
The invention belongs to Control of drug quality field, be specifically related to a kind of semen litchi dripping pill quality determining method, particularly a kind of method measuring semen litchi dripping pill procyanidins content.
Background technology
Procyanidin is the general name of the large class polyphenolic substance extensively existed in plant, form to ten dimer combinations primarily of the monomer of the catechin of varying number and epicatechin, dimer, tripolymer, have the ability of stronger oxidation and scavenging free radicals and improve effect of microcirculation in human body.Modern pharmacological research shows, procyanidin also have anti-inflammatory, antitumor, radioresistance, anti-ageing, improve immunity, improve the formation of bone, anti-diabetic, reducing blood lipid, hypotensive, promote wound healing and organization restoration is active and antitumaous effect (Lexus group etc., the Pharmacological Advancement " Chinese Pharmacological Bulletin " of procyanidin, 2002,18 volumes, the 1st phase: 9-12).Also containing a large amount of procyanidin (Ding Li etc., the research of semen litchi chemical composition, " research and development of natural products ", 18 volumes, the 6th phase: 45-47 in 2006) in preliminary discovery semen litchi.
Chinese patent application CN102836265 discloses one and prepares semen litchi dripping pill preparation technology, and technique is: by semen litchi pulverizing medicinal materials, and the 70v/v% ethanol adding 8 ~ 10 times of medicinal material amounts carries out ultrasonic extraction 2 times, each 1 hour; Merge the extract decompression recycling ethanol of 2 times extremely without using distilled water diluting after alcohol taste with crude drug gauge concentration for 0.4g/ml, hold over night; Filter, filtrate, by AB-8 macroporous resin adsorption, first washes with water, continues and use 75v/v% ethanol elution, collects ethanol eluate; Reclaim ethanol, continue to be concentrated into the low temperature drying of thick paste final vacuum, obtain Semen Litchi extract; Semen Litchi extract and matrix (Macrogol 4000 and Macrogol 6000 proportioning are 1:2 potpourri) is taken respectively by 1:4 amount; Described matrix is heated to melting, adds Semen Litchi extract while stirring, mixing, gained mixed liquor is added pill dripping machine receiver, be incubated 75 DEG C, at the uniform velocity instilled be equipped with in the gatherer of cooling medium by water dropper, the holding temperature of described cooling medium is 5 DEG C, after dripping is complete, take out pill, filter out qualified pill, the agent of removing surface cool, vacuum dry overnight at room temperature, obtains semen litchi dripping pill.For ensureing the safe, effective, stable of medicine, be necessary to monitor dripping pill procyanidins content.
At present, the assay of existing document to semen litchi procyanidin composition is generally adopt " molysite catalytic colorimetry ", and principle utilizes phenolic hydroxyl group feature, produces color and carry out with ferric ion chelating, because more traditional Chinese medicine ingredients all has this kind of phenolic hydroxyl group, therefore this method specificity is poor.Therefore, the meaning that a kind of quality determining method that accurately can detect semen litchi dripping pill procyanidin content has reality is set up.
Summary of the invention
In order to solve the problems of the technologies described above, the object of the present invention is to provide a kind of method that specificity is good, effective, stable, can measure semen litchi dripping pill procyanidins content rapidly, delicately, thus effectively detect the quality of semen litchi dripping pill.
Therefore, the invention provides a kind of semen litchi dripping pill quality determining method, comprise the following steps:
1) get procyanidin reference substance, add methyl alcohol dissolving, constant volume, shake up, make every milliliter of reference substance solution containing procyanidin 1.15mg;
2) semen litchi dripping pill is got, grind, accurately weighed 0.5g, add 40mL volumetric concentration be 70% acetone carry out ultrasonic extraction 30min, filter, residue volumetric concentration is the acetone washing of 70%, and merging filtrate carries out heating water bath and volatilizes acetone, and residue methyl alcohol dissolves and is transferred in 25mL measuring bottle, and with methanol dilution to scale, shake up, filter, using subsequent filtrate as need testing solution with 0.2 μm of miillpore filter;
3) accurate aspiration step 1) reference substance solution 0.5,1,3,5,10,20 μ L sample introduction, measure peak area; Obtain typical curve equation: A=1012.25C-567.89, R
2=0.9986; Accurate aspiration step 2) need testing solution 10 μ L, sample introduction, measures peak area, substitutes into above-mentioned typical curve equation and calculates procyanidin content in semen litchi dripping pill.
According to method of the present invention, preferably, the chromatographic column used is CAPCELL PAK C
18uG120S3, specification is 4.6mm × 100mm, 5 μm; Mobile phase is acetonitrile-0.1% phosphoric acid (10:90), and flow velocity is 1.0mL/min, and column temperature is 30 DEG C; Determined wavelength is 280nm, and theoretical cam curve calculates by procyanidin and is at least 2000.
Compared with traditional detection method of the prior art, the invention provides a kind of new semen litchi dripping pill quality determining method, the method is based on high performance liquid chromatography, step is reasonable in design, by suitable pre-service, effectively can remove the impurity in semen litchi dripping pill, the procyanidin in sample is made to extract more complete, have employed brand-new Stationary liquid and mobile phase simultaneously, in conjunction with rational flow velocity, column temperature, wavelength, make the specificity of whole method better, more effective, more stable, can be rapider, measure the procyanidin content in semen litchi dripping pill delicately, thus effectively detect the quality of semen litchi dripping pill, ensure drug safety.
Embodiment
According to specific embodiment, method of the present invention is described in further detail below, but the present invention is not limited to following examples.It should be noted that the method that the semen litchi dripping pill in the inventive method is recorded according to Chinese invention patent application CN102836265 obtains; The materials such as procyanidin reference substance can obtain (such as the present embodiment is from Shanghai Tauto Biotechnology Co., Ltd.) via commercial channel, used acetonitrile, phosphoric acid and methyl alcohol etc. are chromatographically pure.
Embodiment 1
The chromatographic column that method of the present invention adopts is CAPCELL PAK C
18uG120S3, specification is: 4.6mm × 100mm, 5 μm; Mobile phase is acetonitrile-0.1% phosphoric acid (volume ratio is 10:90), and flow velocity is 1.0mL/min, and column temperature is 30 DEG C; Determined wavelength is 280nm.Theoretical cam curve calculates by procyanidin and is at least 2000.Wherein, 0.1% phosphoric acid refers to the concentration of aqueous solution of phosphoric acid is 0.1% (percent by volume).
According to following steps, quality testing is carried out to semen litchi dripping pill:
1. precision takes procyanidin reference substance, is placed in volumetric flask, and add methyl alcohol and dissolve, constant volume, shakes up, and makes every milliliter of reference substance solution containing procyanidin 1.15mg.
2. get semen litchi dripping pill (about 20), grind, accurately weighed 0.5g, adds the acetone ultrasonic extraction 30min that 40mL volumetric concentration is 70%, filter, residue appropriate volume concentration is the acetone washing of 70%, and merging filtrate carries out heating water bath and volatilizes acetone, and residue methyl alcohol dissolves and is transferred in 25mL measuring bottle, and be diluted to scale with proper amount of methanol, shake up, filter, using subsequent filtrate as need testing solution with 0.2 μm of miillpore filter;
3. precision draws procyanidin reference substance solution 0.5,1,3,5,10,20 μ L sample introduction respectively, measures peak area.Return with the logarithm value of the logarithm value of different sample size C and integrating peak areas value A, obtain typical curve equation: A=1012.25C-567.89, R
2=0.9986.
Accurate absorption reference substance solution 10 μ L, in injection liquid chromatography, measure peak area, repeat sample introduction 6 times, recording procyanidin peak area RSD is 1.69%, shows that instrument precision is good.
In the dripping pill need testing solution of known procyanidin content, quantitatively add reference substance solution, mixing, draws 10 μ L, measures peak area by above condition, calculates the amount of procyanidin reference substance according to typical curve, and calculate the recovery, the recovery is 99.0%.
The accurate absorption reference substance solution 10 μ L at 0,1,2,4,8,24 hour respectively, in injection liquid chromatography, measure peak area, peak area RSD is 2.13%, shows that need testing solution is stable in 24 hours.
4. accurate absorption need testing solution 10 μ L, sample introduction, measures peak area, according to typical curve calculation sample procyanidin content.As calculated, semen litchi dripping pill procyanidins content is 0.392g/g.
Reference examples
" molysite catalytic colorimetry " is adopted to detect the procyanidin content in semen litchi dripping pill (method recorded by Chinese invention patent application CN102836265 obtains).
1. preparation standard curve: precision takes procyanidin reference substance 10mg, dissolves and constant volume with methyl alcohol, is mixed with the standard solution that concentration is 1.0mg/mL; Use methanol dilution again, be mixed with the standard series use liquid that concentration is 0,5,10,20,40,80,160,320 μ g/mL.Drawing variable concentrations standard series respectively uses liquid 1.0mL in 10mL scale test tube, add 6mL n-butyl alcohol-hydrochloric acid (V/V=95:5) and 0.2mL ammonium ferric sulfate hydrochloric acid solution successively, shake up, be stoppered test tube plug, heat 40min in boiling water bath after, take out immediately, with 40 DEG C of water cooling 5min, taking-up is put and measure absorbance A value according to the UV-spectrophotometry of " Chinese Pharmacopoeia " annex at wavelength 550nm place to room temperature, with absorbance A and procyanidin concentration mapping, drawing curve, obtaining typical curve equation is: A=0.0031C+0.0935, R
2=0.9995.
2. sample determination: get semen litchi dripping pill (about 20), grind, accurately weighed 0.2g, adds proper amount of methanol, ultrasonic extraction 30min, and filter, residue methanol wash, merging filtrate is transferred to 10mL volumetric flask, methanol constant volume; The accurate 1ml that draws is in 10ml scale test tube, add 6mL n-butyl alcohol-hydrochloric acid (V/V=95:5) and 0.2mL ammonium ferric sulfate hydrochloric acid solution successively, shake up, be stoppered test tube plug, heat 40min in boiling water bath after, take out immediately, with 40 DEG C of water cooling 5min, taking-up is put and to room temperature, is measured absorbance A value at wavelength 550nm place, according to typical curve calculation sample procyanidin content.The procyanidin content that result display measures by this method is more higher than actual value, and in fact include the content of other impurity with the numerical value that " molysite catalytic colorimetry " records, therefore specificity is poor, is not suitable for the Accurate Measurement of procyanidin content.
Claims (2)
1. a semen litchi dripping pill quality determining method, comprises the following steps:
1) get procyanidin reference substance, add methyl alcohol dissolving, constant volume, shake up, make every milliliter of reference substance solution containing procyanidin 1.15mg;
2) semen litchi dripping pill is got, grind, accurately weighed 0.5g, add 40mL volumetric concentration be 70% acetone carry out ultrasonic extraction 30min, filter, residue volumetric concentration is the acetone washing of 70%, and merging filtrate carries out heating water bath and volatilizes acetone, and residue methyl alcohol dissolves and is transferred in 25mL measuring bottle, and with methanol dilution to scale, shake up, filter, using subsequent filtrate as need testing solution with 0.2 μm of miillpore filter;
3) accurate aspiration step 1) reference substance solution 0.5,1,3,5,10,20 μ L sample introduction, measure peak area; Obtain typical curve equation: A=1012.25C-567.89, R
2=0.9986; Accurate aspiration step 2) need testing solution 10 μ L, sample introduction, measures peak area, substitutes into above-mentioned typical curve equation and calculates procyanidin content in semen litchi dripping pill.
2. method according to claim 1, is characterized in that: the chromatographic column used is CAPCELL PAK C
18uG120S3, specification is 4.6mm × 100mm, 5 μm; Mobile phase is acetonitrile-0.1% phosphoric acid (10:90), and flow velocity is 1.0mL/min, and column temperature is 30 DEG C; Determined wavelength is 280nm, and theoretical cam curve calculates by procyanidin and is at least 2000.
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| CN201410195930.7A CN104820026A (en) | 2014-05-09 | 2014-05-09 | Litchi seed dripping pill quality detection method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105353065A (en) * | 2015-12-22 | 2016-02-24 | 黑龙江大学 | Establishing method of HPLC (high-performance liquid chromatography) fingerprint spectrum of lychee seeds, standard fingerprint spectrum obtained with method and application of standard fingerprint spectrum |
| CN108169384A (en) * | 2018-03-07 | 2018-06-15 | 广西中医药大学 | A kind of method for building up of lichee nuclear particle HPLC finger-prints |
| CN110044832A (en) * | 2019-03-11 | 2019-07-23 | 广西中医药大学 | The measuring method of general flavone content in a kind of semen litchi |
| CN110638896A (en) * | 2019-09-09 | 2020-01-03 | 天津科技大学 | Application of lychee seed extract with effects of reducing blood pressure and improving complications of lychee seed extract and preparation method of preparation |
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| CN1892212A (en) * | 2005-06-30 | 2007-01-10 | 三得利株式会社 | Method for procyanidin analysis |
| CN1911314A (en) * | 2006-08-14 | 2007-02-14 | 程颖 | Plant extractive and its prepn. method |
| CN102836265A (en) * | 2012-09-24 | 2012-12-26 | 广州市中医医院 | Litchi seed dropping pill and preparation method thereof |
| CN103698432A (en) * | 2013-12-26 | 2014-04-02 | 广州市中医医院 | Method for measuring saponin content in lychee seed extract |
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2014
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1892212A (en) * | 2005-06-30 | 2007-01-10 | 三得利株式会社 | Method for procyanidin analysis |
| CN1911314A (en) * | 2006-08-14 | 2007-02-14 | 程颖 | Plant extractive and its prepn. method |
| CN102836265A (en) * | 2012-09-24 | 2012-12-26 | 广州市中医医院 | Litchi seed dropping pill and preparation method thereof |
| CN103698432A (en) * | 2013-12-26 | 2014-04-02 | 广州市中医医院 | Method for measuring saponin content in lychee seed extract |
Non-Patent Citations (6)
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| K. NAGENDRA PRASAD ET AL.: "Identification of phenolic compounds and appraisal of antioxidant and antityrosinase activities from litchi (Litchi sinensis Sonn.) seeds", 《FOOD CHEMISTRY》, vol. 116, no. 1, 1 September 2009 (2009-09-01), XP026053892, DOI: doi:10.1016/j.foodchem.2009.01.079 * |
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| 周玮婧 等: "荔枝皮原花青素提取工艺优化", 《农业工程学报》, vol. 25, 30 June 2009 (2009-06-30) * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105353065A (en) * | 2015-12-22 | 2016-02-24 | 黑龙江大学 | Establishing method of HPLC (high-performance liquid chromatography) fingerprint spectrum of lychee seeds, standard fingerprint spectrum obtained with method and application of standard fingerprint spectrum |
| CN108169384A (en) * | 2018-03-07 | 2018-06-15 | 广西中医药大学 | A kind of method for building up of lichee nuclear particle HPLC finger-prints |
| CN110044832A (en) * | 2019-03-11 | 2019-07-23 | 广西中医药大学 | The measuring method of general flavone content in a kind of semen litchi |
| CN110638896A (en) * | 2019-09-09 | 2020-01-03 | 天津科技大学 | Application of lychee seed extract with effects of reducing blood pressure and improving complications of lychee seed extract and preparation method of preparation |
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Application publication date: 20150805 |