CN105985392A - Preparation method of quercetin-3-O-beta-D-glucuronic acid - Google Patents
Preparation method of quercetin-3-O-beta-D-glucuronic acid Download PDFInfo
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- CN105985392A CN105985392A CN201510053441.2A CN201510053441A CN105985392A CN 105985392 A CN105985392 A CN 105985392A CN 201510053441 A CN201510053441 A CN 201510053441A CN 105985392 A CN105985392 A CN 105985392A
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Abstract
The invention discloses a preparation method of quercetin-3-O-beta-D-glucuronic acid. According to the invention, a traditional Chinese medicine perfoliote knotweed herb is adopted as a raw material and is extracted; an obtained extraction liquid is filtered and concentrated; an ethanol content is regulated to 40-80%; impurities are removed; a supernatant is concentrated to dryness and is dissolved with methanol-water; reverse phase column chromatography is carried out; elution is carried out with 20-70% methanol; thin layer chromatography detection is carried out; a fraction containing a target substance is processed through recrystallization, depigmentation and drying, such that quercetin-3-O-beta-D-glucuronic acid pure products with purity higher than 99% can be obtained in batches. The method provided by the invention has the advantages of low organic solvent dose, low toxicity, simple operation and low cost. The method is suitable for laboratory small-scale preparation and pilot-scale production.
Description
Technical field
The invention belongs to field of medicaments, be specifically related to the preparation method of a kind of Quercetin-3-O-β-D-Glucose aldehydic acid reference substance.
Background technology
Quercetin-3-O-β D-Glucose aldehydic acid (structure is as follows) is the flavonoid active component that a kind of nature exists or generates through Quercetin internal metabolism, has anti-oxidation stress and inflammation [Wang Min, Liu Baolin, state Xu Dan.Quercetin and metabolite inhibited oxidation thereof stress be with inflammation.Food Science.null2013，34 (15): 256-260]、Anti-senile dementia [Ho M，Ferruzzi M.G， Janle E.M，et al.Identification of brain-targeted bioactive dietary quercetin-3-O-glucuronide as a novel intervention for Alzheimer’s disease.FASEB，2013，27，1-13]、Lipotropism oxidation [Janisch K.M，Williamson G， Need P，Et al.Properties of quercetin conjugates:Modulation of LDL oxidation and binding to human serum albumin.Free Radical Research.2004，38(8)，877-884.]、Antiplatelet aggregation [Wright B，Moraes1L.A，Kemp C.F，et al.A structural basis for the inhibition of collagen-stimulated platelet function by quercetin and structurally related flavonoids.British Jounal of Pharmacology，2010，159，1312-1325] etc. biological activity，At present in treatment diseases associated with senescence (patent No.: KR2013/008 031)、Struvite related cardiovascular disease (the patent No.: US 20130109641A1、The aspects such as osteoporosis (patent No.: US 20040162247) all have patent application，Show that it has a extensive future in treatment and health medicine field.But its material source is still its application prospect major obstacle of restriction.Chinese Industrial Standards (CIS) material center not this reference substance at present, and Sigma Co., USA's Quercetin-3-O-β D-Glucose aldehydic acid reference substance price is up to 1400 yuan/milligram.China's natural pharmaceutical resources is the abundantest, therefore needs badly and finds the medical material rich in Quercetin-3-O-β-D-Glucose aldehydic acid from nature, studies simple preparation technology, reduces preparation cost, blank to fill up the domestic wide raw material sources of this prospect in medicine.
Herba Polygoni cymosi, dry aerial parts for polygonaceae plant Herba Polygoni cymosi, have another name called and pass through leaf knotweed, Herba Lespedezae Cuneatae, Herba Polygoni cymosi, Herba Damnacanthi, plough point grass etc., there is heat-clearing and toxic substances removing, inducing diuresis to remove edema, cough-relieving, for diseases such as laryngopharynx swelling and pain, cough due to lung-heat, children's's pertussis, edema oliguria, damp-heat dysentery, eczema, furuncle and phyma, snake bite and insect stings.Herba Polygoni cymosi is distributed all over China, and aboundresources, cheaper starting materials is easy to get.By liquid phase-mass spectrometry on-line analysis technology (see accompanying drawing 1), we find that Herba Polygoni cymosi Main Flavonoids composition is Quercetin-3-O-β-D-Glucose aldehydic acid, but the most relevant Herba Polygoni cymosi flavones ingredient is reported, it mostly is Quercetin 3-O-β-D-Glucose aldehydic acid derivant and aglycon quercetin [5-7] thereof, analyzes the condition during Quercetin 3-O-β-D-Glucose aldehydic acid separates or extracts that is probably and be acutely derivatized or hydrolyze caused by generation artifact.And at present from Herba Polygoni cymosi separation prepare that the preparation method of Quercetin-3-O-β-D-Glucose aldehydic acid (patent No.: CN 101851261A) need to be extracted with high concentration ethanol, organic solvent extracts, the troublesome operation such as silica gel column chromatography repeatedly, and consumption of organic solvent is big, toxicity is big, cost is high.We study employing simple separation technique can prepare Quercetin-3-O-β d-glucuronic acid sterling in a large number from Herba Polygoni cymosi.
Summary of the invention
The present invention seeks to overcome prior art not enough, it is provided that a kind of simple to operate, environmental protection, the preparation method of Quercetin-3-O-β-D-Glucose aldehydic acid that with low cost and products obtained therefrom purity is high.
For solve above-mentioned technical problem, the present invention by the following technical solutions:
A kind of preparation method of Quercetin-3-O-β-D-Glucose aldehydic acid, its Integral Thought is water or Diluted Alcohol extraction medical material, alcohol water precipitation impurity, and reversed phase column chromatography preparation tentatively obtains target substance, recrystallizing and refining, and its concrete preparation process is as follows:
A. take Herba Polygoni cymosi medical material cutting, with water or Diluted Alcohol heating and refluxing extraction 2 times, each 30～90min, filter, united extraction filtrate；
B. extracting filtrate and be concentrated into proper volume, the ethanol content in regulation filtrate is 40～80%, stand at low temperature；Filter or sucking filtration, remove precipitation；
C. supernatant vacuum-concentrcted after precipitate with ethanol, 20-70% methanol-water dissolves the sample Han object, upper ODS reversed phase chromatography post, 20-70% methanol-eluted fractions.
D.30-60% methanol/ethanol eluted fraction concentrates, and methanol redissolves, thin layer analysis detection, containing object flow point stand at low temperature, separates out buff powder.
E. centrifugation or draw mother solution, transfer separates out material, and mother solution continues recrystallization, coarse-grain dehydrated alcohol eluting or gel filtration chromatography depigmentation, until HPLC detection purity is more than 98%, is dried, i.e. prepares Quercetin-3-O-β-D-Glucose aldehydic acid sterling.
The present invention compares with existing Quercetin-3-O-β-D-Glucose aldehydic acid purification preparation technology, have the following advantages, without loaded down with trivial details time-consuming, cost is high, toxicity is big multi-step separates, chemosynthesis or conditioning apparatus is harsh, cost is high method for tissue culture, cheap and easy to get, only need to use alcohol-aqueous solvent, environmental protection and separation filler renewable, energy Reusability, it is simple to operate, it is not necessary to use valuable instrument and equipment, so that sample preparation cost lowers significantly.
Accompanying drawing explanation
With specific embodiment, the present invention is further elaborated below in conjunction with the accompanying drawings.
Fig. 1 is Herba Polygoni cymosi medical material liquid phase-mass spectrometry (LC-MS) chromatogram.A: diode array detector (PDA) response spectrogram；B: extract ion stream (EIC, m/z 477) spectrogram (A, B are two kinds of results under two kinds of detection modes, and side-by-side comparison is not separated).
Fig. 2 is Quercetin-3-O-β-D-Glucose aldehydic acid liquid phase-mass spectrometry (LC-MS) chromatogram.A: diode array detector (PDA) response spectrogram；Total ion current (TIC) spectrogram under B: electron spray ionisation (ESI) negative ion mode.
Fig. 3 is Quercetin-3-O-β-D-Glucose aldehydic acid liquid phase-mass spectrometry (LC-MS) chromatogram.A: diode array detector (PDA) response spectrogram；Total ion current (TIC) spectrogram under B: electron spray ionisation (ESI) positive ion mode.
Fig. 4 is Quercetin-3-O-β-D-Glucose aldehydic acid negative ion mode mass spectrum.A: high-resolution electron spray first mass spectrometric (HR-ESI-MS)；B: high-resolution electron spray second order ms (HR-ESI-MS/MS) mass spectrum.
Detailed description of the invention
Embodiment 1
Take Herba Polygoni cymosi medical material 400g (about 4cm/ section), add 8 times amount water micro-boiling heating and refluxing extraction 2 times, each 1 hour, gauze filters, it is concentrated into and is equivalent to medical material concentration 0.8g/ml, add ethanol adjusting its concentration is 50%, stand at low temperature, to be precipitated completely, supernatant filters, concentrate, sample 20% methanol dissolves, the open chromatographic column (diameter 5cm × long 32cm) of upper ODS, methanol-0.05% acetic acid water (20: 80 → 35: 65 → 45: 55) gradient elution, every half column volume is 1 eluted fraction, 2 column volumes of each ratio eluting, 45% methanol-eluted fractions flow point concentrating under reduced pressure, methanol redissolves, thin layer is analysed, CHCl3-MeOH-H2O-HCOOH (7: 3: 0.5: 0.2) launches, obvious skin dark stain point (Rf value Rf about 0.4) is had under 254nm, flow point stand at low temperature, waits to separate out a large amount of buff powder, removes band pigment mother solution, the a small amount of washing with alcohol of crystalline portion, being dried, weigh, HPLC purity assay is 99.8%, LC-MS electron spray negative ions pattern analysis all only has 1 chromatographic peak (see accompanying drawing 2,3).
Quercetin-3-O-β-D-Glucose aldehydic acid Structural Identification: light yellow amorphous powder (MeOH), UV λmax(MeOH) nm:256,355nm；HR-ESI-MS:m/z [M-H]-477.06616(C21H17O13, value of calculation: 477.06746), molecular formula is C21H18O13；MS/MS (ESI): m/z (%): 301.03442 (100). (see Fig. 4).
[Yao is newborn for Duan Yinghui, Dai Yi, Gao Hao, leaf literary talent for data above and document report.The chemical constitution study of Herba Pileae Scriptae.Chinese herbal medicine, 2010,41 (1): 29-32], basically identical.
Embodiment 2
Take Herba Polygoni cymosi medical material 400g (about 4cm/ section), add 8 times amount 30% ethanol micro-boiling heating and refluxing extraction 2 times, each 1 hour, gauze filters, and is concentrated into and is equivalent to medical material concentration 0.8g/ml, and add ethanol adjusting its concentration is 50%, stand at low temperature, to be precipitated supernatant filters completely, concentrates, sample 20% methanol dissolves, the open chromatographic column (diameter 5cm × long 32cm) of upper ODS, methanol-water (20: 80 → 30: 70 → 40: 60) gradient elution, every half column volume is 1 eluted fraction, each flow point concentrating under reduced pressure, methanol redissolves, and thin layer is analysed, CHCl3-MeOH-H2O-HCOOH (7: 3: 0.5: 0.2) launches, and has obvious skin dark stain point for containing object flow point (Rf value Rf about 0.4), stand at low temperature under 254nm, waiting to separate out a large amount of buff powder, crystallization unit methanol dissolves, upper gel chromatographic columns, methanol-eluted fractions, TLC detects CHCl3-MeOH-H2O-HCOOH (7: 3: 0.5: 0.2) launches, and compares with reference substance, and under 254nm, same position speckle is object.
Embodiment 3
Take Herba Polygoni cymosi medical material 400g (about 4cm/ section), add 8 times amount water micro-boiling heating and refluxing extraction 2 times, each 1 hour, gauze filters, being concentrated into and be equivalent to medical material concentration 0.8g/ml, add ethanol adjusting its concentration is 50%, stand at low temperature, to be precipitated completely, supernatant filters, and concentrates, and sample 20% methanol dissolves, upper ODS mesolow chromatographic column (diameter 4cm × long 33cm), methanol-0.05% acetic acid water (20: 80 → 30: 70 → 40: 60) gradient elution, 40% methanol-eluted fractions flow point concentrating under reduced pressure, methanol redissolves, thin layer is analysed, CHCl3-MeOH-H2O-HCOOH (7: 3: 0.5: 0.2) launches, obvious skin dark stain point (Rf value Rf about 0.4) flow point stand at low temperature is had under 254nm, wait to separate out a large amount of buff powder, remove band pigment mother solution, the a small amount of washing with alcohol of crystalline portion, being dried, weigh, HPLC purity assay is more than 99%.
Embodiment 4
Take Herba Polygoni cymosi medical material 400g (about 4cm/ section), add 8 times amount water micro-boiling heating and refluxing extraction 2 times, each 1 hour, gauze filters, being concentrated into and be equivalent to medical material concentration 0.8g/ml, add ethanol adjusting its concentration is 50%, stand at low temperature, to be precipitated completely, supernatant filters, and concentrates, and sample 20% methanol dissolves, upper ODS mesolow chromatographic column (diameter 4cm × long 33cm), methanol-0.05% acetic acid water (20: 80 → 30: 70 → 40: 60) gradient elution, 40% methanol-eluted fractions flow point concentrating under reduced pressure, methanol redissolves, thin layer is analysed, CHCl3-MeOH-H2O-HCOOH (7: 3: 0.5: 0.2) launches, and has obvious skin dark stain point (Rf value Rf about 0.4) flow point stand at low temperature, wait to separate out a large amount of buff powder under 254nm, removing band pigment mother solution, crystallization unit methanol dissolves, upper gel chromatographic columns, methanol-eluted fractions, TLC detects CHCl3-MeOH-H2O-HCOOH (7: 3: 0.5: 0.2) launches, and compares with reference substance, and under 254nm, same position speckle is object, and HPLC purity assay is more than 99%.
The present invention concrete technical scheme required for protection, is not limited to the concrete of the technical scheme expressed by above-described embodiment and combines.
Claims (3)
1. the preparation method of Quercetin-3-O-β-D-Glucose aldehydic acid, it is characterised in that step is as follows:
A. take Herba Polygoni cymosi medical material cutting, with water or Diluted Alcohol heating and refluxing extraction 2 times, each 30～90min, filter, united extraction filtrate；
B. extracting filtrate and be concentrated into proper volume, the ethanol content in regulation filtrate is 40～80%, stand at low temperature；Filter or sucking filtration, remove precipitation；
C. supernatant vacuum-concentrcted after precipitate with ethanol, 20-70% methanol-water dissolves the sample Han object, reversed phase column chromatography, 20-70% methanol-eluted fractions；
D. methanol-eluted fractions flow point concentrates, and methanol redissolves, thin layer analysis detection, containing object flow point stand at low temperature, separates out buff powder；
E. centrifugation or draw mother solution, transfer separates out material, and mother solution continues recrystallization, coarse-grain dehydrated alcohol eluting or gel filtration chromatography depigmentation, until HPLC detection purity is more than 98%, is dried, i.e. prepares Quercetin-3-O-β-D-Glucose aldehydic acid sterling.
The preparation method of Quercetin-3-O-β-D-Glucose aldehydic acid the most according to claim 1, it is characterised in that: in step 1, column chromatography eluting solvent is methanol-water or methanol-diluted acid aqueous systems, and diluted acid water is formic acid water or the acetic acid water that concentration is less than 1%.
The preparation method of Quercetin-3-O-β-D-Glucose aldehydic acid the most according to claim 1, it is characterised in that: the filler of reversed phase column chromatography is anti-phase C-18 or C-8 filler, and chromatographic column is open or mesolow column chromatography system.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109467581A (en) * | 2018-12-19 | 2019-03-15 | 南京宸翔医药研究有限责任公司 | A kind of extracting method of lotus flavone glycosides and pharmaceutical preparation including lotus flavone glycosides |
| CN109942640A (en) * | 2019-02-21 | 2019-06-28 | 广东省中医院(广州中医药大学第二附属医院广州中医药大学第二临床医学院广东省中医药科学院) | A method of separating phellodendrine glucuronide conjugate from urine |
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| CN101851261A (en) * | 2010-06-10 | 2010-10-06 | 贵州师范大学 | Polygonum perfoliatum medicinal material, method for preparing reference substance of active constituents in preparation thereof as well as content determination method |
| CN102010453A (en) * | 2010-11-03 | 2011-04-13 | 杨达 | Flavonol glycosides compound extracted from loropetalum chinense leaf and medicinal application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109467581A (en) * | 2018-12-19 | 2019-03-15 | 南京宸翔医药研究有限责任公司 | A kind of extracting method of lotus flavone glycosides and pharmaceutical preparation including lotus flavone glycosides |
| CN109942640A (en) * | 2019-02-21 | 2019-06-28 | 广东省中医院(广州中医药大学第二附属医院广州中医药大学第二临床医学院广东省中医药科学院) | A method of separating phellodendrine glucuronide conjugate from urine |
| CN109942640B (en) * | 2019-02-21 | 2021-10-26 | 广东省中医院(广州中医药大学第二附属医院广州中医药大学第二临床医学院广东省中医药科学院) | Method for separating phellodendrine glucuronic acid conjugate from urine |
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