CN105646628A - Method for preparing chemical constituent in pericarpium trichosanthis injection and application of chemical constituents - Google Patents
Method for preparing chemical constituent in pericarpium trichosanthis injection and application of chemical constituents Download PDFInfo
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- CN105646628A CN105646628A CN201610118571.4A CN201610118571A CN105646628A CN 105646628 A CN105646628 A CN 105646628A CN 201610118571 A CN201610118571 A CN 201610118571A CN 105646628 A CN105646628 A CN 105646628A
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- pericarpium trichosanthis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
- C07H19/167—Purine radicals with ribosyl as the saccharide radical
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/06—Benzopyran radicals
- C07H17/065—Benzo[b]pyrans
- C07H17/07—Benzo[b]pyran-4-ones
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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Abstract
The invention discloses a method for preparing a chemical constituent in pericarpium trichosanthis injection and application of the chemical constituent. The method includes steps of (1), mixing concentrate of the pericarpium trichosanthis injection and silica gel with each other to obtain stirred samples, then carrying out silica gel normal phase column chromatography on the stirred samples, eluting the stirred samples by the aid of first eluents, then eluting the stirred samples by the aid of second eluents, collecting elution liquid and concentrating the elution liquid under the vacuum condition to obtain chromatography products; (2), mixing the chromatography products obtained at the step (1) with methanol to obtain first mixtures, stirring the first mixtures at the room temperature, cooling the first mixtures until the temperatures of the first mixtures are 0-4 DEG C, filtering the first mixtures, uniformly mixing filter cakes with water to obtain second mixtures and then filtering the second mixtures to obtain solid which is diosmetin-7-O-beta-D-glucoside. The first eluents comprise methanol and dichloromethane, and a mass ratio of the methanol to the dichloromethane is 1:(3-4). The second eluents comprise methanol and dichloromethane, and a mass ratio of the methanol to the dichloromethane is 1:(1-1.5). The method and the application have the advantages that the content of a chemical compound prepared by the aid of the method is stable and uniform and can be used as an index for controlling the quality of the pericarpium trichosanthis injection.
Description
Technical field
The present invention relates to drug world, the preparation method and its usage being specifically related in Pericarpium Trichosanthis injection chemical composition.
Background technology
The root (Chinese medicine name Radix Trichosanthis) of Fructus Trichosanthis (Classification system: TrichosantheskirilowiiMaxim.), sarcocarp (Chinese medicine name Fructus Trichosanthis is real), peel (the Chinese medicine name fruit-rind of Chinese trichosanthes), seed (Chinese medicine name semen trichosanthis) are all famous Chinese medicine. Wherein Pericarpium Trichosanthis is made into injection (Pericarpium Trichosanthis injection, the biochemical pharmaceutcal corporation, Ltd of Shanghai medicine-feeding first), the quality excellent with it and curative effect, is widely used in angina pectoris, coronary heart disease etc. at home.
As a kind of common Chinese medicine, Pericarpium Trichosanthis chemical composition is extremely complex, its real effective ingredient is so far not clearly, existing document discloses some Pericarpium Trichosanthis chemical compositions, as: Pericarpium Trichosanthis chemical composition has been carried out detailed summary and analysis by " Fructus Trichosanthis peel chemical composition " (Shandong Traditional Chinese Medicine University Ph.D. Dissertation, Li Aifeng, 2011), when doing water-soluble chemical component and separating, it selects 826 type resins separation to obtain the compound that polarity is relatively small; The patent of Li Aifeng et al.: Pericarpium Trichosanthis chemical composition, by anti-phase preparative liquid chromatography, has also been studied by a kind of a kind of method (CN103304613A) of 4 kinds of ucleosides chemical compositions of purification, a kind of method (CN103304490A) of 5 kinds of purine of purification and pyrimidine bases, method (CN103304611A) separating 3 kinds of flavonoid glycosides of purification from Pericarpium Trichosanthis etc. of separating from Pericarpium Trichosanthis of separating from Pericarpium Trichosanthis. But, above-mentioned document method therefor and Pericarpium Trichosanthis injection industrial preparation process greatly differ from each other, thus above-mentioned literature method separates the compound obtained and can not reflect the true chemical composition of Pericarpium Trichosanthis injection; It addition, above-mentioned document does not carry out effectively except sugar and depigmentation operation before reversed phase column chromatography, and containing abundant saccharide and pigment in Fructus Trichosanthis, chromatographic column causing irreversible damage, therefore, such method is not suitable for industrialized production and poor repeatability.
As a kind of Chinese medicine injection, also there are many documents that Pericarpium Trichosanthis injection chemical composition was carried out report, the production technology of Pericarpium Trichosanthis injection is described by Pericarpium Trichosanthis injection and preparation method thereof (patent CN1460493A): 1, water carries, 2,75% ethanol precipitation, 3, cationic resin separates, collect target phase fraction, carry out concentrating and obtain Pericarpium Trichosanthis injection stock solution, specify that in gained stock solution and be mainly composed of aminoacid.The finger printing of Pericarpium Trichosanthis injection is disclosed and has illustrated by the liquid-phase fingerprint method of testing (patent CN101059482A) of Pericarpium Trichosanthis or Pericarpium Trichosanthis injection, and the primary amino acid in Pericarpium Trichosanthis injection has been controlled by author with pre-column derivatization. The chemical constitution study of Pericarpium Trichosanthis injection is also confined to Amino acids by existing document.
Pericarpium Trichosanthis injection application is more and more extensive, and separation and qualification to its chemical composition are of great significance.
Summary of the invention
It is an object of the invention to provide the preparation method and its usage of chemical composition in a kind of Pericarpium Trichosanthis injection. The method of the present invention can prepare adenosine, diosmetin-7-O-��-D-Glucose glycosides, rutin from Pericarpium Trichosanthis simultaneously, and provides the method controlling Pericarpium Trichosanthis injection quality for reference substance with adenosine, diosmetin-7-O-��-D-Glucose glycosides, rutin. The method of the present invention is simple, reproducible, Environmental Safety.
The present invention utilizes normal phase column chromatography method to, when aminoacid is easily separated in Pericarpium Trichosanthis injection, having been surprisingly found that water-insoluble precipitates. To this precipitation recrystallization, and carry out Structural Identification, it is determined that for diosmetin-7-O-��-D-Glucose glycosides. The present invention is to column chromatography method optimization, and carries out further reversed phase chromatographic column separation, finally get back compound adenosine and rutin. Remove pigment and saccharide before carrying out reversed phase chromatography due to the present invention with normal phase column, therefore the present invention can prepare adenosine and rutin in a large number, furnishes ample material for follow-up quality control.
The present invention is achieved through the following technical solutions above-mentioned purpose:
The present invention provides the preparation method of chemical composition in Pericarpium Trichosanthis injection, and it comprises the steps:
(1) Pericarpium Trichosanthis injection concentrate is mixed with silica gel mix sample, then silica normal phase column chromatography is carried out, it is first methanol by volume ratio: dichloromethane=1:(3��4) carry out eluting as eluant, then it is methanol by volume ratio: dichloromethane=1:(1��1.5) carry out eluting as eluant, collecting eluent, concentrating under reduced pressure obtains chromatography thing;
(2) chromatography thing step (1) obtained and methanol mixed, be stirred at room temperature, and is cooled to 0��4 DEG C, filters, and filters after gained filter cake and water mixing, and gained solid is diosmetin-7-O-��-D-Glucose glycosides.
Wherein, described Pericarpium Trichosanthis injection concentrate refers to that Pericarpium Trichosanthis injection is evaporated to dripless to drip the material obtained.
Wherein, described Pericarpium Trichosanthis injection can be the Pericarpium Trichosanthis injection that this area is conventional, it is preferred that prepared by following step: after being mixed with water by Pericarpium Trichosanthis, reflux, extract, 3��4 times under normal pressure, united extraction liquid; Concentrating under reduced pressure, adding mass percent concentration is the ethanol of 90%��95%, and adding to ethanol mass percent is 70%��75%, stands, and filters, obtains supernatant; Cation exchange resin column on supernatant, washes with water, then carries out eluting with ammonia as eluant, by eluent acid for adjusting pH to 3.5��4, refrigerated overnight, filters and obtains Pericarpium Trichosanthis injection.
Wherein, described concentrating under reduced pressure is preferably 0.75��2 times of the raw material weight being evaporated to Pericarpium Trichosanthis.
Wherein, the described cation exchange resin in cation exchange resin column is preferably the 732 type storng-acid cation exchange resins purchased from Shanghai Resin Factory.
Wherein, described water is preferably deionized water.
Wherein, the concentration of described ammonia is preferably 1��2mol/L.
Wherein, the temperature of described cold preservation is preferably 0��3 DEG C.
Wherein, in step (1), Pericarpium Trichosanthis injection concentrate mixes with silica gel to be mixed in sample, and the mass ratio of described Pericarpium Trichosanthis injection concentrate and described silica gel is preferably 1:(1��2).
Wherein, in step (1), in silica normal phase column chromatography, the consumption of described silica gel is 5��20 times of preferably Pericarpium Trichosanthis injection concentrate quality.
Wherein, in step (1), described is methanol by volume ratio: dichloromethane=1:(3��4) carry out eluting as eluant and be preferably 3��5 column volumes of eluting.
In step (1), the order number of described silica gel is preferably 100��200 orders.
Target compound can not only be enriched with by the column chromatography of step (1), moreover it is possible to pigment bigger for adsorptivity and saccharide compound is retained on a silica gel column, in order to avoid the reversed phase chromatographic column damaged in latter acts.
In step (2), the mass ratio of described chromatography thing and methanol is preferably 1:(100��150).
In step (2), described methanol is preferably absolute methanol.
In step (2), described water is preferably the hot water of 50��80 DEG C.
Present invention also offers diosmetin-7-O-��-D-Glucose glycosides as reference substance application of diosmetin-7-O-��-D-Glucose glycosides content in detection medicine.
Wherein, described medicine is preferably Pericarpium Trichosanthis injection.
Wherein, described application is preferably comprised following step: Pericarpium Trichosanthis injection and diosmetin-7-O-��-D-Glucose glycosides reference substance solution are carried out high performance liquid chromatography detection respectively, retention time according to diosmetin-7-O-��-D-Glucose glycosides reference substance chromatogram determines the peak of related substances in Pericarpium Trichosanthis injection, and according to the content of diosmetin-7-O-��-D-Glucose glycosides in calculated by peak area Pericarpium Trichosanthis injection.
Wherein, described diosmetin-7-O-��-D-Glucose glycosides reference substance solution is preferably diosmetin-7-O-��-D-Glucose glycosides and water, ethanol and is mixed to form the solution that concentration is 0.3mg/mL, and the volume ratio of water and ethanol is 1:1.
Wherein, described Pericarpium Trichosanthis injection is the Pericarpium Trichosanthis injection that aforementioned preparation process prepares.
Wherein, the condition of described high performance liquid chromatography is preferably as follows: chromatographic column is octadecyl silane chromatographic column, and sample size is 20 �� L, and elution requirement is as follows:
| Time (min) | A water (%) | B acetonitrile (%) | Flow velocity (mL/min) | Detection wavelength |
| 0 | 90 | 10 | 1 | 254nm |
| 5 | 90 | 10 | 1 | 254nm |
| 35 | 0 | 100 | 1 | 254nm |
| 40 | 0 | 100 | 1 | 254nm |
��
Meeting on the basis of this area general knowledge, above-mentioned each optimum condition, can combination in any, obtain the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material are all commercially.
The actively progressive effect of the present invention is in that:
(1) present invention finds and separates to be purified into a monomeric compound first from Pericarpium Trichosanthis injection: diosmetin-7-O-��-D-Glucose glycosides.
(2) it have also been found that a kind of detection method, it is possible to for reference substance with diosmetin-7-O-��-D-Glucose glycosides the diosmetin-7-O-��-D-Glucose glycosides in Pericarpium Trichosanthis injection is detected and controls. Research finds, in five batches of injection, and this compounds content stable uniform, illustrate that the compound diosmetin-7-O-beta-glucosidase that present invention separation obtains can as Pericarpium Trichosanthis injection quality control a index.
Accompanying drawing explanation
Fig. 1 is the HPLC chromatogram of diosmetin-7-O-��-D-Glucose glycosides that embodiment 4 prepares.
Fig. 2 is the HPLC chromatogram of the adenosine that embodiment 5 prepares.
Fig. 3 is the HPLC chromatogram of the rutin that embodiment 5 prepares.
Detailed description of the invention
Mode by the examples below further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments. The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.
Prepared by embodiment 1 Pericarpium Trichosanthis injection stock solution
After 5kg Pericarpium Trichosanthis is cut into small pieces, addition 25L deionized water, reflux, extract, 3 times under normal pressure, each 2 hours, united extraction liquid. Gained extracting solution being concentrated into 10kg, adds 90% ethanol while hot, adding to alcohol content is 75%, and room temperature is placed 48 hours so that it is fully precipitate. Cross leaching supernatant. By gained supernatant concentration to 5L, reclaim ethanol. On 5L concentrated solution, (its resin is purchased from the strong acidic ion resin of Shanghai Resin Factory for 732 type cation exchange resin columns, load the chromatographic column of diameter 40cm length 1.5m, with 25L distilled water prewashing, flow velocity is 500mL/min), adsorption of effective component, discard effluent, 50L deionized water wash is used after end of the sample, then with the ammonia of 1.5mol/L as eluent, enrichment amino acids, concentration, except injecting water to 1L after ammonia, adjusts pH to 3.5-4 with hydrochloric acid, 0 DEG C of refrigerated overnight, filters and obtains Pericarpium Trichosanthis injection stock solution 1L.
Ibid legal system obtains Pericarpium Trichosanthis injection stock solution 20L, takes this stock solution 20L and is evaporated to dripless and drips, obtains Pericarpium Trichosanthis injection extractum 350g.
Embodiment 2 column chromatography enrichment target compound
Taking Pericarpium Trichosanthis injection extractum 100g, mix sample with purchased from the 100-200 order silica gel 200g of Qingdao Marine Chemical Co., Ltd., the 100-200 order silica gel dress post of Ling Qu 1.5kg Qingdao Marine Chemical Co., Ltd., silicagel column specification is diameter 12 centimeters, length 1.0 meters. Select the analytical pure methanol of Chemical Reagent Co., Ltd., Sinopharm Group, dichloromethane as eluant, carry out column chromatography eluting.
Methanol: dichloromethane (volume ratio)=4 column volumes of 1:3 elder generation eluting, discards eluent. Use methanol instead: dichloromethane (volume ratio)=1:1 eluting, collect eluent, and monitor for reference substance thin layer chromatography with rutin, after speckle shown by rutin is thin out, terminating to collect, collect eluent 6L altogether, concentrating under reduced pressure removes the solvent collected in liquid and obtains chromatography thing 0.8g.
Embodiment 3 column chromatography enrichment target compound
Taking Pericarpium Trichosanthis injection extractum 150g, mix sample with purchased from the 100-200 order silica gel 200g of Qingdao Marine Chemical Co., Ltd., the 100-200 order silica gel dress post of Ling Qu 1.5kg Qingdao Marine Chemical Co., Ltd., silicagel column specification is diameter 12 centimeters, length 1.0 meters. Select the analytical pure methanol of traditional Chinese medicines reagent, dichloromethane as eluant, carry out column chromatography eluting.
Methanol: dichloromethane (volume ratio)=5 column volumes of 1:3 elder generation eluting, discards eluent. Use methanol instead: dichloromethane=1:1 (volume ratio), collect eluent, and monitor for reference substance thin layer chromatography with rutin, after speckle shown by rutin is thin out, terminating to collect, collect eluent 8L altogether, concentrating under reduced pressure removes the solvent collected in liquid and obtains chromatography thing 1.0g.
Embodiment 4 recrystallization separation purification diosmetin-7-O-��-D-Glucose glycosides
Taking the chromatography thing 2.5g prepared by embodiment 2 and 3, add the analytical pure methanol 500mL of traditional Chinese medicines groupings of the world economy reagent company limited, stir 10 minutes, be then cooled to 0 DEG C under room temperature, filtration after standing 12 hours, filtrate collection is standby.Filter cake mixes with 80 DEG C of hot water, filtered while hot after stirring 10 minutes, obtain solids 25mg, detecting purity through HPLC is 98% (see figure 1), nuclear-magnetism identifies that (Avance III 600MHzBruker) (for diosmetin-7-O-��-D-Glucose glycosides, will collect the filtrate obtained simultaneously and merge.
Gained diosmetin-7-O-��-D-Glucose glycosides nuclear magnetic data is as follows:1H-NMR(400MHz,DMSO-d6) �� ppm:12.97 (1H, s), 10.01 (1H, br), 7.61 (2H, m), 7.00 (1H, s), 6.95 (1H, d, J=5.6Hz), 6.88 (1H, d, J=1Hz), 6.45 (1H, d, J=1Hz), 5.41 (1H, d, J=3.2Hz), 5.15 (1H, d, J=3.2hz), 5.08 (2H, m), 4.64 (1H, m), 3.90 (3H, s), and 3.1-3.7 (6H, m).13C-NMR(100MHz,DMSO-d6) �� ppm:182.5,164.6,163.5,161.5,157.4,151.4,148.5,121.8,120.9,116.3,110.8,105.8,103.9,100.5,99.9,95.5,77.7,76.9,73.6,70.1,61.0,56.5.
Embodiment 5 reversed phase high-pressure chromatograph prepares adenosine and rutin
Filtrate reduced in volume embodiment 4 filtered, selects VARIANprostarSD-1 to prepare liquid phase systems and is prepared, select 2 inch chromatography column, be prepared by following condition:
| Time (min) | A water (%) | B acetonitrile (%) | Flow velocity (mL/min) | Detection wavelength |
| 0 | 90 | 10 | 80 | 254nm 4 --> |
| 5 | 90 | 10 | 80 | 254nm |
| 35 | 0 | 100 | 80 | 254nm |
| 40 | 0 | 100 | 80 | 254nm |
Collecting the eluent of 10min-11min, concentrating under reduced pressure obtains compound adenosine 208mg, and detecting purity through HPLC was 99% (as shown in Figure 2); Collecting the eluent of 21min-22min, namely concentrating under reduced pressure obtains compound rutin 35mg, and detecting purity through HPLC was 99% (as shown in Figure 3).
Gained rutin nuclear magnetic data following (Avance III 600MHzBruker):1H-NMR(600MHz,DMSO-d6) �� ppm:12.61 (1H, s), 10.70 (1H, brs), 9.55 (1H, brs), 7.55 (1H, s), 7.53 (1H, s), 6.84 (1H, s), 6.39 (1H, s), 6.19 (1H, s), 5.34 (2H, m), 4.39 (1H, s), 1.0 (3H, dJ=5.6Hz).13C-NMR(100MHz,DMSO-d6) �� ppm:177.3,164.0,161.1,156.5,156.3,148.3,144.6,133.2,121.5,116.2,115.1,1.3.9,101.1,100.6,98.6,93.5,76.4,75.8,74.0,71.8,70.5,70.3,69.9,68.1,66.9,17.6.
Gained adenosine nuclear magnetic data following (Avance III 400MHzBruker):1H-NMR(600MHz,DMSO-d6) �� ppm:8.36 (1H, s, 2-H), 8.14 (1H, s, 8-H), 5.88 (1H, d, J=6.4Hz, 1 '-H), 4.61 (1H, t, J=5.6Hz, 2 '-H), 4.15 (1H, m, 3 '-H), 4.11 (1H, m, 3 '-H).13C-NMR(150MHz,DMSO-d6) �� ppm:156.6,152.8,149.6,140.4,119.9,88.4,86.4,73.9,71.1,61 .2.
Embodiment 6 carries out Pericarpium Trichosanthis injection Quality Control with adenosine, diosmetin-7-O-��-D-Glucose glycosides, rutin for reference substance
Accurate preparation adenosine concentration is that 0.3mg/mL aqueous solution, diosmetin-7-O-��-D-Glucose glycosides 0.3mg/mL water/ethanol (volume ratio 1:1) solution and rutin 0.3mg/mL methanol solution are as reference substance, test sample selects five batches of Pericarpium Trichosanthis injections (the biochemical pharmaceutcal corporation, Ltd of Shanghai medicine-feeding first), chromatographic condition is as follows: chromatographic column selects octadecyl silane Diamonsil, C18 (4.6 �� 250mm, 5 ��m), elution requirement is as follows:
| Time (min) | A water (%) | B acetonitrile (%) | Flow velocity (mL/min) | Detection wavelength |
| 0 | 90 | 10 | 1 | 254nm |
| 5 | 90 | 10 | 1 | 254nm |
| 35 | 0 | 100 | 1 | 254nm |
| 40 | 0 | 100 | 1 | 254nm |
Test sample and reference substance by each sample introduction 20 �� L of above-mentioned condition, according to reference substance adenosine, diosmetin-7-O-��-D-Glucose glycosides, rutin the retention time at peak determine tie substance and content thereof in test sample.The stable content of adenosine, diosmetin-7-O-��-D-Glucose glycosides, rutin in the Pericarpium Trichosanthis injection of five batches, and conformance with standard after measured. As shown in the table:
Claims (10)
1. the preparation method of chemical composition in Pericarpium Trichosanthis injection, it comprises the steps:
(1) Pericarpium Trichosanthis injection concentrate is mixed with silica gel mix sample, then silica normal phase column chromatography is carried out, it is first methanol by volume ratio: dichloromethane=1:(3��4) carry out eluting as eluant, then it is methanol by volume ratio: dichloromethane=1:(1��1.5) carry out eluting as eluant, collecting eluent, concentrating under reduced pressure obtains chromatography thing;
(2) chromatography thing step (1) obtained and methanol mixed, be stirred at room temperature, and is cooled to 0��4 DEG C, filters, and filters after gained filter cake and water mixing, and gained solid is diosmetin-7-O-��-D-Glucose glycosides.
2. preparation method as claimed in claim 1, it is characterised in that described Pericarpium Trichosanthis injection is prepared by following step: after being mixed with water by Pericarpium Trichosanthis, reflux, extract, 3��4 times under normal pressure, united extraction liquid; Concentrating under reduced pressure, adding mass percent concentration is the ethanol of 90%��95%, and adding to ethanol mass percent is 70%��75%, stands, and filters, obtains supernatant; Cation exchange resin column on supernatant, washes with water, then carries out eluting with ammonia as eluant, by eluent acid for adjusting pH to 3.5��4, refrigerated overnight, filters and obtains Pericarpium Trichosanthis injection.
3. preparation method as claimed in claim 2, it is characterised in that described concentrating under reduced pressure is 0.75��2 times of the raw material weight being evaporated to Pericarpium Trichosanthis;
And/or, the described cation exchange resin in cation exchange resin column is the 732 type storng-acid cation exchange resins purchased from Shanghai Resin Factory;
And/or, described water is deionized water;
And/or, the concentration of described ammonia is 1��2mol/L;
And/or, the temperature of described cold preservation is 0��3 DEG C.
4. preparation method as claimed in claim 1, it is characterised in that in step (1), Pericarpium Trichosanthis injection concentrate mixes with silica gel to be mixed in sample, and the mass ratio of described Pericarpium Trichosanthis injection concentrate and described silica gel is 1:(1��2).
5. preparation method as claimed in claim 1, it is characterised in that in step (1), in silica normal phase column chromatography, the consumption of described silica gel is 5��20 times of Pericarpium Trichosanthis injection concentrate quality;
And/or, in step (1), described is methanol by volume ratio: dichloromethane=1:(3��4) to carry out eluting as eluant be 3��5 column volumes of eluting;
And/or, in step (1), the order number of described silica gel is 100��200 orders.
6. preparation method as claimed in claim 1, it is characterised in that in step (2), the mass ratio of described chromatography thing and methanol is 1:(100��150);
And/or, in step (2), described methanol is absolute methanol;
And/or, in step (2), described water is the hot water of 50��80 DEG C.
7. diosmetin-7-O-��-D-Glucose glycosides is as reference substance application of diosmetin-7-O-��-D-Glucose glycosides content in detection medicine.
8. apply as claimed in claim 7, it is characterised in that described medicine is Pericarpium Trichosanthis injection.
9. apply as claimed in claim 8, it is characterized in that, described application comprises the steps: Pericarpium Trichosanthis injection and diosmetin-7-O-��-D-Glucose glycosides reference substance solution are carried out high performance liquid chromatography detection respectively, retention time according to diosmetin-7-O-��-D-Glucose glycosides reference substance chromatogram determines the peak of related substances in Pericarpium Trichosanthis injection, and according to the content of diosmetin-7-O-��-D-Glucose glycosides in calculated by peak area Pericarpium Trichosanthis injection.
10. apply as claimed in claim 9, it is characterized in that, described diosmetin-7-O-��-D-Glucose glycosides reference substance solution is diosmetin-7-O-��-D-Glucose glycosides and water, ethanol are mixed to form the solution that concentration is 0.3mg/mL, and the volume ratio of water and ethanol is 1:1;
And/or, the condition of described high performance liquid chromatography is as follows: chromatographic column is octadecyl silane chromatographic column, and sample size is 20 �� L, and elution requirement is as follows:
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| CN110376294A (en) * | 2019-05-28 | 2019-10-25 | 神威药业集团有限公司 | A kind of construction method of Snakegourd Fruit granule finger-print |
| CN110376294B (en) * | 2019-05-28 | 2021-07-23 | 神威药业集团有限公司 | Method for constructing fingerprint spectrum of snakegourd fruit formula particles |
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