CN101334390A - Determination method for morinda root oligosacchride of morinda root Chinese herb or its extract - Google Patents
Determination method for morinda root oligosacchride of morinda root Chinese herb or its extract Download PDFInfo
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- CN101334390A CN101334390A CNA2008101340927A CN200810134092A CN101334390A CN 101334390 A CN101334390 A CN 101334390A CN A2008101340927 A CNA2008101340927 A CN A2008101340927A CN 200810134092 A CN200810134092 A CN 200810134092A CN 101334390 A CN101334390 A CN 101334390A
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- 239000000284 extract Substances 0.000 title claims abstract description 36
- 241000157491 Morinda Species 0.000 title claims description 68
- 235000017524 noni Nutrition 0.000 title claims description 68
- 238000000034 method Methods 0.000 title claims description 65
- 229920001542 oligosaccharide Polymers 0.000 claims abstract description 159
- 150000002482 oligosaccharides Chemical class 0.000 claims abstract description 157
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 57
- 239000013558 reference substance Substances 0.000 claims abstract description 47
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 44
- 241000096284 Gynochthodes officinalis Species 0.000 claims abstract description 39
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 17
- 238000010812 external standard method Methods 0.000 claims abstract description 10
- 229920001202 Inulin Polymers 0.000 claims description 130
- 229940029339 inulin Drugs 0.000 claims description 130
- JYJIGFIDKWBXDU-MNNPPOADSA-N inulin Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@]1(OC[C@]2(OC[C@]3(OC[C@]4(OC[C@]5(OC[C@]6(OC[C@]7(OC[C@]8(OC[C@]9(OC[C@]%10(OC[C@]%11(OC[C@]%12(OC[C@]%13(OC[C@]%14(OC[C@]%15(OC[C@]%16(OC[C@]%17(OC[C@]%18(OC[C@]%19(OC[C@]%20(OC[C@]%21(OC[C@]%22(OC[C@]%23(OC[C@]%24(OC[C@]%25(OC[C@]%26(OC[C@]%27(OC[C@]%28(OC[C@]%29(OC[C@]%30(OC[C@]%31(OC[C@]%32(OC[C@]%33(OC[C@]%34(OC[C@]%35(OC[C@]%36(O[C@@H]%37[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O%37)O)[C@H]([C@H](O)[C@@H](CO)O%36)O)[C@H]([C@H](O)[C@@H](CO)O%35)O)[C@H]([C@H](O)[C@@H](CO)O%34)O)[C@H]([C@H](O)[C@@H](CO)O%33)O)[C@H]([C@H](O)[C@@H](CO)O%32)O)[C@H]([C@H](O)[C@@H](CO)O%31)O)[C@H]([C@H](O)[C@@H](CO)O%30)O)[C@H]([C@H](O)[C@@H](CO)O%29)O)[C@H]([C@H](O)[C@@H](CO)O%28)O)[C@H]([C@H](O)[C@@H](CO)O%27)O)[C@H]([C@H](O)[C@@H](CO)O%26)O)[C@H]([C@H](O)[C@@H](CO)O%25)O)[C@H]([C@H](O)[C@@H](CO)O%24)O)[C@H]([C@H](O)[C@@H](CO)O%23)O)[C@H]([C@H](O)[C@@H](CO)O%22)O)[C@H]([C@H](O)[C@@H](CO)O%21)O)[C@H]([C@H](O)[C@@H](CO)O%20)O)[C@H]([C@H](O)[C@@H](CO)O%19)O)[C@H]([C@H](O)[C@@H](CO)O%18)O)[C@H]([C@H](O)[C@@H](CO)O%17)O)[C@H]([C@H](O)[C@@H](CO)O%16)O)[C@H]([C@H](O)[C@@H](CO)O%15)O)[C@H]([C@H](O)[C@@H](CO)O%14)O)[C@H]([C@H](O)[C@@H](CO)O%13)O)[C@H]([C@H](O)[C@@H](CO)O%12)O)[C@H]([C@H](O)[C@@H](CO)O%11)O)[C@H]([C@H](O)[C@@H](CO)O%10)O)[C@H]([C@H](O)[C@@H](CO)O9)O)[C@H]([C@H](O)[C@@H](CO)O8)O)[C@H]([C@H](O)[C@@H](CO)O7)O)[C@H]([C@H](O)[C@@H](CO)O6)O)[C@H]([C@H](O)[C@@H](CO)O5)O)[C@H]([C@H](O)[C@@H](CO)O4)O)[C@H]([C@H](O)[C@@H](CO)O3)O)[C@H]([C@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@H](O)[C@@H](CO)O1 JYJIGFIDKWBXDU-MNNPPOADSA-N 0.000 claims description 128
- 239000000243 solution Substances 0.000 claims description 58
- 238000012360 testing method Methods 0.000 claims description 43
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 36
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 36
- 230000014759 maintenance of location Effects 0.000 claims description 26
- 239000003814 drug Substances 0.000 claims description 24
- 229910052799 carbon Inorganic materials 0.000 claims description 21
- 239000000463 material Substances 0.000 claims description 21
- 238000010828 elution Methods 0.000 claims description 19
- 238000002360 preparation method Methods 0.000 claims description 18
- 229940079593 drug Drugs 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 14
- 239000000047 product Substances 0.000 claims description 14
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 13
- 238000004440 column chromatography Methods 0.000 claims description 11
- 239000000706 filtrate Substances 0.000 claims description 10
- 239000012141 concentrate Substances 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 9
- 238000001914 filtration Methods 0.000 claims description 7
- 239000011259 mixed solution Substances 0.000 claims description 7
- 238000000105 evaporative light scattering detection Methods 0.000 claims description 6
- 239000012982 microporous membrane Substances 0.000 claims description 6
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 238000005303 weighing Methods 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 2
- 238000003809 water extraction Methods 0.000 claims description 2
- 238000005259 measurement Methods 0.000 abstract description 5
- 239000000126 substance Substances 0.000 abstract description 5
- 150000001875 compounds Chemical class 0.000 abstract description 2
- 238000000691 measurement method Methods 0.000 abstract 2
- 229920000642 polymer Polymers 0.000 abstract 2
- 241000411851 herbal medicine Species 0.000 abstract 1
- 239000004615 ingredient Substances 0.000 abstract 1
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- 239000000523 sample Substances 0.000 description 38
- 239000012071 phase Substances 0.000 description 27
- 239000000203 mixture Substances 0.000 description 16
- 239000003153 chemical reaction reagent Substances 0.000 description 15
- 238000003556 assay Methods 0.000 description 9
- 238000000926 separation method Methods 0.000 description 8
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- 238000007803 cold maceration Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- -1 inulin oligosaccharides Chemical class 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 238000012372 quality testing Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000005464 sample preparation method Methods 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 1
- LPQOADBMXVRBNX-UHFFFAOYSA-N ac1ldcw0 Chemical compound Cl.C1CN(C)CCN1C1=C(F)C=C2C(=O)C(C(O)=O)=CN3CCSC1=C32 LPQOADBMXVRBNX-UHFFFAOYSA-N 0.000 description 1
- 239000010975 amethyst Substances 0.000 description 1
- 230000001430 anti-depressive effect Effects 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
Classifications
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/047—Standards external
-
- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8836—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving saccharides
-
- 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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Saccharide Compounds (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention provides a measurement method of the content of morinda officinalis oligosaccharides, which comprises the steps as follows: the high-performance liquid chromatography is adopted for carrying out the measurement; water solution containing acetonitrile is taken as mobile phase, the contents of 3 to 9 polymers of inulin-typed oligosaccharides are respectively calculated by peak areas according to the external standard method, and the contents of the 3 to 9 polymers of the inulin-typed oligosaccharides are summed to obtain the content of the morinda officinalis oligosaccharides; the measurement method takes a single reference substance for measuring the total content of compounds with similar structures in the morinda officinalis oligosaccharides in morinda officinalis Chinese herbal medicine or morinda officinalis extract, and simultaneously measuring the ingredients and the relative contents thereof in the measured chemical part, thereby simplifying the operation steps, improving the accuracy of the content measurement, reducing the number of the reference substances, shortening the measurement time, and having good repeatability and high applicability.
Description
Technical field
The invention provides a kind of detection method of morinda root oligosacchride, it is a kind of content assaying method that contains the oligosaccharide mixture of a plurality of oligosaccharides compositions, refers in particular to the high performance liquid chromatogram content assaying method of the morinda root oligosacchride in the extract of Morinda officinalis Chinese crude drug (comprising the prepared slices of Chinese crude drugs) or Morinda officinalis Chinese crude drug.
Background technology
At pharmaceutical field, assay for certain composition, adopting the stratographic analysis of high performance liquid chromatogram is very conventional method, it is reference substance that those skilled in the art usually set a certain composition, in subject matter, carry out the assay determination of certain composition with reference to this reference substance, when containing plurality of Chinese effective constituent and needs and simultaneously this plurality of Chinese composition carried out qualitative and quantitative measurement, often find out its multiple one to one reference substance according to this multiple composition, measure successively again, this shortcoming that unified subject matter is measured one by one is inevitable, complex operation, time and effort consuming, inefficiency, each composition relative content standard is independent separately in the same chemical position, uncorrelated mutually, so can cause deviation to a certain degree unavoidably, can't be more effective, control of quality accurately.
For the Morinda officinalis medicinal material that contains morinda root oligosacchride or the extract of medicine materical crude slice or its Morinda officinalis Chinese crude drug of making, its effective constituent is oligosaccharides class material, this oligosaccharides material is the potpourri of 3-9 sugar, if adopt the chemical colour reaction reaction, show and other carbohydrate (monose, disaccharide, polysaccharide) identical response feature, therefore, digital proof is for detecting morinda root oligosacchride, select the chromatogram discriminating of HPLC to have more specificity, and, need develop and a kind ofly can when measuring this chemistry position total amount, can also identify wherein each composition for the result that measures science and rigorous more, and provide each composition relative content, reduce the kind of reference substance as far as possible, high performance liquid chromatogram content assaying method simple to operate.
Summary of the invention
At present in Morinda officinalis medicinal material (comprising medicine materical crude slice) that contains morinda root oligosacchride or the extract made with the Morinda officinalis medicinal material, what its quality monitoring aspect did not have a standard maturation contains the survey method, the content assaying method that the purpose of this invention is to provide a kind of morinda root oligosacchride, use the technical indicator in this method and this method, can well monitor the Morinda officinalis medicinal material that contains morinda root oligosacchride or the quality of its extract of making, make the traditional Chinese medicine quality stable and controllable, ensure curative effect, service society better.
The objective of the invention is to develop a kind of Morinda officinalis medicinal material (comprising medicine materical crude slice) of morinda root oligosacchride composition or method of quality control of its extract of making of containing, this method is easy, efficient, is fit to the needs of quality testing in scientific research, market sale, market surveillance, clinical practice and the suitability for industrialized production.
The invention provides a kind of content assaying method of morinda root oligosacchride, this morinda root oligosacchride contains inulin type oligosaccharides 5 aggressiveness at least, and is present in Morinda officinalis Chinese crude drug (comprising medicine materical crude slice) or its extract, and this content assaying method comprises:
(1) adopt high performance liquid chromatography to measure; Chromatographic condition is to be moving phase with the aqueous solution that contains acetonitrile, adopts differential detecting device or evaporative light-scattering detector to detect;
(2) preparation of reference substance solution: take by weighing inulin type oligosaccharides 5 aggressiveness product in contrast, add the moving phase described in water or the step (1) and make reference substance solution;
(3) preparation of need testing solution: get the morinda root oligosacchride sample, add the moving phase dissolving in water or the step (1), get supernatant, filter, get subsequent filtrate, promptly get need testing solution;
Described morinda root oligosacchride sample is made by following method, get Morinda officinalis medicinal material (or medicine materical crude slice), water extracts extract, this extract is the extract of Morinda officinalis Chinese crude drug, carbon column chromatography on this extract, first water are eluted to reducing sugar and detect (sulfuric acid-phynol method) and be negative, and use the 20-50% ethanol elution again, collect the ethanol elution part, obtain the morinda root oligosacchride sample through concentrate drying;
(4) measure: draw reference substance solution and need testing solution respectively, inject liquid chromatograph, measure;
(5) calculate, press each chromatographic peak and inulin type oligosaccharides 5 aggressiveness reference substance relative retention times in the test sample collection of illustrative plates, identify the existence of each oligosaccharides in total oligosaccharides; By the content of external standard method with the inulin type oligosaccharides n aggressiveness that contained in this morinda root oligosacchride of calculated by peak area, n is arbitrary or its mixing of 3～9, and then determine the relative content ratio of each inulin type oligosaccharides n aggressiveness in this morinda root oligosacchride, content with inulin type oligosaccharides n aggressiveness sums up again, promptly gets the content of the total oligosaccharides of described Morinda officinalis.
The above-mentioned Morinda officinalis Chinese crude drug that adopts had both comprised the Chinese crude drug raw material without any processing, also comprised the medicine materical crude slice through processing, and also comprised the Morinda officinalis medicine materical crude slice that obtains through processing procedure well known in the art.
Above-mentioned morinda root oligosacchride is the active component with antidepressant effect, comprise inulin type oligosaccharides 3 sugar～9 sugar, promptly, morinda root oligosacchride of the present invention is the oligosaccharides that contains inulin type oligosaccharides 5 aggressiveness (5 sugar) at least, or contains inulin type oligosaccharides 5 aggressiveness (5 sugar) and inulin type oligosaccharides 3 aggressiveness (3 sugar), inulin type oligosaccharides 4 aggressiveness (4 sugar), inulin type oligosaccharides 6 aggressiveness (6 sugar), inulin type oligosaccharides 7 aggressiveness (7 sugar), inulin type oligosaccharides 8 aggressiveness (8 sugar) and inulin type oligosaccharides 9 aggressiveness (9 sugar) simultaneously arbitrarily or the potpourri of its combination.
In a preferred embodiment of the invention, the middle chromatographic column of step (1) preferably adopts nh 2 column (amino chromatographic column) or C18 reversed-phase column in the said method; Inertsil NH for example
2Post (5 μ m, 4.6 * 250mm), the detecting device of employing is differential detecting device or evaporative light-scattering detector, the general selection of described moving phase contains the aqueous solution of the acetonitrile of 40-80% (volume); Be preferably volume ratio 3: 1～1: 1 the acetonitrile and the mixed solution of water.
Above-mentioned morinda root oligosacchride preferably also contains arbitrary oligosaccharides or its potpourri in inulin type oligosaccharides 3～4,6～9 aggressiveness when containing inulin type oligosaccharides 5 aggressiveness (5 sugar).
Preferably, morinda root oligosacchride of the present invention is the potpourri that contains inulin type oligosaccharides 5 aggressiveness (5 sugar), inulin type oligosaccharides 3 aggressiveness (3 sugar), inulin type oligosaccharides 4 aggressiveness (4 sugar), inulin type oligosaccharides 6 aggressiveness (6 sugar), inulin type oligosaccharides 7 aggressiveness (7 sugar), inulin type oligosaccharides 8 aggressiveness (8 sugar) and inulin type oligosaccharides 9 aggressiveness (9 sugar).Generally speaking, can abbreviate inulin type oligosaccharides n aggressiveness as n sugar, n is 3～9, and for example, inulin type oligosaccharides 5 aggressiveness are 5 sugar, and inulin type oligosaccharides 9 aggressiveness are 9 sugar.
The inventor finds, in order to improve the conditions of streaking of chromatogram, make the peak type of the chromatogram that obtains complete attractive in appearance, the moving phase of the aqueous solution that contains acetonitrile that adopts in the content assaying method of the present invention preferably also contains triethylamine, and described triethylamine shared volume in this moving phase generally is no more than 0.5%; Generally can be 0.01%-0.5%.
What the step of the filtration described in the preparation of need testing solution of the present invention preferably adopted is miillpore filter, and the aperture of this miillpore filter generally can be 0.3,0.45,0.5,0.75um, more preferably 0.45um.
In a preferred embodiment of the invention, the described moving phase of above-mentioned content assaying method step (1) most preferably is the mixed solution of 68: 32 acetonitrile of volume ratio and water, also contains 0.5% the triethylamine that is no more than this mobile phase volume in this mixed solution; For example, the mixed solution that can be 68: 32: 0.1 acetonitrile of volume ratio, water and triethylamine is as moving phase, and collection of illustrative plates that obtains and peak type present the amazing perfect effect that is tending towards.
In the detection method of the present invention, carbon column chromatography on the water extraction that step (3) is adopted, preferred first water wash-out, use about 20-50% ethanol elution again, more preferably behind the water elution,, obtain preferable morinda root oligosacchride sample through concentrate drying with about 30% ethanol elution.Then, get morinda root oligosacchride sample 50-200mg, the accurate title, decide, and puts in the 10ml measuring bottle, and the moving phase dissolving in water or the step (1) is diluted to scale, preferably again through filtering with microporous membrane, gets subsequent filtrate, can obtain need testing solution.
Reference substance of the present invention is selected inulin type oligosaccharides 5 aggressiveness for use, selecting inulin type oligosaccharides 5 aggressiveness is because morinda root oligosacchride is the potpourri of 3～9 aggressiveness oligosaccharides in contrast, under the HPLC chromatographic condition of determining, 5 sugared relative retention times are placed in the middle, chromatographic peak peak height or peak area are moderate, demarcate other 3-4,6-9 sugar with it, each sugar is more even with 5 sugared peak relative position differences, the system deviation that exists in the time of can reducing with chromatogram peak area or peak height integral and calculating relative content and total oligosaccharide content.Simultaneously, adopt 5 sugar can make the peak type of HPLC of other sugar (that is 3,4,6,7,8 and 9 sugar) clear, degree of separation is better, accuracy is high.
The need testing solution of step (3) is preferably every 50-200mg morinda root oligosacchride sample and adds the solution that entry or moving phase 10ml obtain, in the process of preparation need testing solution, also experienced ultrasonic in the course of dissolution, the dissolving back is got subsequent filtrate and is need testing solution with the process that the 0.45um miillpore filter filters;
Described test sample then directly prepares need testing solution as being the morinda root oligosacchride bulk drug; As be Chinese crude drug or Chinese crude drug extract, then handle as follows and obtain the test sample morinda root oligosacchride: to the Chinese crude drug (or prepared slices of Chinese crude drugs) that contains morinda root oligosacchride, water extracts extract, carbon column chromatography on the extract, elder generation's water is eluted to reducing sugar and detects (sulfuric acid-phynol method) and be negative, use the 20-50% ethanol elution again, preferred 30% ethanol elution.Collect the ethanol elution part, obtain the morinda root oligosacchride sample through concentrate drying; To containing the Chinese medical extract of morinda root oligosacchride, then directly go up activated carbon column chromatography, handle as stated above then.
In another embodiment of the present invention, described content assaying method comprises:
(1) adopt high performance liquid chromatography to measure; Chromatographic condition is to adopt nh 2 column; With acetonitrile: water: the volume ratio of triethylamine is a moving phase at 68: 32: 0.1; Detecting device is the differential detecting device; 40 ℃ of column temperatures, 40 ℃ of detector temperatures; Number of theoretical plate calculates by inulin type oligosaccharides 5 aggressiveness and is not less than 2000;
(2) preparation of reference substance solution takes by weighing inulin type oligosaccharides 5 aggressiveness product in contrast, adds water and makes the reference substance solution that every 1ml contains 1mg;
(3) preparation of need testing solution, the above-mentioned employing Morinda officinalis medicinal material extract of learning from else's experience obtains containing the morinda root oligosacchride sample 50-200mg that the extract of morinda root oligosacchride obtains via activated carbon column chromatography again, put in the tool plug conical flask, add entry 10ml, under the power 100W frequency 40KHz ultrasonic 10 minutes, leave standstill, get supernatant, filter with the 0.45um miillpore filter, get subsequent filtrate, promptly get need testing solution;
(4) measure, draw reference substance solution and need testing solution 20 μ l respectively, inject liquid chromatograph, measure;
(5) calculate, press each chromatographic peak and inulin type oligosaccharides 5 aggressiveness reference substance relative retention times in the test sample collection of illustrative plates, identify the existence of each oligosaccharides in total oligosaccharides; Calculate the content of inulin type oligosaccharides 3～9 aggressiveness by external standard method respectively with peak area, and then determine the relative content ratio of each oligosaccharides in total oligosaccharides, the content with inulin type oligosaccharides 3～9 aggressiveness sums up again, promptly gets the content of the total oligosaccharides of Morinda officinalis.
The present invention with wherein this composition of inulin type oligosaccharides 5 aggressiveness product in contrast, adopts high performance liquid chromatography in oligosaccharide mixture, utilize differential detecting device or evaporative light-scattering detector to measure the method for oligosaccharide content.Its innovative point is: with this composition of these inulin type oligosaccharides 5 aggressiveness is benchmark, the retention time of this composition is decided to be 1, the corresponding relative retention time that is converted into of the retention time of other condensate chromatographic peaks, differentiate the existence of other oligosaccharides that 5 sugar are outer with relative retention time, calculate the content of each composition and total oligosaccharides by external standard method respectively with peak area again, can obtain comparatively stable and the content of morinda root oligosacchride accurately.Can measure the content of the compound of a plurality of similar like this with a kind of reference substance, improve the accuracy of assay, simplify operation steps, reduce reference substance quantity, shorten minute, good reproducibility, applicability height.
Illustrate with following example but do not limit this patent:
The retention time of 5 sugared chromatographic peaks in the test sample collection of illustrative plates in this liquid chromatogram, should be consistent with inulin type oligosaccharides 5 aggressiveness reference substance retention times, compare with inulin oligosaccharides 5 aggressiveness, relative retention time also should occur and be 0.50～0.70 inulin type oligosaccharides 3 aggressiveness peaks; 0.70～0.90 inulin type oligosaccharides 4 aggressiveness peaks; 1.10～1.40 inulin type oligosaccharides 6 aggressiveness peaks; 1.50～1.83 inulin type oligosaccharides 7 aggressiveness peaks; 1.83～2.50 inulin type oligosaccharides 8 aggressiveness peaks; 1.85～3.40 inulin type oligosaccharides 9 aggressiveness peaks calculate the content of each oligosaccharides more respectively with peak area by external standard method.
The selection of chromatographic condition of the present invention is based on great deal of experiment data, selects the each side index to comprise peak type, degree of separation, peak area, reappearance or the like, conditions such as the chromatographic column of the best that comprehensive evaluation draws, moving phase, detecting device and column temperature.Specific as follows:
The HPLC of reference substance inulin type oligosaccharides 5 aggressiveness (5 sugar) analyzes:
Get 5 sugared reference substance test liquid 20 μ l under the assay item, under chromatographic condition of the present invention, carry out HPLC and analyze, record chromatogram, data: relative retention time 17.502, peak area 118018, peak area (%) 100.0000.
The HPLC of morinda root oligosacchride analyzes:
Get morinda root oligosacchride test liquid 20 μ l under the assay item, carrying out HPLC under above-mentioned chromatographic condition analyzes, the record chromatogram, peak among the figure is followed successively by chromatographic peak from left to right 1～No. 7, determines that through method of spectroscopy such as HPLC-MS-MS (from left to right) is successively: inulin type oligosaccharides 3 aggressiveness (C
18H
320
163 sugar), inulin type oligosaccharides 4 aggressiveness (C, be called for short:
24H
42O
214 sugar), inulin type oligosaccharides 5 aggressiveness (C, be called for short:
30H
52O
265 sugar), inulin type oligosaccharides 6 aggressiveness (C, be called for short:
36H
62O
316 sugar), inulin type oligosaccharides 7 aggressiveness (C, be called for short:
42H
72O
367 sugar), inulin type oligosaccharides 8 aggressiveness (C, be called for short:
48H
82O
418 sugar) and inulin type oligosaccharides 9 aggressiveness (C, be called for short:
54H
92O
46, be called for short: 9 sugar).Wherein the retention time of 5 sugared chromatographic peaks is consistent with the reference substance retention time.The retention time of 3～9 condensate chromatographic peaks, spectrum analysis is as follows, and data see Table 1.(Resolution in the table: degree of separation, T.Plate# theoretical cam curve, Ret.Time: retention time, Area: area)
Table 1
| ID | Ret.Time | Area | Area％ | Resolution | T.Plate# |
| 1 | 10.099 | 84373 | 8.9974 | 2.908 | 4428.619 |
| 2 | 13.259 | 142746 | 15.2222 | 4.496 | 4409.893 |
| 3 | 17.405 | 163729 | 17.4599 | 4.554 | 4637.704 |
| 4 | 22.773 | 174663 | 18.6258 | 4.613 | 4873.221 |
| 5 | 30.173 | 152323 | 16.2435 | 4.902 | 4956.905 |
| 6 | 39.836 | 121337 | 12.9392 | 4.801 | 4754.490 |
| 7 | 52.705 | 94798 | 10.1092 | 5.042 | 5691.831 |
The chromatogram identification result:
The above results is carried out statistical study, obtain maximal value, minimum value and the mean value of 3～9 each chromatographic peak relative retention time of sugar, see Table 2.
The relative retention time statistical value of each chromatographic peak of table 2 morinda root oligosacchride
The HPLC of solvent analyzes
Get the pure water solvent, or get moving phase solution, handle, get 20 μ l respectively, inject HPLC, measure, carry out HPLC under the above-mentioned chromatographic condition and analyze, the record chromatogram according to the need testing solution preparation method.Chromatogram with contain Morinda officinalis sample chromatogram figure contrast, the result shows in the blank sample chromatogram, with the corresponding retention time of morinda root oligosacchride 5 sugared reference substance chromatograms place, and and contain corresponding each oligosaccharides relative retention time place in the chromatogram of morinda root oligosacchride sample, there is no chromatographic peak.Show and do not exist blank solvent to disturb.
Morinda root oligosacchride is inulin type oligosaccharides 3 aggressiveness (C
18H
32O
163 sugar), inulin type oligosaccharides 4 aggressiveness (C, be called for short:
24H
42O
214 sugar), inulin type oligosaccharides 5 aggressiveness (C, be called for short:
30H
52O
265 sugar), inulin type oligosaccharides 6 aggressiveness (C, be called for short:
36H
62O
316 sugar), inulin type oligosaccharides 7 aggressiveness (C, be called for short:
42H
72O
367 sugar), inulin type oligosaccharides 8 aggressiveness (C, be called for short:
48H
82O
418 sugar) and inulin type oligosaccharides 9 aggressiveness (C, be called for short:
54H
92O
46, be called for short: potpourri 9 sugar).The present invention has set up the HPLC content assaying method of this morinda root oligosacchride.Description of test is as follows:
1 instrument and reagent:
Tianjin, island 20AT highly effective liquid phase chromatographic system
RID-10A differential detecting device
The LCsolution chromatographic work station
Chromatographic column: Inertsil NH
2Post (5 μ m, 4.5 * 250mm)
Reagent: acetonitrile-chromatographically pure (Tian Jinsi friend biomedical technology development company), it is pure that water-high purity water, all the other reagent are analysis
Reference substance: inulin type oligosaccharides 5 aggressiveness, Nat'l Pharmaceutical ﹠ Biological Products Control Institute provides, lot number 200701, purity 99.99%.
2. method and result:
2.1. chromatographiccondition
Chromatographic column: Inertsil NH
2Post (5 μ m, 4.5 * 250mm)
Moving phase: acetonitrile: water: triethylamine (68: 32: 0.1)
Detecting device: RID-10A differential detecting device
Temperature: 40 ℃ of column temperatures, 40 ℃ of detector temperatures
Flow velocity: 1.2mL/min;
With this understanding, each component reaches baseline separation in the sample.Record inulin type oligosaccharides 5 aggressiveness peak number of theoretical plates and be not less than 2000.
2.2. linear relationship is investigated: accurate inulin type oligosaccharides 5 aggressiveness reference substance solution (2.37mg/ml) 5,10,15,20,25, the 30 μ l that draw, inject liquid chromatograph, measure according to above-mentioned chromatographic condition, with the peak area integrated value is ordinate, inulin type oligosaccharides 5 aggressiveness sample sizes are horizontal ordinate, the drawing standard curve gets regression equation: y=5555523.8X-6336r=0.9999.The result shows, inulin type oligosaccharides 5 aggressiveness tool good linear relationship in 0.0119～0.0711mg scope.See Table 3.
The investigation of table 3 linear relationship
2.3 precision experiment: accurate inulin type oligosaccharides 5 aggressiveness reference substance solution (2.37mg/ml) the 10 μ l that draw, continuous sample introduction 6 times is measured according to above-mentioned chromatographic condition, calculates average peak area and relative standard deviation, sees Table 4.The result shows: the precision of this method is good.
Table 4 precision measurement result
2.4. blank test: get the pure water solvent, or moving phase solution, according to test sample disposal methods under " assay " item, get 20 μ l, under above-mentioned chromatographic condition, carry out HPLC and analyze, the record chromatogram, the result shows noiseless.
2.5. need testing solution study on the stability: need testing solution (lot number 0762), after sample preparation 0,2,4,8,10,22 hour, measure in accordance with the law, the result shows that need testing solution is basicly stable in 22 hours.The results are shown in Table 5.
Table 5 stability test result
2.6. replica test: press the text method, get with batch (0762) sample, 6 parts of parallel sample preparations are measured, and the results are shown in Table 6, and the result shows that repeatability better.
Table 6 replica test result
2.7. recovery test: adopt the application of sample absorption method, precision takes by weighing same crowd (0762) (5 sugared content 54.1mg/g) sample 150mg of known content, the accurate respectively 5 sugared reference substances (7.716mg) that add, 6 parts of parallel sample preparations, measure, be calculated as follows the recovery, the result shows that this method has good accuracy, and data see Table 7.
Table 7 recovery test result
2.8. sample determination result: measure 3 batch samples, the results are shown in Table 8.
Table 8, sample determination result
3 method durability researchs
3.1
Chromatographic column: MERK Lichrosorb NH
2Post (5 μ m, 4.5 * 250mm)
Moving phase: acetonitrile: water: triethylamine (68: 32: 0.1)
Detecting device: RID-10A differential detecting device
Temperature: 40 ℃ of column temperatures, 40 ℃ of detector temperatures
Flow velocity: 1.2mL/min;
Result: see Table 9,10.
Table 9, durability result 1
The relative retention time of table 10, each chromatographic peak of morinda root oligosacchride
3.2
Chromatographic column: SEPAX Amethyst amino post (5 μ m, 4.6 * 250mm)
Moving phase: acetonitrile: water: triethylamine (68: 32: 0.1)
Detecting device: RID-10A differential detecting device
Temperature: 40 ℃ of column temperatures, 40 ℃ of detector temperatures
Flow velocity: 1.2mL/min;
Result: see Table 11,12.
Table 11, durability result 2
The relative retention time of table 12, each chromatographic peak of morinda root oligosacchride
4, determining of relative retention time scope: many batches of morinda root oligosacchrides are measured, the retention time of inulin type oligosaccharides 3～9 aggressiveness is comparatively fixing, inulin type oligosaccharides 3～9 aggressiveness are also comparatively stable with respect to the relative retention time of 5 aggressiveness, see Table 13.
Table 13, relative retention time scope
Through systematic study, it is reference substance that the present invention adopts inulin oligosaccharides 5 aggressiveness, and the method for calculating morinda root oligosacchride content with the peak area sum of 3-9 sugar is practicable.Accurate, sensitive, single-minded, the blank auxiliary material of this method is noiseless to measuring, and can effectively control this product content.Method of quality control rapid and convenient of the present invention, repeatability is good, is fit to the quality testing of breadboard sample detection and industrialized production.
Embodiment
Describe the present invention in detail below in conjunction with embodiment, but do not limit practical range of the present invention.
Embodiment one. the assay of Morinda officinalis medicinal material
1 instrument and reagent:
Tianjin, island 20AT highly effective liquid phase chromatographic system
The LCsolution chromatographic work station
Reagent: acetonitrile-chromatographically pure (Tian Jinsi friend biomedical technology development company), it is pure that water-high purity water, all the other reagent are analysis
Reference substance: inulin type oligosaccharides 5 aggressiveness, Nat'l Pharmaceutical ﹠ Biological Products Control Institute provides, lot number 200701, purity 99.99%.
2. method and result:
2.1. chromatographiccondition
Chromatographic column: Inertsil NH
2Post (5 μ m, 4.5 * 250mm)
Moving phase: acetonitrile: water: triethylamine (68: 32: 0.1)
Detecting device: RID-10A differential detecting device
Temperature: 40 ℃ of column temperatures, 40 ℃ of detector temperatures
Flow velocity: 1.2mL/min;
With this understanding, each component reaches baseline separation in the sample.Record inulin type oligosaccharides 5 aggressiveness peak number of theoretical plates and be not less than 2000.
It is an amount of that inulin type oligosaccharides 5 aggressiveness (5 sugar) reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds water and makes the solution that every 1ml contains 1mg, promptly.
Morinda officinalis medicinal material 20g is got in the preparation of need testing solution, according to cold-maceration under an appendix XA of Chinese Pharmacopoeia version in 2005 item, extract extract, evaporate to dryness, dry thing water dissolving makes into the solution that concentration is 1g/ml, last carbon column chromatography, elder generation's water is eluted to reducing sugar and detects (sulfuric acid-phynol method) and be negative, and uses 30% ethanol elution of about 6 times of column volumes again.Collect 30% ethanol elution part, obtain morinda root oligosacchride (sample) through concentrate drying.The about 150mg of sample thief, the accurate title, decide, and puts in the 10ml measuring bottle, is diluted with water to scale, through 0.45 μ m filtering with microporous membrane, gets subsequent filtrate, promptly.
Accurate respectively reference substance solution and each the 20 μ l of need testing solution of drawing of determination method, inject liquid chromatograph, measure, calculate the content of inulin type oligosaccharides 3 aggressiveness (3 sugar), inulin type oligosaccharides 4 aggressiveness (4 sugar), inulin type oligosaccharides 5 aggressiveness (5 sugar), inulin type oligosaccharides 6 aggressiveness (6 sugar), inulin type oligosaccharides 7 aggressiveness (7 sugar), inulin type oligosaccharides 8 aggressiveness (8 sugar) and inulin type oligosaccharides 9 aggressiveness (9 sugar) respectively with peak area by external standard method, the content of 3～9 sugar is summed up, promptly get the morinda root oligosacchride content of effective.
The Morinda officinalis medicinal material is pressed dry product and is calculated, and contains inulin type oligosaccharides 5 aggressiveness (C
30H
52O
26) must not be less than 2.0%, contain morinda root oligosacchride (being the total amount of inulin type oligosaccharides 3 aggressiveness-9 aggressiveness) with inulin type oligosaccharides 5 aggressiveness (C
30H
52O
26) meter, must not be less than 10.0%.
Embodiment two. the extract of Morinda officinalis medicinal material
1 instrument and reagent:
Tianjin, island 20AT highly effective liquid phase chromatographic system
The LCsolution chromatographic work station
Reagent: acetonitrile-chromatographically pure (Tian Jinsi friend biomedical technology development company), it is pure that water-high purity water, all the other reagent are analysis
Reference substance: inulin type oligosaccharides 5 aggressiveness, Nat'l Pharmaceutical ﹠ Biological Products Control Institute provides, lot number 200701, purity 99.99%.
2. method and result:
2.1. chromatographiccondition
Chromatographic column: NH
2Post (5 μ m, 4.5 * 250mm)
Moving phase: acetonitrile: water: triethylamine (68: 32: 0.1)
Detecting device: RID-10A differential detecting device
Temperature: 40 ℃ of column temperatures, 40 ℃ of detector temperatures
Flow velocity: 1.2mL/min;
With this understanding, each component reaches baseline separation in the sample.Record inulin type oligosaccharides 5 aggressiveness peak number of theoretical plates and be not less than 2000.
It is an amount of that inulin type oligosaccharides 5 aggressiveness (5 sugar) reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds moving phase and makes the solution that every 1ml contains 1mg, promptly.
Morinda officinalis medicinal substances extract 5g is got in the preparation of need testing solution, and water dissolving makes into the solution that concentration is 1g/ml, and last carbon column chromatography, first water are eluted to reducing sugar and detect (sulfuric acid-phynol method) and be negative, and use 30% ethanol elution again.Collect 30% ethanol elution part, obtain morinda root oligosacchride (sample) through concentrate drying.The about 150mg of sample thief, the accurate title, decide, and puts in the 10ml measuring bottle, is diluted to scale with moving phase, through 0.45 μ m filtering with microporous membrane, gets subsequent filtrate, promptly.
Accurate respectively reference substance solution and each the 20 μ l of need testing solution of drawing of determination method, inject liquid chromatograph, measure, calculate the content of inulin type oligosaccharides 3 aggressiveness (3 sugar), inulin type oligosaccharides 4 aggressiveness (4 sugar), inulin type oligosaccharides 5 aggressiveness (5 sugar), inulin type oligosaccharides 6 aggressiveness (6 sugar), inulin type oligosaccharides 7 aggressiveness (7 sugar), inulin type oligosaccharides 8 aggressiveness (8 sugar) and inulin type oligosaccharides 9 aggressiveness (9 sugar) respectively with peak area by external standard method, the content of 3～9 sugar is summed up, promptly get the morinda root oligosacchride content of effective.
The Morinda officinalis medicinal substances extract is pressed dry product and is calculated, and contains inulin type oligosaccharides 5 aggressiveness (C
30H
52O
26) must not be less than 3.0%, contain morinda root oligosacchride (being the total amount of inulin type oligosaccharides 3 aggressiveness-9 aggressiveness) with inulin type oligosaccharides 5 aggressiveness (C
30H
52O
26) meter, must not be less than 20.0%.
Embodiment three. the assay of Morinda officinalis medicinal material
1 instrument and reagent:
Tianjin, island 20AT highly effective liquid phase chromatographic system
The LCsolution chromatographic work station
Reagent: acetonitrile-chromatographically pure (Tian Jinsi friend biomedical technology development company), it is pure that water-high purity water, all the other reagent are analysis
Reference substance: inulin type oligosaccharides 5 aggressiveness, Nat'l Pharmaceutical ﹠ Biological Products Control Institute provides, lot number 200701, purity 99.99%.
2. method and result:
2.1. chromatographiccondition
Chromatographic column: NH
2Post (5 μ m, 4.5 * 250mm)
Moving phase: acetonitrile: water (40: 60)
Detecting device: RID-10A differential detecting device
Temperature: 40 ℃ of column temperatures, 40 ℃ of detector temperatures
Flow velocity: 1.0mL/min;
With this understanding, each component reaches baseline separation in the sample.Record inulin type oligosaccharides 5 aggressiveness peak number of theoretical plates and be not less than 2500.
It is an amount of that inulin type oligosaccharides 5 aggressiveness (5 sugar) reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds moving phase and makes the solution that every 1ml contains 1mg, promptly.
Morinda officinalis medicinal material 20g is got in the preparation of need testing solution, according to cold-maceration under an appendix X of Chinese Pharmacopoeia version in 2005 the A item, extract extract, evaporate to dryness, dry thing water dissolving makes into the solution that concentration is 1g/ml, last carbon column chromatography, elder generation's water is eluted to reducing sugar and detects (sulfuric acid-phynol method) and be negative, and uses 20% ethanol elution of about 8 times of column volumes again.Collect 20% ethanol elution part, obtain morinda root oligosacchride (sample) through concentrate drying.The about 150mg of sample thief, the accurate title, decide, and puts in the 10ml measuring bottle, is diluted to scale with moving phase, through 0.45 μ m filtering with microporous membrane, gets subsequent filtrate, promptly.
Accurate respectively reference substance solution and each the 20 μ l of need testing solution of drawing of determination method, inject liquid chromatograph, measure, calculate the content of inulin type oligosaccharides 3 aggressiveness (3 sugar), inulin type oligosaccharides 4 aggressiveness (4 sugar), inulin type oligosaccharides 5 aggressiveness (5 sugar), inulin type oligosaccharides 6 aggressiveness (6 sugar), inulin type oligosaccharides 7 aggressiveness (7 sugar), inulin type oligosaccharides 8 aggressiveness (8 sugar) and inulin type oligosaccharides 9 aggressiveness (9 sugar) respectively with peak area by external standard method, the content of 3～9 sugar is summed up, promptly get the morinda root oligosacchride content of effective.
The Morinda officinalis medicinal material is pressed dry product and is calculated, and contains inulin type oligosaccharides 5 aggressiveness (C
30H
52O
26) must not be less than 2.0%, contain morinda root oligosacchride (being the total amount of inulin type oligosaccharides 3 aggressiveness-9 aggressiveness) with inulin type oligosaccharides 5 aggressiveness (C
30H
52O
26) meter, must not be less than 10.0%.
Embodiment four. the assay of Morinda officinalis medicinal material
1 instrument and reagent:
Tianjin, island 20AT highly effective liquid phase chromatographic system
The LCsolution chromatographic work station
Reagent: acetonitrile-chromatographically pure (Tian Jinsi friend biomedical technology development company), it is pure that water-high purity water, all the other reagent are analysis
Reference substance: inulin type oligosaccharides 5 aggressiveness, Nat'l Pharmaceutical ﹠ Biological Products Control Institute provides, lot number 200701, purity 99.99%.
2. method and result:
2.1. chromatographiccondition
Chromatographic column: C18 post (5 μ m, 4.5 * 250mm)
Moving phase: acetonitrile: water: triethylamine (78: 22: 0.2)
Detecting device: evaporative light-scattering detector
Temperature: 35 ℃ of column temperatures, 35 ℃ of detector temperatures
Flow velocity: 1.2mL/min;
With this understanding, each component reaches baseline separation in the sample.Record inulin type oligosaccharides 5 aggressiveness peak number of theoretical plates and be not less than 3000.
It is an amount of that inulin type oligosaccharides 5 aggressiveness (5 sugar) reference substance is got in the preparation of reference substance solution, and accurate the title decides, and adds water and makes the solution that every 1ml contains 1mg, promptly.
Morinda officinalis medicinal material 20g is got in the preparation of need testing solution, according to cold-maceration under an appendix XA of Chinese Pharmacopoeia version in 2005 item, extract extract, evaporate to dryness, dry thing water dissolving makes into the solution that concentration is 1g/ml, last carbon column chromatography, elder generation's water is eluted to reducing sugar and detects (sulfuric acid-phynol method) and be negative, and uses 50% ethanol elution of about 4 times of column volumes again.Collect 50% ethanol elution part, obtain morinda root oligosacchride (sample) through concentrate drying.The about 150mg of sample thief, the accurate title, decide, and puts in the 10ml measuring bottle, is diluted with water to scale, through 0.45 μ m filtering with microporous membrane, gets subsequent filtrate, promptly.
Accurate respectively reference substance solution and each the 20 μ l of need testing solution of drawing of determination method, inject liquid chromatograph, measure, calculate the content of inulin type oligosaccharides 3 aggressiveness (3 sugar), inulin type oligosaccharides 4 aggressiveness (4 sugar), inulin type oligosaccharides 5 aggressiveness (5 sugar), inulin type oligosaccharides 6 aggressiveness (6 sugar), inulin type oligosaccharides 7 aggressiveness (7 sugar), inulin type oligosaccharides 8 aggressiveness (8 sugar) and inulin type oligosaccharides 9 aggressiveness (9 sugar) respectively with peak area by external standard method, the content of 3～9 sugar is summed up, promptly get the morinda root oligosacchride content of effective.
The Morinda officinalis medicinal material is pressed dry product and is calculated, and contains inulin type oligosaccharides 5 aggressiveness (C
30H
52O
26) must not be less than 2.O%, contain morinda root oligosacchride (being the total amount of inulin type oligosaccharides 3 aggressiveness-9 aggressiveness) with inulin type oligosaccharides 5 aggressiveness (C
30H
52O
26) meter, must not be less than 10.0%.
Claims (9)
1. the content assaying method of a morinda root oligosacchride, this morinda root oligosacchride contains inulin type oligosaccharides 5 aggressiveness at least, and is present in Morinda officinalis Chinese crude drug or its extract, and this content assaying method comprises:
(1) adopt high performance liquid chromatography to measure, chromatographic condition is to be moving phase with the aqueous solution that contains acetonitrile, adopts differential detecting device or evaporative light-scattering detector to detect;
(2) preparation of reference substance solution takes by weighing inulin type oligosaccharides 5 aggressiveness product in contrast, adds the moving phase described in water or the step (1) and makes reference substance solution;
(3) the morinda root oligosacchride sample is got in the preparation of need testing solution, adds the moving phase dissolving in water or the step (1), gets supernatant, filters, and gets subsequent filtrate, promptly gets need testing solution;
Described morinda root oligosacchride sample is made by following method, get the Morinda officinalis medicinal material, water extracts extract, this extract is the extract of Morinda officinalis Chinese crude drug, carbon column chromatography on this extract, first water are eluted to reducing sugar and detect and be negative, and use the 20-50% ethanol elution again, collect the ethanol elution part, obtain the morinda root oligosacchride sample through concentrate drying;
(4) measure, draw reference substance solution and need testing solution respectively, inject liquid chromatograph, measure;
(5) calculate, press each chromatographic peak and inulin type oligosaccharides 5 aggressiveness reference substance relative retention times in the test sample collection of illustrative plates; By the content of external standard method with the inulin type oligosaccharides n aggressiveness that contained in this morinda root oligosacchride of calculated by peak area, n is arbitrary or its mixing of 3～9, and then determine the relative content ratio of each inulin type oligosaccharides n aggressiveness in this morinda root oligosacchride, content with inulin type oligosaccharides n aggressiveness sums up again, promptly gets the content of the total oligosaccharides of described Morinda officinalis.
2. the described content assaying method of claim 1, the described chromatographic condition of step in this method (1) comprise and adopt nh 2 column or C18 reversed-phase column that described moving phase contains the acetonitrile of 40-80% volume.
3. the described content assaying method of claim 1, the described moving phase of step in this method (1) is volume ratio 3: 1～1: 1 the acetonitrile and the mixed solution of water.
4. the described content assaying method of claim 1, wherein, described morinda root oligosacchride also contains arbitrary oligosaccharides or its potpourri in inulin type oligosaccharides 3～4,6～9 aggressiveness.
5. the described content assaying method of claim 1, wherein, described morinda root oligosacchride is the potpourri that contains inulin type oligosaccharides 5 aggressiveness, inulin type oligosaccharides 3～4 aggressiveness, inulin type oligosaccharides 6～9 aggressiveness.
6. the described content assaying method of claim 1, wherein, the described moving phase of step in this method (1) is the mixed solution of 68: 32 acetonitrile of volume ratio and water, also contains 0.5% the triethylamine that is no more than this mobile phase volume in this mixed solution.
7. the described content assaying method of claim 1, wherein, carbon column chromatography on the described water extraction of step in this method (3), first water wash-out is used 30% ethanol elution again, obtains described morinda root oligosacchride sample through concentrate drying.
8. the described content assaying method of claim 1, step in this method (3) comprising:
Get morinda root oligosacchride sample 50-200mg, the accurate title, decide, and puts in the 10ml measuring bottle, and the moving phase dissolving in water or the step (1) is diluted to scale, through filtering with microporous membrane, gets subsequent filtrate, promptly gets need testing solution.
9. the described content assaying method of claim 1, wherein, the detecting device that adopts in this method step (1) is differential detecting device or evaporative light-scattering detector.
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| WO2009155756A1 (en) * | 2008-06-26 | 2009-12-30 | 北京同仁堂股份有限公司 | Method for determining the contents of oligosaccharides in morinda officinalis chinese medicine or extraction thereof |
| CN112730635A (en) * | 2019-10-28 | 2021-04-30 | 中国科学院大连化学物理研究所 | Liquid chromatography analysis method of oligosaccharide |
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| CN111198241A (en) * | 2020-03-17 | 2020-05-26 | 乐斯福(明光)有限公司 | Sugar content detection method for baking powder used in cold climate |
| CN115980200A (en) * | 2021-10-14 | 2023-04-18 | 绿谷(上海)医药科技有限公司 | Method for measuring content of polysaccharide or oligosaccharide medicine |
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| CN1253462C (en) * | 2004-07-30 | 2006-04-26 | 广州中医药大学 | Process for preparing morinda officinalis total oligosaccharide |
| CN100342859C (en) * | 2004-08-20 | 2007-10-17 | 中国人民解放军军事医学科学院毒物药物研究所 | Method for preparing formulation containing Indianmulberry extract |
| TWI285262B (en) * | 2005-06-20 | 2007-08-11 | Dev Center Biotechnology | Characteristic mass spectrum fingerprinting setting method and rapid identification method of Chinese herb medicine and prescription |
| CN101334389B (en) * | 2008-06-26 | 2011-06-22 | 北京同仁堂股份有限公司 | Morinda root oligosacchride content determination method |
-
2008
- 2008-07-24 CN CN2008101340912A patent/CN101334389B/en active Active
- 2008-07-24 CN CN2008101340927A patent/CN101334390B/en active Active
- 2008-09-12 WO PCT/CN2008/072344 patent/WO2009155755A1/en active Application Filing
- 2008-09-12 WO PCT/CN2008/072346 patent/WO2009155756A1/en active Application Filing
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009155756A1 (en) * | 2008-06-26 | 2009-12-30 | 北京同仁堂股份有限公司 | Method for determining the contents of oligosaccharides in morinda officinalis chinese medicine or extraction thereof |
| CN112730635A (en) * | 2019-10-28 | 2021-04-30 | 中国科学院大连化学物理研究所 | Liquid chromatography analysis method of oligosaccharide |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101334390B (en) | 2011-06-22 |
| CN101334389A (en) | 2008-12-31 |
| CN101334389B (en) | 2011-06-22 |
| HK1122102A1 (en) | 2009-05-08 |
| HK1122101A1 (en) | 2009-05-08 |
| WO2009155755A1 (en) | 2009-12-30 |
| WO2009155756A1 (en) | 2009-12-30 |
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