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WO2009155755A1 - Method for determining the contents of oligosaccharides in morinda officinalis - Google Patents

Method for determining the contents of oligosaccharides in morinda officinalis Download PDF

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Publication number
WO2009155755A1
WO2009155755A1 PCT/CN2008/072344 CN2008072344W WO2009155755A1 WO 2009155755 A1 WO2009155755 A1 WO 2009155755A1 CN 2008072344 W CN2008072344 W CN 2008072344W WO 2009155755 A1 WO2009155755 A1 WO 2009155755A1
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WIPO (PCT)
Prior art keywords
oligosaccharide
morinda
mer
content
mobile phase
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Application number
PCT/CN2008/072344
Other languages
French (fr)
Chinese (zh)
Inventor
张绍来
顾海鸥
李志猛
李银
邱落
杜菁
彭鹏
刘柏刚
张学著
张维钧
张薇
励华
Original Assignee
北京同仁堂股份有限公司
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Publication of WO2009155755A1 publication Critical patent/WO2009155755A1/en

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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

Definitions

  • the present invention provides a method for detecting Morinda oligosaccharide, which is a method for determining the content of a mixture of oligosaccharides containing a plurality of oligosaccharide components, in particular, a Morinda citrifolia in a preparation or an extract or a drug substance.
  • chromatographic analysis using high-performance liquid phase is a very conventional method, and those skilled in the art often set a certain component as a reference substance, and refer to the reference substance in the target substance for a certain component.
  • Analytical determination when a plurality of traditional Chinese medicine active ingredients are included and it is necessary to simultaneously determine qualitative and quantitative determinations of the plurality of traditional Chinese medicine ingredients, a plurality of reference materials corresponding to the one-to-one correspondence are often found according to the plurality of ingredients, and then measured sequentially.
  • the active ingredient is an oligosaccharide substance
  • the oligosaccharide material is a mixture of 3-9 sugars. If chemical color reaction is used, it shows the same reaction characteristics as other sugars (monosaccharides, disaccharides, polysaccharides).
  • the data proves that for the detection of Morinda officinalis, the selection
  • the chromatographic identification of HPLC is more specific, and in order to make the results of the determination more scientific and rigorous, it is necessary to develop a method for determining the total amount of the chemical part while identifying each component and giving the relative content of each component. Minimize the type of reference substance and operate a simple HPLC method.
  • the object of the present invention is to develop a quality control method for a preparation containing a Morinda oligosaccharide component, a raw material drug, and a Chinese herbal medicine extract containing Morinda oligosaccharide, using the method And the technical indicators in the method, can well monitor the quality of the Morinda preparation containing Morinda officinalis, especially the oral preparation, and monitor the raw material containing Morinda oligosaccharides or extracted from natural plants.
  • the extract containing Morinda oligosaccharides makes the quality of traditional Chinese medicine stable and controllable, guarantees curative effect, and can better serve the society.
  • the present invention provides a method for determining the content of Morinda officinalis oligosaccharides, which comprises at least a chitosan-type oligosaccharide 5-mer, and is present in a raw material of Morinda officinolate or extracted from a natural plant (Chinese herbal medicine)
  • the content determination method includes: (1) Determination by high performance liquid chromatography, using an aqueous solution containing acetonitrile as a mobile phase, using a differential detector or an evaporative light scattering detector;
  • the above-mentioned Morinda oligosaccharide preferably contains, in addition to the inulin-type oligosaccharide 5-mer, any of the oligosaccharides of the inulin type oligosaccharide 3 ⁇ 4, 6 ⁇ 9-mer, or a mixture thereof.
  • the above continuous filtrate is the intermediate stage filtrate collected during the filtration process.
  • the above morinda officinalis oligosaccharides are active ingredients having antidepressant effects, including chrysanthemum-type oligosaccharide 3 saccharides to 9 saccharides, that is, the Morinda oligosaccharides of the present invention contain at least a chrysanthemum type Oligosaccharide of oligosaccharide 5-mer (1F-Fructofuranosyl nystose), or both of the amylose-type oligosaccharide 5-mer and the inulin-type oligosaccharide trimer, inulin-type oligosaccharide 4-mer (Nice saccharide n ysto Se), inulin oligosaccharides, 6-mers ((2 ⁇ 1) If the furanosyl sugar hexasaccharide), inulin oligosaccharides 7 mer (hep tasaccharide), inulin type A mixture of oligosacc
  • the column in the step (1) in the above method is preferably an amino column (amino column) or a C18 reverse phase column; for example, an Inertsil NH 2 column (5 ⁇ , 4.6 x 250 mm), which is used for detection.
  • the mobile phase generally selects an aqueous solution containing 40-80% by volume of acetonitrile; preferably a volume ratio of 3:1 to 1:1. A mixed solution of acetonitrile and water.
  • the Morinda oligosaccharide of the present invention comprises a chrysanthemum-type oligosaccharide 5-mer, a chrysanthemum-type oligosaccharide trimer, a chrysanthemum-type oligosaccharide tetramer, a chrysanthemum-type oligosaccharide 6-mer, a chrysanthemum-type oligosaccharide At least one or a mixture of one or more of a sugar 7-mer, a chrysanthemum-type oligosaccharide 8-mer, and a chrysanthemum-type oligosaccharide 9-mer.
  • the mobile phase of the aqueous solution containing acetonitrile used in the content determination method of the present invention preferably further contains triethylamine, said three
  • the volume of amine in the mobile phase generally does not exceed 0.5%; typically it can range from 0.01% to 0.5%.
  • the mobile phase described in the step (1) of the content determination method is most preferably a volume ratio.
  • 68: 32 a mixed solution of acetonitrile and water, the mixed solution further containing not more than 0.5% by volume of the mobile phase of triethylamine; for example, acetonitrile, water and triethylamine in a volume ratio of 68:32:0.1
  • the mixed solution acts as a mobile phase, and the resulting map and peak shape present a surprisingly perfect effect.
  • the reference substance of the present invention is a chrysanthemum-type oligosaccharide 5-mer, because the Morinda oligosaccharide is a mixture of 3 ⁇ 9-mer oligosaccharides, and the amylose-type oligosaccharide 5-mer is determined under the HPLC chromatographic conditions determined by the study. Relative retention time is centered, peak height or peak area is moderate, and other oligosaccharides are calibrated. The relative position difference between each oligosaccharide and the amylose oligosaccharide 5-mer peak is relatively uniform, which can reduce the peak area of the chromatogram or The peak height integral calculates the relative deviation of the relative content and total oligosaccharide content. At the same time, the use of the amylose-type oligosaccharide 5-mer can make other oligosaccharides have clear peak shape, good resolution and high accuracy.
  • the test subject of the present invention is Morinda oligosaccharide, which is present in a raw material medicine containing Morinda oligosaccharide, or in an extract containing Morinda officinose extracted from a natural plant (Chinese herbal medicine).
  • the preparation includes a solid preparation and a liquid preparation, and the solid preparation may be a tablet, a capsule, a granule, a pill or the like, and the liquid preparation may be an oral solution, a syrup, an injection or the like.
  • the raw material drug containing Morinda oligosaccharide theoretically includes both a solid raw material drug and a liquid raw material drug, and similarly, a natural plant (Chinese herbal medicine) extracted extract theory containing Morinda officinalis
  • the upper layer may also be an extract in a solid state, or may be an extract in a liquid state.
  • the extract containing the Morinda oligosaccharide extracted from the above natural plant may be an extract containing Morinda citrifolia extracted from Morinda citrifolia, or a plurality of natural plants (at least at least Including Basil, it can also be a plant belonging to the genus Basil, such as the genus Morinda cochinchinensis DC, the Morinda parvifolia Benth, the Morinda umbellata L., etc.
  • the extract of the oligosaccharide may also be an extract containing Morinda oligosaccharide extracted from a mixed plant including the above plants.
  • the preparation of the test solution includes: taking a solid preparation containing Morinda oligosaccharide or a solid Basil oligosaccharide raw material.
  • the drug substance or extract herein should contain Morinda oligosaccharides.
  • the content of Morinda oligosaccharides which is determined by the extraction of the Morinda oligosaccharide content in natural plants (Chinese herbal medicines), the extraction and refining process, and the application purpose of the extract.
  • the types and amounts of other ingredients or impurities in natural plants (Chinese herbal medicines) containing Morinda oligosaccharides are more complicated than those of the drug substances.
  • the above filtration is a microporous membrane, and the pore size of the microporous membrane may generally be 0.3, 0.45, 0.5, 0.75 um, preferably 0.45 um.
  • the collection of the continuous filtrate is the filtrate from the intermediate stage of the filtration process.
  • every 0.2-0.5 g of the solid preparation or the solid Morinda oligosaccharide bulk drug or solid extract is added to the stoppered conical flask, water and/or mobile phase 10 ml, ultrasonication, supernatant, microporous
  • the filter membrane is filtered, and the filtrate is taken to obtain a test solution.
  • the preparation of the test solution includes: taking a liquid preparation containing Morinda oligosaccharide or a liquid raw material containing Morinda oligosaccharide or a natural plant ( Chinese herbal medicine) Extracted liquid extract containing Morinda oligosaccharide, diluted with water or mobile phase, take the supernatant, filter, and take the filtrate to obtain the test solution.
  • each of the above 0.1-10 ml of the liquid preparation or the liquid Morinda oligosaccharide raw material or liquid extract is added to the stoppered conical flask by adding water and/or a mobile phase to 10 ml, and filtering through a microporous membrane. The filtrate is taken to obtain the test solution.
  • the ultrasonic wave is also subjected to dissolution during the dissolution process, and after filtration, it is filtered through a microporous membrane having a pore diameter of not more than 0.75 ⁇ m.
  • the microporous membrane in the preferred embodiment is a microporous membrane of 0.45 ⁇ m.
  • the filter membrane and the continuous filtrate are the test solution; the solid preparation test solution is more preferably a solution obtained by adding water or a mobile phase of 10 ml per 0.3 g of the test sample.
  • the preparation scheme of the most preferred test solution includes: each 0.3 g of solid containing Morinda oligosaccharide The preparation is added with 10 ml of water to obtain a test solution; the solid preparation is a capsule (the main object of which is the content of the capsule), a powder injection, a tablet or a granule.
  • the detector used in the high performance liquid chromatography in the measuring method of the present invention is a differential detector or an evaporative light scattering detector.
  • the method for determining content includes:
  • test solution taking the contents of the capsule containing Morinda oligosaccharide 0.25-0.35g, placing it in a conical flask, adding 10 ml of water, and aspirating at a frequency of 100 W at 40 KHz for 10 minutes. Set, take the supernatant, filter with 0.45um microporous membrane, take the filtrate, then get the test solution;
  • the invention uses the inulin oligosaccharide 5mer in the Morinda oligosaccharide as a reference substance, and uses HPLC method to determine the content of Morinda oligosaccharide in various preparations or extracts or raw materials, and the innovation lies in:
  • the composition of the inulin-type oligosaccharide 5-mer is based on the composition, and the retention time of the component is set to 1, and the retention time of the chromatographic peaks of other polymers is converted into relative retention time, and the relative retention time is used to identify other than 5 sugars.
  • oligosaccharides The presence of oligosaccharides, and the content of each component and total oligosaccharides were calculated by the external standard method according to the external standard method, and the stable and accurate content of Morinda oligosaccharides was obtained.
  • a reference substance can be used to characterize a plurality of structurally similar compounds, and the relative content of a plurality of structurally similar compounds and the total content of the mixture can be determined, the accuracy of the content determination is improved, and the operation steps are simplified.
  • the number of reference materials is reduced, the measurement time is shortened, the repeatability is good, and the applicability is high.
  • the retention time of the 5-sugar chromatographic peak in the chromatogram of the sample should be consistent with the retention time of the inulin-type oligosaccharide 5-mer reference, and should be relative to the inulin oligosaccharide 5-mer.
  • the chromatographic conditions of the present invention are selected based on a large amount of experimental data, and various indicators including peak shape, resolution, peak area, reproducibility, etc. are selected, and the optimal column, mobile phase, detector, and column temperature are comprehensively evaluated. And other conditions.
  • the details are as follows: HPLC analysis of the reference product of the inulin-type oligosaccharide 5-mer (5 sugar):
  • a 5 saccharide control solution was prepared for 20 ⁇ l, and subjected to HPLC analysis under the chromatographic conditions of the present invention, and the chromatogram was recorded. Data: relative retention time 17.502, peak area 118018, peak area (%) 100.0000.
  • chrysanthemum-type oligosaccharide trimer (C 18 3 ⁇ 4 2 0 16 , abbreviated as: 3 sugar), chrysanthemum-type oligosaccharide 4-mer (C 24 H 42 0 21 , abbreviation: 4 Sugar), chrysanthemum-type oligosaccharide 5-mer (C 3 .H 52 0 26 , abbreviated as: 5 sugar), chrysanthemum-type oligosaccharide 6-mer (C 36 H 62 0 31 , abbreviated as 6 sugar), chrysanthemum starch Type oligosaccharide 7-mer (C 42 H 72 0 36 , abbreviated as: 7 sugar), inulin-type oligosaccharide 8-mer (C 48 H 82 0 41 , abbreviated as: 8 sugar) and inulin-type oligosaccharide 9-mer (C 54 H
  • the retention time of the 5 sugar peaks was consistent with the retention time of the control. 3 ⁇ 9
  • the retention time of the peak of the polymer, which is converted to the relative retention time by the standard of 5 sugars, and the spectrum is resolved as follows. The data is shown in Table 1.
  • Morinda officinalis oligosaccharide which is a chrysanthemum-type oligosaccharide trimer (C 18 H 32 0 16 , abbreviated as: 3 sugar), and a chamomile-type oligosaccharide 4-mer (C 24 H 42 0 21 , abbreviation: 4 sugar), chrysanthemum-type oligosaccharide 5-mer (C 3 .H 52 0 26 , abbreviated as: 5 sugar), chrysanthemum-type oligosaccharide 6-mer (C 36 H 62 0 31 , Abbreviation: 6 sugar), chrysanthemum-type oligosaccharide 7-mer (C 42 H 72 0 36 , abbreviated as 7 sugar), chrysanthemum-type oligosaccharide 8-mer (C 48 H 82 0 41 , abbreviation
  • acetonitrile is chromatographically pure, 7j-pure water, and the remaining reagents are of analytical grade;
  • the column is an Inertsil N3 ⁇ 4 column (5 ⁇ m, 4. 5 X 250 mm);
  • the mobile phase is acetonitrile: water: triethylamine (68: 32: 0.1);
  • the detector is a RID-10A differential detector
  • Blank test Weigh the excipient according to the proportion of the prescription, take the treatment method of the test sample, take 20 ⁇ 1, perform HPLC analysis under the above chromatographic conditions, record the chromatogram, and the result shows no interference.
  • Detector RID-10A differential detector
  • the present invention uses the amyloid oligosaccharide 5-mer as a reference substance, and calculates the Bayu day by the sum of the peak areas of 3-9 sugars.
  • the method of oligosaccharide content is practical.
  • the method is accurate, sensitive, specific, and blank excipients have no interference to the determination, and can effectively control the content of the product.
  • the quality control method of the invention is fast, convenient and reproducible, and is suitable for laboratory sample detection and quality inspection of industrialized large production. Specific implementation
  • Example 1 Determination of the content of Morinda oligosaccharide capsules
  • Reagents Acetonitrile-chromatographic pure (Tianjin Siyou Biomedical Technology Development Company), 7j-pure water, the rest are of analytical grade; Reference: Chrysanthemum-type oligosaccharide 5-mer, supplied by China National Institute for the Control of Pharmaceutical and Biological Products, purity 99.99 %.
  • Detector RID-10A differential detector
  • each component reached baseline separation.
  • the number of theoretical plates of the amylose-type oligosaccharide 5-mer peak was determined to be not less than 2000.
  • the preparation of the reference solution is taken from the appropriate amount of the amylose-type oligosaccharide 5-mer (5 sugar) reference substance, accurately weighed, and water is added to make each Lml contains 1mg of solution, that is.
  • the measurement method accurately absorbs the reference solution and the test solution 20 ⁇ 1, respectively, and injects into the liquid chromatograph, and determines the content of the amylose-type oligosaccharide 3-mer-9-mer by the external standard method.
  • the content of each oligosaccharide is added, that is, the content of the active ingredient of the Morinda oligosaccharide is obtained.
  • Each of the capsules containing the inulin-type oligosaccharide 5 mer is not less than 9.0 mg, and the total amount of the stearyl amylose-containing oligosaccharide 3-9 mer is determined by the amylose-type oligosaccharide 5-mer. Less than 75.0 mg.
  • Example 2 Determination of the content of Morinda granules
  • Detector RID-10A differential detector
  • test solution Take 0.25g sample of Morinda granules granules, accurately weighed, set 10ml volumetric flask, add mobile phase 10ml, ultrasonic, take the supernatant, dilute to the mark with mobile phase, pass 0.45 ⁇ The microporous membrane was filtered and the filtrate was taken.
  • Determination method respectively, accurately draw the reference solution and the test solution 20 ⁇ 1, inject into the liquid chromatograph, calculate the content of the amylose-type oligosaccharide 3-mer-9-mer by the external standard method, respectively.
  • the content of the oligosaccharide is added, that is, the content of the active ingredient of the Morinda oligosaccharide is obtained.
  • Bayer oligosaccharide granules should not be less than 6.0mg per gram of inulin-containing oligosaccharide 5g, and the total amount of chrysanthemum-type oligosaccharide 3 ⁇ 9-mers
  • the sugar 5 polymer meter shall not be less than 50.0 mg.
  • Example 3 Determination of the content of Morinda oligosaccharide tablets
  • Detector RID-10A differential detector
  • Preparation of the reference solution Take the amylose-type oligosaccharide 5-mer (5 sugar). The appropriate amount of the reference substance, accurately weighed, add water to make a solution containing lmg per lml, that is.
  • test solution Take 0.4g of Morinda oligosaccharide tablet sample, accurately weigh it, put it into a 10ml volumetric flask, add 10ml of water, sonicate, take the supernatant, dilute with water to the mark, pass 0.45 ⁇ microporous Filter the membrane and take the filtrate to obtain.
  • the measurement method accurately absorbs the reference solution and the test solution for each 20 ⁇ 1, and injects into the liquid chromatograph, and calculates the content of the amylose-type oligosaccharide 3-mer-9-mer by the external standard method.
  • the content of the sugar is added, that is, the content of the active ingredient of the Morinda oligosaccharide is obtained.
  • Each tablet (0.4g/tablet) of the Morinda oligosaccharide tablets should not be less than 9.0mg, and the total amount of the chrysanthemum-type oligosaccharide 3 ⁇ 9-mers should be The sugar 5 polymer meter shall not be less than 75.0 mg.
  • Example 4 Determination of the content of freeze-dried powder of Morinda officinalis
  • Detector evaporative light scattering detector
  • Preparation of the test solution Take 0.3g of the Morinda oligosaccharide freeze-dried powder sample, accurately weigh it, put it into a 10ml volumetric flask, add 10ml of mobile phase to the mark, filter through 0.45 ⁇ microporous membrane, and take the filtrate. That is.
  • the measurement method was the same as in Example 1.
  • the gluten-free oligosaccharide freeze-dried powder needle should not be less than 60.0 mg per gram of inulin-containing oligosaccharide, and the total amount of inulin-containing oligosaccharide 3-9 mer is in the form of inulin-type oligosaccharide 5-mer. No less than 500.0mg.
  • Example 5 Determination of the content of Morinda oligosaccharide oral solution
  • Detector RID-10A differential detector
  • Preparation of the test solution Take 10 ml of the sample of Morinda oligosaccharide oral solution, accurately weigh it, place it in a 10 ml volumetric flask, dilute to the mark with water, filter through a 0.45 ⁇ microporous membrane, and take the filtrate to obtain.
  • the measurement method was the same as in Example 1.
  • the 5 mg of the inulin-containing oligosaccharide-containing oligosaccharide per 10 ml of the Morinda oligosaccharide oral solution shall not be less than 9.0 mg, and the total amount of the chrysanthemum-type oligosaccharide 3-9-mer is determined by the amylose-type oligosaccharide 5-mer. Not less than 75.0mg.
  • Example 6 Determination of the content of Morinda oligosaccharide syrup
  • Detector evaporative light scattering detector
  • Preparation of the reference solution Take the amylose-type oligosaccharide 5-mer (5 sugar). The appropriate amount of the reference substance, accurately weighed, add water to make a solution containing lmg per lml, that is.
  • test solution Take 5 ml of Morinda oligosaccharide syrup sample, accurately weigh it, place it in a 10 ml volumetric flask, dilute with water to the mark, filter through 0.45 ⁇ microporous membrane, and take the filtrate to obtain.
  • Bayan oligosaccharide syrup containing 5 mg of inulin-containing oligosaccharide per ml, containing amylose-type oligosaccharide 3 ⁇ 9-mer The total amount is not less than 7.5 mg based on the amylose-type oligosaccharide 5-mer.
  • Example 7 Determination of the content of Morinda oligosaccharide tablets
  • Detector RID-10A differential detector
  • Preparation of the reference solution Take the amylose-type oligosaccharide 5-mer (5 sugar). The appropriate amount of the reference substance, accurately weighed, and add the mobile phase to prepare a solution containing lmg per lml.
  • test solution Take 0.4g of Morinda oligosaccharide tablet sample, accurately weigh it, put it in a 10ml volumetric flask, add 10ml of mobile phase, sonicate, take the supernatant, dilute to the mark with mobile phase, pass 0.45 Filter through ⁇ microporous membrane and take the filtrate to obtain.
  • the measurement method was the same as in Example 1.
  • Each of the Morinda oligosaccharide tablets contains not less than 9.0 mg of the amylose-type oligosaccharide, and the total amount of the chrysanthemum-type oligosaccharide 3-9-mer is calculated by the amylose-type oligosaccharide 5-mer. Not less than 75.0mg.
  • Example 8 Determination of the content of Morinda oligosaccharide capsules
  • Detector RID-10A differential detector
  • the measurement method was the same as in Example 1.
  • Each of the capsules containing the inulin-type oligosaccharide 5 mer can be no less than 9.0 mg, containing the amylose-type oligosaccharide 3 ⁇ 9-mer. The total amount is not less than 75 mg based on the amylose-type oligosaccharide 5-mer.
  • Example 9 Determination of the content of raw materials of Morinda officinalis
  • Preparation of the reference solution Take the amylose-type oligosaccharide 5-mer (5 sugar). The appropriate amount of the reference substance, accurately weighed, add water to make a solution containing lmg per lml, that is.
  • test solution Take the raw material of Morinda oligosaccharide, stir it well, take about 150mg, accurately weigh it, place it in a conical flask, add 10ml of water precisely, sonicate for 10 minutes, filter with 0.45um microporous The membrane is filtered. The filtrate is taken and obtained.
  • the measurement method was the same as in Example 1.
  • the Morinda oligosaccharide bulk drug is calculated as a dry product, and the amylose-type oligosaccharide 5-mer is not less than 6.0%, and the total amount of the chrysanthemum-type oligosaccharide 3 ⁇ 9-mer is in the form of the chrysanthemum-type oligosaccharide 5 0% ⁇ The polymer meter, not less than 50.0%.
  • Example 10 Determination of the content of the raw material of Morinda oligosaccharide
  • Detector evaporative light scattering detector
  • Preparation of the test solution Take 1 ml of the raw material medicine of Morinda oligosaccharide liquid, place it in a stoppered conical flask, precisely add 10 ml of the mobile phase, filter it with a 0.44 ⁇ m microporous membrane, and take the filtrate to obtain.
  • the measurement method was the same as in Example 1.
  • the amount of the amylose-type oligosaccharide 5-mer of the Morinda oligosaccharide raw material is not less than 6.0%, and the total amount of the 3-9-mer of the amylose-type oligosaccharide is determined by the amylose-type oligosaccharide 5-mer. 50.0%.
  • Example 11 Determination of extracts containing Morinda offigo oligosaccharides extracted from natural plants
  • the preparation of the reference solution is taken from the appropriate amount of the amylose-type oligosaccharide 5-mer (5 sugar) reference substance, accurately weighed, and water is added to make each Lml contains 1mg of solution, that is.
  • the measurement method was the same as in Example 1.
  • the extract containing Morinda officinalis oligosaccharides extracted from natural plants is calculated as a dry product, and the amylose-containing oligosaccharide 5-mer (C 30 H 52 O 26 ) is not less than 0.12%, containing Morinda officinalis ( That is, the total amount of the chrysanthemum-type oligosaccharide 3-mer-9-mer is not less than 1.0% in terms of the chrysanthemum-type oligosaccharide 5-mer (C 3 .H 52 0 26 ).
  • Example 12 Determination of liquid extract containing Morinda officinalis extracted from natural plants
  • Detector RID-10A differential detector
  • test solution 10 ml of the extract containing Morinda officinose extracted from natural plants, subjected to activated carbon column chromatography, first eluted with water to reduce sugar (sulfuric acid-phenol method) was negative, It was then eluted with about 4 column volumes of 30% ethanol. The fraction eluted with 30% ethanol was collected and concentrated to obtain Morinda oligosaccharides (sample). Take about 150mg of sample, accurately weigh it, put it into a 10ml volumetric flask, dilute it to the mark with water, filter through 0.45 ⁇ microporous membrane, and take the filtrate to obtain.
  • the extract containing Morinda officinalis oligosaccharides extracted from natural plants is calculated as dry product, and the amylose-containing oligosaccharide 5-mer (C 30 H 52 O 26 ) is not less than 6%, containing Morinda officinalis ( That is, the total amount of the chrysanthemum-type oligosaccharide 3-mer-9-mer is not less than 50%, based on the amylose-type oligosaccharide 5-mer (C 3 .H 52 0 26 ).

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Abstract

A method for determining the content of oligosaccharides in morinda officinalis comprises the following steps: inspecting by high performance liquid chromatography (HPLC); using the aqueous solution of acetonitrile as the mobile phase; using inulin-type oligosaccharides (5) as the control sample, calculating respectively the content of inulin-type oligosaccharides (3~9) by the peak area based on the external standard method; obtaining the content of the morinda officinalis oligosaccharides by adding the contents of inulin-type oligosaccharides (3~9). In the present invention, while the total content of compounds with similar construction in chemical part is determined by using a single control sample, the single constituent and the respective content thereof are determined.

Description

巴戟天寡糖的含量测定方法 技术领域  Method for determining content of sorghum oligosaccharide
本发明提供了一种巴戟天寡糖的检测方法， 其为一种含有多个寡糖成分的寡糖混合物的 含量测定方法， 尤其是指在制剂或提取物或原料药中的巴戟天寡糖的高效液相含量测定方法。 背景技术  The present invention provides a method for detecting Morinda oligosaccharide, which is a method for determining the content of a mixture of oligosaccharides containing a plurality of oligosaccharide components, in particular, a Morinda citrifolia in a preparation or an extract or a drug substance. A method for determining the high-performance liquid phase content of oligosaccharides. Background technique
在制药领域， 对于某种成分的含量测定， 采用高效液相的色谱分析是很常规的方法， 本 领域技术人员常常设定某一成分为对照品， 在标的物中参照该对照品进行某成分的分析测定， 对于含有多种中药有效成分且需要同时对该多种中药成分进行定性和定量测定的时候， 往往 根据该多种成分找出其一一对应的多种对照品， 再依次进行测定， 这种对含有多个同类近似 成分的一个标的物进行逐一测定的缺点是不可避免的， 操作繁琐、 耗时费力、 效率低下， 同 一化学部位中各成分相对含量标准各自独立、 互不相关， 如此难免会造成一定程度的偏差， 无法更有效、 精确地控制质量。  In the pharmaceutical field, for the determination of the content of a certain component, chromatographic analysis using high-performance liquid phase is a very conventional method, and those skilled in the art often set a certain component as a reference substance, and refer to the reference substance in the target substance for a certain component. Analytical determination, when a plurality of traditional Chinese medicine active ingredients are included and it is necessary to simultaneously determine qualitative and quantitative determinations of the plurality of traditional Chinese medicine ingredients, a plurality of reference materials corresponding to the one-to-one correspondence are often found according to the plurality of ingredients, and then measured sequentially. The disadvantage of one-by-one measurement of a target containing a plurality of similar components is inevitable, cumbersome, time-consuming, and inefficient, and the relative content standards of the components in the same chemical part are independent and irrelevant. This will inevitably lead to a certain degree of deviation, and it is impossible to control the quality more effectively and accurately.
对含巴戟天寡糖的制剂而言， 以及对巴戟天寡糖原料药或由天然植物提取得到的含有巴 戟天寡糖的提取物而言， 其有效成分为寡糖类物质， 该寡糖物质是 3— 9糖的混合物， 若采用 化学显色反应， 显示与其它糖类（单糖、 双糖、 多糖）相同的反应特征， 因此， 数据证明对 于检测巴戟天寡糖， 选择 HPLC的色谱鉴别更具有专属性， 而且为了测定的结果更加科学和严 谨， 需要研发出一种能在测定该化学部位总量的同时还能鉴别出其中各个成分、 并给出各个 成分相对含量、 尽量减少对照品的种类、 操作简单的高效液相含量测定方法。  For the preparation containing the sorghum oligosaccharide, and the extract of the Morinda oligosaccharide bulk drug or the extract containing the natural plant, the active ingredient is an oligosaccharide substance, The oligosaccharide material is a mixture of 3-9 sugars. If chemical color reaction is used, it shows the same reaction characteristics as other sugars (monosaccharides, disaccharides, polysaccharides). Therefore, the data proves that for the detection of Morinda officinalis, the selection The chromatographic identification of HPLC is more specific, and in order to make the results of the determination more scientific and rigorous, it is necessary to develop a method for determining the total amount of the chemical part while identifying each component and giving the relative content of each component. Minimize the type of reference substance and operate a simple HPLC method.
发明内容 Summary of the invention
目前在含有巴戟天寡糖的中药制剂中， 以及含有巴戟天寡糖的原料药或由天然植物提取 得到的含有巴戟天寡糖的提取物中， 尤其是制剂及提取物生产的质量监控方面， 没有规范成 熟的含测方法， 本发明的目的是开发一种含有巴戟天寡糖成分的制剂、 原料药以及含有巴戟 天寡糖的中药提取物的质量控制方法， 使用该方法以及该方法中的技术指标， 可以很好的监 控含有巴戟天寡糖的巴戟天制剂、 尤其是口服制剂的质量， 以及监控含有巴戟天寡糖的原料 药或由天然植物提取得到的含有巴戟天寡糖的提取物， 使中药质量稳定可控， 保障疗效， 能 更好地服务社会。  Currently in the preparation of traditional Chinese medicines containing Morinda oligosaccharides, as well as the raw materials containing Morinda oligosaccharides or the extracts containing Morinda oligosaccharides extracted from natural plants, especially the quality of preparations and extracts. In terms of monitoring, there is no standardized and mature test method, and the object of the present invention is to develop a quality control method for a preparation containing a Morinda oligosaccharide component, a raw material drug, and a Chinese herbal medicine extract containing Morinda oligosaccharide, using the method And the technical indicators in the method, can well monitor the quality of the Morinda preparation containing Morinda officinalis, especially the oral preparation, and monitor the raw material containing Morinda oligosaccharides or extracted from natural plants. The extract containing Morinda oligosaccharides makes the quality of traditional Chinese medicine stable and controllable, guarantees curative effect, and can better serve the society.
本发明提供一种巴戟天寡糖的含量测定方法， 该巴戟天寡糖至少含有菊淀粉型寡糖 5聚 体， 并存在于巴戟天寡糖原料药或由天然植物（中药材）提取得到的含有巴戟天寡糖的提取 物或含有巴戟天寡糖的制剂中， 该含量测定方法包括： ( 1 )采用高效液相色谱法进行测定， 以含有乙腈的水溶液为流动相， 采用示差检测器或 蒸发光散射检测器检测； The present invention provides a method for determining the content of Morinda officinalis oligosaccharides, which comprises at least a chitosan-type oligosaccharide 5-mer, and is present in a raw material of Morinda officinolate or extracted from a natural plant (Chinese herbal medicine) In the obtained extract containing Morinda oligosaccharide or the preparation containing Morinda oligosaccharide, the content determination method includes: (1) Determination by high performance liquid chromatography, using an aqueous solution containing acetonitrile as a mobile phase, using a differential detector or an evaporative light scattering detector;
(2)对照品溶液的制备： 称取菊淀粉型寡糖 5聚体作为对照品， 加水或步骤 （1 ) 中所述 的流动相制成对照品溶液；  (2) Preparation of reference solution: Weigh the inulin-type oligosaccharide 5-mer as a control, add water or the mobile phase described in step (1) to prepare a reference solution;
(3 )供试品溶液的制备： 取含有巴戟天寡糖的制剂或含有巴戟天寡糖的原料药或由天然 植物 （中药材） 提取得到的含有巴戟天寡糖的提取物， 力 M-100重量倍数的水或者流动相使其 混合或溶解， 取上清液， 滤过， 取续滤液， 即得供试品溶液；  (3) Preparation of the test solution: Take a preparation containing Morinda oligosaccharide or a raw material containing Morinda oligosaccharide or an extract containing Morinda oligosaccharides extracted from natural plants (Chinese herbal medicine), The M-100 weight multiple of water or mobile phase is mixed or dissolved, the supernatant is taken, filtered, and the filtrate is taken to obtain the test solution;
(4) 测定： 分别吸取对照品溶液与供试品溶液， 注入液相色谱仪， 测定；  (4) Determination: separately draw the reference solution and the test solution, inject into the liquid chromatograph, and measure;
(5 )计算： 按供试品图谱中各个色谱峰与菊淀粉型寡糖 5聚体对照品相对保留时间， 鉴 定总寡糖中各寡糖的存在； 按外标法以峰面积计算该巴戟天寡糖中含有的菊淀粉型寡糖 3〜9 聚体中任一寡糖聚体或其混合物的含量， 进而确定该巴戟天寡糖中各菊淀粉型寡糖聚体的相 对含量比例， 再将菊淀粉型寡糖 3〜9聚体的含量加和， 得所述的巴戟天总寡糖的含量。  (5) Calculation: According to the relative retention time of each chromatographic peak in the test sample and the amylose oligosaccharide 5-mer reference substance, identify the existence of each oligosaccharide in the total oligosaccharide; calculate the bar by peak area according to the external standard method The content of any oligosaccharide or a mixture thereof in the inulin oligosaccharide 3~9 polymer contained in the oligosaccharide, thereby determining the relative content of each of the inulin oligosaccharides in the Morinda oligosaccharide In proportion, the content of the chrysanthemum-type oligosaccharide 3~9-mer is added to obtain the content of the total oligosaccharide of the Morinda officinalis.
上述的巴戟天寡糖在含有菊淀粉型寡糖 5聚体的同时， 优选还含有菊淀粉型寡糖 3〜4、 6〜9聚体中任一寡糖或其混合物。  The above-mentioned Morinda oligosaccharide preferably contains, in addition to the inulin-type oligosaccharide 5-mer, any of the oligosaccharides of the inulin type oligosaccharide 3~4, 6~9-mer, or a mixture thereof.
上述续滤液是过滤过程中收集到的中间阶段的滤液。  The above continuous filtrate is the intermediate stage filtrate collected during the filtration process.
上述巴戟天寡糖（morinda officinalis oligosaccharides)为具有抗抑郁作用的活性成分， 包 括菊淀粉型寡糖 3糖〜 9糖，即，本发明所述的巴戟天寡糖是至少含有菊淀粉型寡糖 5聚体（ 1F 一果呋喃糖基耐斯糖 lF-fructofuranosyl nystose) 的寡糖， 或同时含有菊淀粉型寡糖 5聚体以 及菊淀粉型寡糖 3聚体、菊淀粉型寡糖 4聚体（耐斯糖 nystoSe)、菊淀粉型寡糖 6聚体（(2→1) 果呋喃糖基蔗糖 hexasaccharide)、 菊淀粉型寡糖 7聚体 （hep tasaccharide), 菊淀粉型寡糖 8 聚体 （8糖） 和菊淀粉型寡糖 9聚体 （9糖） 任意或其组合的混合物。 The above morinda officinalis oligosaccharides are active ingredients having antidepressant effects, including chrysanthemum-type oligosaccharide 3 saccharides to 9 saccharides, that is, the Morinda oligosaccharides of the present invention contain at least a chrysanthemum type Oligosaccharide of oligosaccharide 5-mer (1F-Fructofuranosyl nystose), or both of the amylose-type oligosaccharide 5-mer and the inulin-type oligosaccharide trimer, inulin-type oligosaccharide 4-mer (Nice saccharide n ysto Se), inulin oligosaccharides, 6-mers ((2 → 1) If the furanosyl sugar hexasaccharide), inulin oligosaccharides 7 mer (hep tasaccharide), inulin type A mixture of oligosaccharide 8-mer (8 sugar) and chrysanthemum-type oligosaccharide 9-mer (9 sugar) in any combination or combination thereof.
在本发明的优选实施例中，上述方法中步骤（1 )中色谱柱优选采用氨基柱（氨基色谱柱) 或 C18反相柱； 例如 Inertsil NH2柱 （5μηι， 4.6x250 mm ), 采用的检测器为示差检测器或蒸 发光散射检测器， 针对这样的色谱条件， 所述的流动相一般选择含有 40-80% (体积） 的乙腈 的水溶液； 优选为体积比 3: 1〜1： 1的乙腈与水的混合溶液。 In a preferred embodiment of the present invention, the column in the step (1) in the above method is preferably an amino column (amino column) or a C18 reverse phase column; for example, an Inertsil NH 2 column (5 μηι, 4.6 x 250 mm), which is used for detection. For the chromatographic conditions, the mobile phase generally selects an aqueous solution containing 40-80% by volume of acetonitrile; preferably a volume ratio of 3:1 to 1:1. A mixed solution of acetonitrile and water.
本发明的巴戟天寡糖是含有菊淀粉型寡糖 5聚体、 菊淀粉型寡糖 3聚体、 菊淀粉型寡糖 4聚体、 菊淀粉型寡糖 6聚体、 菊淀粉型寡糖 7聚体、 菊淀粉型寡糖 8聚体和菊淀粉型寡糖 9 聚体的至少一种或者一种以上的混合物。  The Morinda oligosaccharide of the present invention comprises a chrysanthemum-type oligosaccharide 5-mer, a chrysanthemum-type oligosaccharide trimer, a chrysanthemum-type oligosaccharide tetramer, a chrysanthemum-type oligosaccharide 6-mer, a chrysanthemum-type oligosaccharide At least one or a mixture of one or more of a sugar 7-mer, a chrysanthemum-type oligosaccharide 8-mer, and a chrysanthemum-type oligosaccharide 9-mer.
发明人发现， 为了改善拖尾， 使得到的色谱图的峰型完整美观， 本发明所述的含量测定 方法中采用的含有乙腈的水溶液的流动相优选还含有三乙胺， 所述的三乙胺在该流动相中所 占的体积一般不超过 0.5 %； 一般可为 0.01%-0.5%。  The inventors have found that in order to improve the tailing, the peak shape of the obtained chromatogram is intact and beautiful, and the mobile phase of the aqueous solution containing acetonitrile used in the content determination method of the present invention preferably further contains triethylamine, said three The volume of amine in the mobile phase generally does not exceed 0.5%; typically it can range from 0.01% to 0.5%.
在本发明的优选实施例中， 上述含量测定方法步骤 （1 ) 所述的流动相最优选为体积比 68： 32的乙腈和水的混合溶液， 该混合溶液中还含有不超过该流动相体积的 0.5%的三乙胺； 例如， 可以是体积比 68: 32： 0.1的乙腈、 水和三乙胺的混合溶液作为流动相， 得到的图谱和 峰型呈现令人惊喜的趋于完美效果。 In a preferred embodiment of the present invention, the mobile phase described in the step (1) of the content determination method is most preferably a volume ratio. 68: 32 a mixed solution of acetonitrile and water, the mixed solution further containing not more than 0.5% by volume of the mobile phase of triethylamine; for example, acetonitrile, water and triethylamine in a volume ratio of 68:32:0.1 The mixed solution acts as a mobile phase, and the resulting map and peak shape present a surprisingly perfect effect.
本发明对照品选用菊淀粉型寡糖 5聚体，这是因为巴戟天寡糖是 3〜9聚体寡糖的混合物， 在研究确定的 HPLC色谱条件下， 菊淀粉型寡糖 5聚体相对保留时间居中、 色谱峰峰高或峰 面积适中， 以其标定其它寡糖聚体， 各寡糖与菊淀粉型寡糖 5聚体峰相对位置差较均匀， 可 减少以色谱图峰面积或峰高积分计算相对含量和总寡糖含量时存在的系统偏差。 同时， 采用 菊淀粉型寡糖 5聚体能够使得其他寡糖的 HPLC的峰型清晰、 分离度较好、 准确度高。  The reference substance of the present invention is a chrysanthemum-type oligosaccharide 5-mer, because the Morinda oligosaccharide is a mixture of 3~9-mer oligosaccharides, and the amylose-type oligosaccharide 5-mer is determined under the HPLC chromatographic conditions determined by the study. Relative retention time is centered, peak height or peak area is moderate, and other oligosaccharides are calibrated. The relative position difference between each oligosaccharide and the amylose oligosaccharide 5-mer peak is relatively uniform, which can reduce the peak area of the chromatogram or The peak height integral calculates the relative deviation of the relative content and total oligosaccharide content. At the same time, the use of the amylose-type oligosaccharide 5-mer can make other oligosaccharides have clear peak shape, good resolution and high accuracy.
本发明的检测对象是巴戟天寡糖， 其存在于含有巴戟天寡糖的原料药中， 或存在于由天 然植物 （中药材） 提取得到的含有巴戟天寡糖的提取物中， 或存在于含有巴戟天寡糖的制剂 中， 该制剂包括固体制剂和液体制剂， 固体制剂可是片剂、 胶囊剂、 颗粒剂、 丸剂等等， 液 体制剂可是口服液、 糖浆剂、 注射剂等。  The test subject of the present invention is Morinda oligosaccharide, which is present in a raw material medicine containing Morinda oligosaccharide, or in an extract containing Morinda officinose extracted from a natural plant (Chinese herbal medicine). Or in the preparation containing Morinda oligosaccharide, the preparation includes a solid preparation and a liquid preparation, and the solid preparation may be a tablet, a capsule, a granule, a pill or the like, and the liquid preparation may be an oral solution, a syrup, an injection or the like.
其中， 含有巴戟天寡糖的原料药理论上既包括固体的原料药， 也可以是液体的原料药， 同样地， 天然植物 （中药材） 提取得到的含有巴戟天寡糖的提取物理论上也可以是固体状态 的提取物， 还可以是液体状态的提取物。  Among them, the raw material drug containing Morinda oligosaccharide theoretically includes both a solid raw material drug and a liquid raw material drug, and similarly, a natural plant (Chinese herbal medicine) extracted extract theory containing Morinda officinalis The upper layer may also be an extract in a solid state, or may be an extract in a liquid state.
上述天然植物 （中药材） 提取得到的含有巴戟天寡糖的提取物既可以是巴戟天中药材提 取得到的含有巴戟天寡糖的提取物， 也可以是多种天然植物 （其中至少包括巴戟天， 还可以 是与巴戟天相近科属的植物， 例如茜草科的大果巴戟 Morinda cochinchinensis DC、 百眼藤 Morinda parvifolia Benth,羊角藤 Morinda umbellata L.等）提取到的含有巴戟天寡糖的提取物， 还可是包括上述植物在内的混合植物提取到的含有巴戟天寡糖的提取物。  The extract containing the Morinda oligosaccharide extracted from the above natural plant (Chinese herbal medicine) may be an extract containing Morinda citrifolia extracted from Morinda citrifolia, or a plurality of natural plants (at least at least Including Basil, it can also be a plant belonging to the genus Basil, such as the genus Morinda cochinchinensis DC, the Morinda parvifolia Benth, the Morinda umbellata L., etc. The extract of the oligosaccharide may also be an extract containing Morinda oligosaccharide extracted from a mixed plant including the above plants.
不管本发明的检测对象巴戟天寡糖存在于巴戟天寡糖原料药中， 还是存在于由天然植物 提取到的含有巴戟天寡糖的提取物中， 抑或存在于含有巴戟天寡糖的制剂中， 以该巴戟天寡 糖的存在形式为固体状态为例， 所述的供试品溶液的制备包括： 取含有巴戟天寡糖的固体制 剂或固体巴戟天寡糖原料药或由天然植物 （中药材） 提取得到的含有巴戟天寡糖的固体提取 物， 加水或者流动相使其溶解或混合， 取上清液， 滤过， 取续滤液， 即得供试品溶液。  Regardless of whether the test object of the present invention is present in the Morinda oligosaccharide bulk drug, it is present in the extract containing the Morinda oligosaccharide extracted from the natural plant, or is present in the extract containing Morinda citrifolia. In the preparation of the sugar, taking the form of the Morinda oligosaccharide as a solid state, the preparation of the test solution includes: taking a solid preparation containing Morinda oligosaccharide or a solid Basil oligosaccharide raw material. A solid extract containing Morinda officinalis extracted from natural plants (Chinese herbal medicines), added with water or mobile phase to dissolve or mix, and the supernatant is taken, filtered, and the filtrate is taken to obtain the test sample. Solution.
业内公知， 此处的原料药或提取物均应含有巴戟天寡糖。 而含有巴戟天寡糖的原料药， 其中的巴戟天寡糖含量至少占原料药总量的 50%以上； 而由天然植物 （中药材） 提取得到的 含有巴戟天寡糖的提取物， 其中的巴戟天寡糖含量则没有限制， 具体由提取天然植物 （中药 材） 中的巴戟天寡糖含量和提取精制工艺、 以及提取物应用目的而定。 同样地， 含有巴戟天 寡糖的天然植物 （中药材） 中的其它成分或杂质的种类和量也较原料药复杂。  It is well known in the art that the drug substance or extract herein should contain Morinda oligosaccharides. The raw material medicine containing Morinda oligosaccharide, wherein the Morinda oligosaccharide content accounts for at least 50% of the total amount of the raw material medicine; and the extract containing the Morinda oligosaccharide extracted from the natural plant (Chinese herbal medicine) There is no restriction on the content of Morinda oligosaccharides, which is determined by the extraction of the Morinda oligosaccharide content in natural plants (Chinese herbal medicines), the extraction and refining process, and the application purpose of the extract. Similarly, the types and amounts of other ingredients or impurities in natural plants (Chinese herbal medicines) containing Morinda oligosaccharides are more complicated than those of the drug substances.
上述滤过优选采用的是微孔滤膜，该微孔滤膜的孔径一般可以是 0.3、 0.45、 0.5、 0.75um， 优选 0.45um。 收集续滤液是过滤过程中收集中间阶段的滤液。 优选地， 上述每 0.2-0.5g固体制剂或固体巴戟天寡糖原料药或固体提取物， 于具塞锥形 瓶中加入水和 /或流动相 10ml， 超声， 取上清液， 微孔滤膜滤过， 取续滤液， 即得供试品溶液。 Preferably, the above filtration is a microporous membrane, and the pore size of the microporous membrane may generally be 0.3, 0.45, 0.5, 0.75 um, preferably 0.45 um. The collection of the continuous filtrate is the filtrate from the intermediate stage of the filtration process. Preferably, every 0.2-0.5 g of the solid preparation or the solid Morinda oligosaccharide bulk drug or solid extract is added to the stoppered conical flask, water and/or mobile phase 10 ml, ultrasonication, supernatant, microporous The filter membrane is filtered, and the filtrate is taken to obtain a test solution.
以该巴戟天寡糖为液体状态为例， 所述的供试品溶液的制备包括： 取含有巴戟天寡糖的 液体制剂或含有巴戟天寡糖的液体原料药或由天然植物 （中药材） 提取得到的含有巴戟天寡 糖的液体提取物， 加水或流动相稀释， 取上清液， 滤过， 取续滤液， 即得供试品溶液。  Taking the Morinda oligosaccharide as a liquid state, the preparation of the test solution includes: taking a liquid preparation containing Morinda oligosaccharide or a liquid raw material containing Morinda oligosaccharide or a natural plant ( Chinese herbal medicine) Extracted liquid extract containing Morinda oligosaccharide, diluted with water or mobile phase, take the supernatant, filter, and take the filtrate to obtain the test solution.
优选地， 上述每 0.1-10ml的液体制剂或液体巴戟天寡糖原料药或液体提取物， 于具塞锥 形瓶中加入水和 /或流动相至 10ml， 用微孔滤膜滤过， 取续滤液， 即得供试品溶液。  Preferably, each of the above 0.1-10 ml of the liquid preparation or the liquid Morinda oligosaccharide raw material or liquid extract is added to the stoppered conical flask by adding water and/or a mobile phase to 10 ml, and filtering through a microporous membrane. The filtrate is taken to obtain the test solution.
在制备供试品溶液的过程中， 溶解过程中还经历了超声， 溶解后用孔径为不超过 0.75um 的微孔滤膜滤过，优选实施例中的微孔滤膜为 0.45um的微孔滤膜，取续滤液即为供试品溶液； 所述的固体制剂供试品溶液更优选为每 0.3g供试品加入水或流动相 10ml得到的溶液。  In the process of preparing the test solution, the ultrasonic wave is also subjected to dissolution during the dissolution process, and after filtration, it is filtered through a microporous membrane having a pore diameter of not more than 0.75 μm. The microporous membrane in the preferred embodiment is a microporous membrane of 0.45 μm. The filter membrane and the continuous filtrate are the test solution; the solid preparation test solution is more preferably a solution obtained by adding water or a mobile phase of 10 ml per 0.3 g of the test sample.
再以该巴戟天寡糖的存在于含有巴戟天寡糖的固体制剂中为例， 所述的最优选的供试品 溶液的制备方案包括： 每 0.3g含有巴戟天寡糖的固体制剂加入水 10ml， 即得供试品溶液； 该 固体制剂为胶囊剂 （主要检测对象是胶囊剂的内容物)、 粉针剂、 片剂或颗粒剂。  Taking the presence of the Morinda oligosaccharide as an example in a solid preparation containing Morinda oligosaccharides, the preparation scheme of the most preferred test solution includes: each 0.3 g of solid containing Morinda oligosaccharide The preparation is added with 10 ml of water to obtain a test solution; the solid preparation is a capsule (the main object of which is the content of the capsule), a powder injection, a tablet or a granule.
本发明的测定方法中高效液相色谱法采用的检测器为示差检测器或蒸发光散射检测器。 在本发明的另一实施例中， 所述的含量测定方法包括：  The detector used in the high performance liquid chromatography in the measuring method of the present invention is a differential detector or an evaporative light scattering detector. In another embodiment of the present invention, the method for determining content includes:
( 1 )采用高效液相色谱法进行测定； 色谱条件是采用氨基柱； 以乙腈： 水： 三乙胺的体 积比 68: 32： 0.1为流动相； 检测器为示差检测器； 柱温 40°C， 检测器温度 40°C ; 理论板数按 菊淀粉型寡糖 5聚体计算不低于 2000;  (1) Determination by high performance liquid chromatography; chromatographic conditions using an amino column; acetonitrile: water: triethylamine volume ratio of 68: 32: 0.1 as mobile phase; detector is a differential detector; column temperature 40 ° C, the detector temperature is 40 ° C; the number of theoretical plates is not less than 2000 according to the amylose-type oligosaccharide 5-mer;
(2)对照品溶液的制备， 取菊淀粉型寡糖 5聚体为对照品， 加水制成 lmg /ml对照品溶液； (2) Preparation of the reference solution, taking the amylose-type oligosaccharide 5-mer as a reference substance, adding water to make a solution of 1 mg / ml of the reference substance;
(3 )供试品溶液的制备， 取含有巴戟天寡糖的胶囊剂的内容物 0.25-0.35g， 置具塞锥形瓶 中， 加入水 10ml， 功率 100W 频率 40KHz下超声 10分钟， 静置， 取上清液， 用 0.45um微孔滤 膜滤过， 取续滤液， 即得供试品溶液； (3) Preparation of the test solution, taking the contents of the capsule containing Morinda oligosaccharide 0.25-0.35g, placing it in a conical flask, adding 10 ml of water, and aspirating at a frequency of 100 W at 40 KHz for 10 minutes. Set, take the supernatant, filter with 0.45um microporous membrane, take the filtrate, then get the test solution;
(4) 进行测定， 分别吸取对照品溶液与供试品溶液 20μ1， 注入液相色谱仪， 测定； (4) Perform the measurement, respectively, take the reference solution and the test solution 20μ1, inject into the liquid chromatograph, and measure;
(5 )计算， 按供试品图谱中各个色谱峰与菊淀粉型寡糖五聚体对照品相对保留时间， 鉴 定总寡糖中各个寡糖的存在； 按外标法以峰面积分别计算菊淀粉型寡糖 3〜9聚体的含量， 进 而确定总寡糖中各个寡糖的相对含量比例， 再将菊淀粉型寡糖 3〜9聚体的含量进行加和， 即 得巴戟天总寡糖的含量。 (5) Calculate, according to the relative retention time of each chromatographic peak in the test sample and the amylose oligosaccharide pentamer reference substance, identify the existence of each oligosaccharide in the total oligosaccharide; calculate the chrysanthemum by the external standard method The content of the starch-type oligosaccharide 3~9-mer, and further determining the relative content ratio of each oligosaccharide in the total oligosaccharide, and then adding the content of the chrysanthemum-type oligosaccharide 3~9-mer, The content of oligosaccharides.
巴戟天寡糖的结构  Basil oligosaccharide structure
Figure imgf000005_0001
其中 =l为 3糖； n=2为 4糖； n=3为 5糖； n=4为 6糖； n=5为 7糖； n=6为 8糖； n=7为 9糖。 本发明以巴戟天寡糖中的菊淀粉型寡糖 5聚体作为对照品， 采用 HPLC法来测定各种制 剂或提取物或原料药中巴戟天寡糖的含量， 创新点在于： 以该菊淀粉型寡糖 5聚体这种成分 为基准，将该成分的保留时间定为 1，其他聚合体色谱峰的保留时间相应换算成相对保留时间， 用相对保留时间鉴别 5糖外的其它寡糖的存在， 再按外标法以峰面积分别计算各成分以及总 寡糖的含量， 即可得到较为稳定且准确的巴戟天寡糖的含量。 ΒΡ， 以一种对照品即可给多个 结构类似的化合物定性， 并能测定多个结构类似的化合物的相对含量及其混合物的总含量， 提高了含量测定的精确性， 简化了操作步骤， 减少了对照品数量， 缩短了测定时间， 重复性 好， 适用性高。
Figure imgf000005_0001
Wherein =1 is 3 sugars; n=2 is 4 sugars; n=3 is 5 sugars; n=4 is 6 sugars; n=5 is 7 sugars; n=6 is 8 sugars; n=7 is 9 sugars. The invention uses the inulin oligosaccharide 5mer in the Morinda oligosaccharide as a reference substance, and uses HPLC method to determine the content of Morinda oligosaccharide in various preparations or extracts or raw materials, and the innovation lies in: The composition of the inulin-type oligosaccharide 5-mer is based on the composition, and the retention time of the component is set to 1, and the retention time of the chromatographic peaks of other polymers is converted into relative retention time, and the relative retention time is used to identify other than 5 sugars. The presence of oligosaccharides, and the content of each component and total oligosaccharides were calculated by the external standard method according to the external standard method, and the stable and accurate content of Morinda oligosaccharides was obtained. ΒΡ, a reference substance can be used to characterize a plurality of structurally similar compounds, and the relative content of a plurality of structurally similar compounds and the total content of the mixture can be determined, the accuracy of the content determination is improved, and the operation steps are simplified. The number of reference materials is reduced, the measurement time is shortened, the repeatability is good, and the applicability is high.
以下述实例来说明但不限制该专利。  The following examples are illustrative but not limiting.
该液相色谱图中供试品图谱中 5糖色谱峰的保留时间， 应与菊淀粉型寡糖 5聚体对照品 保留时间一致， 与菊淀粉寡糖 5聚体相比， 还应出现相对保留时间为 0.50~0.70的菊淀粉型寡 糖 3聚体峰 ; 0.70~0.90的菊淀粉型寡糖 4聚体峰；1.10~1.40的菊淀粉型寡糖 6聚体峰；1.50~1.83 的菊淀粉型寡糖 7聚体峰； 1.83~2.50的菊淀粉型寡糖 8聚体峰； 1.85~3.40的菊淀粉型寡糖 9 聚体峰， 再按外标法以峰面积分别计算各寡糖的含量。 The retention time of the 5-sugar chromatographic peak in the chromatogram of the sample should be consistent with the retention time of the inulin-type oligosaccharide 5-mer reference, and should be relative to the inulin oligosaccharide 5-mer. Chrysanthemum-type oligosaccharide 3-mer peak with retention time of 0.50-0.70 ; inulin-type oligosaccharide 4-mer peak of 0.70-0.90; inulin-type oligosaccharide 6-mer peak of 1.10~1.40; chrysanthemum of 1.50~1.83 Amyloid oligosaccharide 7-mer peak; 1.83~2.50 inulin-type oligosaccharide 8-mer peak; 1.85~3.40 inulin-type oligosaccharide 9-mer peak, and then calculate the oligosaccharide by peak area according to external standard method The content.
本发明的色谱条件的选择是基于大量的实验数据， 选择各方面指标包括峰型、 分离度、 峰面积、 重现性等， 综合评价得出最佳色谱柱、 流动相、 检测器以及柱温等条件。 具体如下: 对照品菊淀粉型寡糖 5聚体 （5糖） 的 HPLC分析：  The chromatographic conditions of the present invention are selected based on a large amount of experimental data, and various indicators including peak shape, resolution, peak area, reproducibility, etc. are selected, and the optimal column, mobile phase, detector, and column temperature are comprehensively evaluated. And other conditions. The details are as follows: HPLC analysis of the reference product of the inulin-type oligosaccharide 5-mer (5 sugar):
取 5糖对照品供试液 20μ1，在本发明所述的色谱条件下进行 HPLC分析，记录色谱图，数据： 相对保留时间 17.502， 峰面积 118018， 峰面积 （％) 100.0000。  A 5 saccharide control solution was prepared for 20 μl, and subjected to HPLC analysis under the chromatographic conditions of the present invention, and the chromatogram was recorded. Data: relative retention time 17.502, peak area 118018, peak area (%) 100.0000.
巴戟天寡糖胶囊 （含有巴戟天寡糖及辅料） 的 HPLC分析：  HPLC analysis of Morinda capsules (containing Morinda citrifolia and excipients):
取巴戟天寡糖胶囊供试液 20μ1， 在上述色谱条件下进行 HPLC分析， 记录色谱图， 图中峰 由左至右依次为 1〜7号色谱峰， 经 HPLC-MS-MS等波谱学方法确定由左至右依次为： 菊淀粉 型寡糖 3聚体 （C18¾2016， 简称 :3糖）、 菊淀粉型寡糖 4聚体 （C24H42021， 简称 :4糖）、 菊淀粉型 寡糖 5聚体 （C3。H52026， 简称 :5糖）、 菊淀粉型寡糖 6聚体 （ C36H62031， 简称 :6糖）、 菊淀粉型 寡糖 7聚体 （C42H72036， 简称 :7糖）、 菊淀粉型寡糖 8聚体 （C48H82041， 简称: 8糖） 和菊淀粉型 寡糖 9聚体 （C54H92046， 简称: 9糖）。 其中 5糖色谱峰的保留时间与对照品保留时间一致。 3〜9 聚合体色谱峰的保留时间， 以 5糖为标准， 换算成相对保留时间， 图谱解析如下。 数据见表 1。 Take the test solution of Morinda oligosaccharide capsules for 20μ1, perform HPLC analysis under the above chromatographic conditions, and record the chromatogram. The peaks in the figure are from 1 to 7 peaks from left to right, and the spectrum is analyzed by HPLC-MS-MS. The method was determined from left to right: chrysanthemum-type oligosaccharide trimer (C 18 3⁄4 2 0 16 , abbreviated as: 3 sugar), chrysanthemum-type oligosaccharide 4-mer (C 24 H 42 0 21 , abbreviation: 4 Sugar), chrysanthemum-type oligosaccharide 5-mer (C 3 .H 52 0 26 , abbreviated as: 5 sugar), chrysanthemum-type oligosaccharide 6-mer (C 36 H 62 0 31 , abbreviated as 6 sugar), chrysanthemum starch Type oligosaccharide 7-mer (C 42 H 72 0 36 , abbreviated as: 7 sugar), inulin-type oligosaccharide 8-mer (C 48 H 82 0 41 , abbreviated as: 8 sugar) and inulin-type oligosaccharide 9-mer (C 54 H 92 0 46 , referred to as: 9 sugar). The retention time of the 5 sugar peaks was consistent with the retention time of the control. 3~9 The retention time of the peak of the polymer, which is converted to the relative retention time by the standard of 5 sugars, and the spectrum is resolved as follows. The data is shown in Table 1.
ID Ret. Time Area Area% Resolution T. Plate# ID Ret. Time Area Area% Resolution T. Plate#
1 10.099 84373 8.9974 2.908 4428.6191 10.099 84373 8.9974 2.908 4428.619
2 13.259 142746 15.2222 4.496 4409.8932 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 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 5 30.173 152323 16.2435 4.902 4956.905
6 39.836 121337 12.9392 4.801 4754.4906 39.836 121337 12.9392 4.801 4754.490
7 52.705 94798 10.1092 5.042 5691.831 表 1 巴戟天寡糖胶囊中各色谱峰的相对保留时间 7 52.705 94798 10.1092 5.042 5691.831 Table 1 Relative retention times of peaks in Morinda capsules
各色谱峰的相对保留时间  Relative retention time of each chromatographic peak
样品批号 序号  Sample lot number
3糖 4糖 5糖 6糖 7糖 8糖 9糖  3 sugar 4 sugar 5 sugar 6 sugar 7 sugar 8 sugar 9 sugar
1 0. 58 0. 76 1. 00 1. 31 1. 73 2. 29 3. 03  1 0. 58 0. 76 1. 00 1. 31 1. 73 2. 29 3. 03
2 0. 58 0. 76 1. 00 1. 31 1. 73 2. 29 3. 03  2 0. 58 0. 76 1. 00 1. 31 1. 73 2. 29 3. 03
3 0. 58 0. 76 1. 00 1. 31 1. 73 2. 29 3. 03  3 0. 58 0. 76 1. 00 1. 31 1. 73 2. 29 3. 03
4 0. 58 0. 76 1. 00 1. 31 1. 73 2. 29 3. 03  4 0. 58 0. 76 1. 00 1. 31 1. 73 2. 29 3. 03
5 0. 58 0. 76 1. 00 1. 31 1. 73 2. 29 3. 02  5 0. 58 0. 76 1. 00 1. 31 1. 73 2. 29 3. 02
6 0. 58 0. 76 1. 00 1. 31 1. 73 2. 29 3. 02  6 0. 58 0. 76 1. 00 1. 31 1. 73 2. 29 3. 02
070601  070601
7 0. 58 0. 76 1. 00 1. 31 1. 73 2. 28 3. 02  7 0. 58 0. 76 1. 00 1. 31 1. 73 2. 28 3. 02
8 0. 58 0. 76 1. 00 1. 31 1. 73 2. 28 3. 02  8 0. 58 0. 76 1. 00 1. 31 1. 73 2. 28 3. 02
9 0. 58 0. 76 1. 00 1. 31 1. 73 2. 28 3. 01  9 0. 58 0. 76 1. 00 1. 31 1. 73 2. 28 3. 01
10 0. 58 0. 76 1. 00 1. 31 1. 73 2. 28 3. 02  10 0. 58 0. 76 1. 00 1. 31 1. 73 2. 28 3. 02
11 0. 58 0. 76 1. 00 1. 31 1. 73 2. 28 3. 01  11 0. 58 0. 76 1. 00 1. 31 1. 73 2. 28 3. 01
12 0. 58 0. 76 1. 00 1. 31 1. 73 2. 28 3. 01  12 0. 58 0. 76 1. 00 1. 31 1. 73 2. 28 3. 01
1 0. 58 0. 76 1. 00 1. 30 1. 72 2. 27 2. 99  1 0. 58 0. 76 1. 00 1. 30 1. 72 2. 27 2. 99
2 0. 58 0. 76 1. 00 1. 30 1. 72 2. 27 2. 99  2 0. 58 0. 76 1. 00 1. 30 1. 72 2. 27 2. 99
070602  070602
3 0. 58 0. 76 1. 00 1. 30 1. 72 2. 27 2. 99  3 0. 58 0. 76 1. 00 1. 30 1. 72 2. 27 2. 99
4 0. 58 0. 76 1. 00 1. 30 1. 72 2. 27 2. 99  4 0. 58 0. 76 1. 00 1. 30 1. 72 2. 27 2. 99
1 0. 59 0. 76 1. 00 1. 30 1. 72 2. 27 2. 99  1 0. 59 0. 76 1. 00 1. 30 1. 72 2. 27 2. 99
2 0. 59 0. 77 1. 00 1. 30 1. 72 2. 26 2. 99  2 0. 59 0. 77 1. 00 1. 30 1. 72 2. 26 2. 99
070603  070603
3 0. 59 0. 76 1. 00 1. 30 1. 72 2. 26 2. 98  3 0. 59 0. 76 1. 00 1. 30 1. 72 2. 26 2. 98
4 0. 59 0. 76 1. 00 1. 30 1. 72 2. 26 2. 98  4 0. 59 0. 76 1. 00 1. 30 1. 72 2. 26 2. 98
(表中 Resolution: 分离度， T.Plate#理论塔板数， Ret.Time: 保留时间， Area: 面积） 色谱鉴别结果：对上述结果进行统计分析，得到 3〜9糖各色谱峰相对保留时间的最大值、 最小值及平均值， 见表 2。  (Resolution in the table: Separation, T.Plate# theoretical plate number, Ret.Time: retention time, Area: area) Chromatographic identification results: statistical analysis of the above results, to obtain relative retention times of each peak of 3~9 sugars The maximum, minimum and average values are shown in Table 2.
表 2 巴戟天寡糖胶囊各色谱峰的相对保留时间统计值  Table 2 Relative retention time statistics of each chromatographic peak of Morinda oligosaccharide capsules
相对保留时间  Relative retention time
糖名称  Sugar name
最大值 最小值 平均值 3糖 0. 59 0. 58 0. 58 Maximum value 3 sugar 0. 59 0. 58 0. 58
4糖 0. 77 0. 76 0. 76  4 sugar 0. 77 0. 76 0. 76
5糖 1. 00 1. 00 1. 00  5 sugar 1. 00 1. 00 1. 00
6糖 1. 31 1. 30 1. 31 6 sugar 1. 31 1. 30 1. 31
7糖 1. 73 1. 72 1. 73 7 sugar 1. 73 1. 72 1. 73
8糖 2. 29 2. 26 2. 28  8 sugar 2. 29 2. 26 2. 28
9糖 3. 03 2. 98 3. 01 溶剂及巴戟天寡糖制剂空白样品 （辅料） 的 HPLC分析  9 Sugar 3. 03 2. 98 3. 01 HPLC and analysis of blank samples (auxiliaries) of solvent and Morinda oligosaccharide preparation
按照处方比例称取辅料， 取纯水溶剂， 或取流动相溶液， 照供试品溶液制备方法处理， 分别取 20 μ 1， 注入 HPLC, 测定， 上述色谱条件下进行 HPLC分析， 记录色谱图。 色谱图与含 巴戟天样品色谱图对照， 结果显示空白样品色谱图中， 在与巴戟天寡糖 5糖对照品色谱图相 应的保留时间处、 及与含巴戟天寡糖制剂样品的色谱图中相应各寡糖相对保留时间处， 均未 见色谱峰。 表明不存在空白溶剂及辅料干扰。  Weigh the excipients according to the proportion of the prescription, take the pure water solvent, or take the mobile phase solution, and treat it according to the preparation method of the test solution. Take 20 μl of each, inject into HPLC, measure, perform HPLC analysis under the above chromatographic conditions, and record the chromatogram. The chromatogram is compared with the chromatogram of the Morinda citrifolia sample. The results show that in the chromatogram of the blank sample, at the retention time corresponding to the chromatogram of the Morinda oligosaccharide 5 sugar reference substance, and the sample containing the Morinda oligosaccharide preparation There were no chromatographic peaks at the relative retention times of the corresponding oligosaccharides in the chromatogram. It indicates that there is no white solvent and auxiliary material interference.
巴戟天寡糖胶囊的有效成分是巴戟天寡糖，为菊淀粉型寡糖 3聚体（C18H32016，简称： 3糖)、 菊淀粉型寡糖 4聚体 （C24H42021， 简称： 4糖）、 菊淀粉型寡糖 5聚体 （C3。H52026， 简称： 5糖）、 菊淀粉型寡糖 6聚体 （ C36H62031， 简称： 6糖）、 菊淀粉型寡糖 7聚体 （C42H72036， 简称： 7糖）、 菊淀粉型寡糖 8聚体 （C48H82041， 简称： 8糖） 和菊淀粉型寡糖 9聚体 （C54H92046， 简称： 9糖） 的混合物。 本发明建立了该巴戟天寡糖胶囊的 HPLC含量测定方法。 实验说明如下： The active ingredient of Morinda oligosaccharide capsule is Morinda officinalis oligosaccharide, which is a chrysanthemum-type oligosaccharide trimer (C 18 H 32 0 16 , abbreviated as: 3 sugar), and a chamomile-type oligosaccharide 4-mer (C 24 H 42 0 21 , abbreviation: 4 sugar), chrysanthemum-type oligosaccharide 5-mer (C 3 .H 52 0 26 , abbreviated as: 5 sugar), chrysanthemum-type oligosaccharide 6-mer (C 36 H 62 0 31 , Abbreviation: 6 sugar), chrysanthemum-type oligosaccharide 7-mer (C 42 H 72 0 36 , abbreviated as 7 sugar), chrysanthemum-type oligosaccharide 8-mer (C 48 H 82 0 41 , abbreviation: 8 sugar) and A mixture of inulin type oligosaccharide 9-mer (C 54 H 92 0 46 , abbreviated: 9 sugar). The invention establishes a HPLC content determination method of the Morinda oligosaccharide capsule. The experiment is described as follows:
1仪器与试剂：  1 instruments and reagents:
岛津 20AT高效液相色谱系统  Shimadzu 20AT High Performance Liquid Chromatography System
LCsolution色谱工作站；  LCsolution chromatography workstation;
试剂： 乙腈为色谱纯， 7j—纯水， 其余试剂均为分析纯；  Reagents: acetonitrile is chromatographically pure, 7j-pure water, and the remaining reagents are of analytical grade;
对照品：菊淀粉型寡糖 5聚体，中国药品生物制品检定所提供，批号 200701，纯度 99. 99%。 2. 方法与结果：  Reference substance: Chrysanthemum starch type oligosaccharide 5-mer, provided by China National Institute for the Control of Pharmaceutical and Biological Products, batch number 200701, purity 99. 99%. 2. Methods and results:
2. 1.色谱分析条件  2. 1. Chromatographic conditions
色谱柱为 Inertsil N¾柱 （5 μ m， 4. 5 X 250 mm )；  The column is an Inertsil N3⁄4 column (5 μm, 4. 5 X 250 mm);
流动相为乙腈： 水： 三乙胺 （68: 32： 0. 1 )；  The mobile phase is acetonitrile: water: triethylamine (68: 32: 0.1);
检测器为 RID-10A示差检测器；  The detector is a RID-10A differential detector;
温 度: 柱温 40°C， 检测器温度 40°C ; 流 速： 1. 2mL/min;  Temperature: column temperature 40 ° C, detector temperature 40 ° C; flow rate: 1. 2mL / min;
在此条件下各组分达到基线分离。 测得菊淀粉型寡糖 5聚体峰理论板数不低于 2000。 2. 2.线性关系考察： 精密吸取菊淀粉型寡糖 5聚体对照品溶液（2. 37mg/ml ) 5、 10、 15、 20、 25、 30 μ 1 , 注入液相色谱仪， 照上述色谱条件测定， 以峰面积积分值为纵坐标， 5糖进 样量为横坐标， 绘制标准曲线， 得回归方程： y=5555523. 8X-6336 r=0. 9999。 结果表明， 菊 淀粉型寡糖 5聚体 （5糖） 在 0. 0119〜0. 0711mg范围内具良好的线性关系。 见表 3。 表 3 线性关系的考察 Under these conditions, each component reached baseline separation. The number of theoretical plates of the amylose-type oligosaccharide 5-mer peak was determined to be not less than 2000. 2. 2. Linear relationship investigation: Precision extraction of the amylose-type oligosaccharide 5-mer reference solution (2.37mg/ml) 5, 10, 15, 20, 25, 30 μ 1 , injected into the liquid chromatograph, according to the above Determination of chromatographic conditions, with the peak area integral value as the ordinate, 5 sugar into The sample is plotted on the abscissa and the standard curve is drawn. The regression equation is obtained: y=5555523. 8X-6336 r=0. 9999. The results showed that the amyloid oligosaccharide 5-mer (5 sugar) had a good linear relationship in the range of 0. 0119~0. 0711mg. See Table 3. Table 3 : Investigation of linear relationship
5糖进样量 （mg) 峰面积  5 sugar injection amount (mg) peak area
0. 0119 60532. 5  0. 0119 60532. 5
0. 0237 123563  0. 0237 123563
0. 0356 190649  0. 0356 190649
0. 0474 259123  0. 0474 259123
0. 0593 321999. 5  0. 0593 321999. 5
0. 0711 388606. 5  0. 0711 388606. 5
2. 3精密度实验： 精密吸取菊淀粉型寡糖 5聚体对照品溶液 （2. 37mg/ml ) ΙΟ μ Ι , 连续 进样 6次， 照上述色谱条件测定， 计算平均峰面积和相对标准偏差， 见表 4。 结果表明： 该方 法的精密度良好。 2. 3 precision experiment: Precision extraction of the amylose oligosaccharide 5-mer reference solution (2.37mg/ml) ΙΟ μ Ι , continuous injection 6 times, according to the above chromatographic conditions, calculate the average peak area and relative standard Deviation, see Table 4. The results show that the precision of the method is good.
表 4 精密度 定结果  Table 4 Precision Results
峰面积 均值 RSD (%)  Peak area mean RSD (%)
1 123774  1 123774
2 122453  2 122453
3 123119 123267 0. 4  3 123119 123267 0. 4
4 123130  4 123130
5 123760  5 123760
6 123366  6 123366
2. 4.空白试验： 按照处方比例称取辅料， 照供试品处理方法处理， 取 20μ1， 在上述的色 谱条件下进行 HPLC分析， 记录色谱图， 结果表明无干扰。  2. 4. Blank test: Weigh the excipient according to the proportion of the prescription, take the treatment method of the test sample, take 20μ1, perform HPLC analysis under the above chromatographic conditions, record the chromatogram, and the result shows no interference.
2. 5.供试品溶液稳定性考察： 取同批供试品溶液， 于制样后 0、 2、 4、 8、 10、 22小时， 依法测定， 结果表明， 供试品溶液在 22小时内基本稳定。 结果见表 5。  2. Investigation of the stability of the test solution: Take the same batch of test solution, after 0, 2, 4, 8, 10, 22 hours after sample preparation, according to the law, the results show that the test solution is in 22 hours It is basically stable inside. The results are shown in Table 5.
表 5 稳定性试验结果  Table 5 Stability test results
时间（小时） 5糖含量 (mg/g) ~~均值 (mg/g) RSD (%) 总糖含量 (mg/g) ~~均值 (mg/g) ~~ RSD (%)Time (hours) 5 Sugar content (mg/g) ~~mean (mg/g) RSD (%) Total sugar content (mg/g) ~~mean (mg/g) ~~ RSD (%)
0 55. 8 311. 5 0 55. 8 311. 5
2 55. 2 308. 7  2 55. 2 308. 7
4 54. 5 311. 2  4 54. 5 311. 2
54. 6 1. 8 310. 4 0. 6 54. 6 1. 8 310. 4 0. 6
8 53. 8 310. 3 8 53. 8 310. 3
10 53. 2 307. 5  10 53. 2 307. 5
22 54. 8 313. 0  22 54. 8 313. 0
2. 6. 重复性试验： 按上述方法， 取同批样品， 平行制样 6份， 测定， 表明重复性较好 见表 6。 表 6 重复性试验结果 2. 6. Repeatability test: According to the above method, the same batch of samples were taken, and 6 samples were prepared in parallel, which showed that the repeatability was better as shown in Table 6. Table 6 Repeatability test results
No 5糖含量 (mg/g) 均值 (mg/g) RSD (%) 总糖含量 (mg/g) 均值（mg/g) RSD (%) No 5 Sugar content (mg/g) Mean (mg/g) RSD (%) Total sugar content (mg/g) Mean (mg/g) RSD (%)
1 54. 3 308. 4 1 54. 3 308. 4
2 56. 0 319. 6  2 56. 0 319. 6
3 53. 2 305. 1  3 53. 2 305. 1
54. 1 1. 9 310. 9 1. 8 54. 1 1. 9 310. 9 1. 8
4 53. 5 308. 3 4 53. 5 308. 3
5 54. 3 315. 4  5 54. 3 315. 4
6 53. 3 308. 6  6 53. 3 308. 6
2. 7. 回收率试验： 采用加样回收法， 精密称取已知含量的同批（5糖含量 54.1mg/g)样 品 0.15g， 分别精密加入 5糖对照品 （7.716mg)， 平行制样 6份， 测定， 按下式计算回收率， 结果表明本方法具有良好的准确度， 数据见表 7。 2. 7. Recovery test: Using the sample recovery method, we accurately weigh 0.15g of the same batch (5 sugar content 54.1mg/g) of known content, and accurately add 5 sugar reference substance (7.716mg), parallel system The sample was sampled and the recovery rate was calculated by the following formula. The results show that the method has good accuracy. The data is shown in Table 7.
出 5糖总量-样品中 5糖的含  5 total sugar - in the sample 5 sugar containing
回收率 (％) = · x l00%  Recovery rate (%) = · x l00%
加入 5糖对照品量 表 7 回收率试验结果  Add 5 sugar control product amount Table 7 Recovery rate test result
No 样品取 样品中 5糖 添加 5糖量 测得 5糖 回收率 均值 RSD 样量 （g) 量 (mg) (mg) 总量 (mg) (%) (%) (%)No sample taken in the sample 5 sugar added 5 sugar amount measured 5 sugar recovery mean RSD sample amount (g) amount (mg) (mg) total amount (mg) (%) (%) (%)
1 0. 1502 8. 128 15. 876 100. 42 1 0. 1502 8. 128 15. 876 100. 42
2 0. 1505 8. 144 15. 802 99. 24  2 0. 1505 8. 144 15. 802 99. 24
3 0. 1511 8. 177 3 0. 1511 8. 177
Figure imgf000010_0001
Figure imgf000010_0001
4 0. 1506 8. 150 15. 765 98. 69  4 0. 1506 8. 150 15. 765 98. 69
5 0. 1509 8. 166 16. 142 103. 38 卜 5 0. 1509 8. 166 16. 142 103. 38
6 0. 1517 8. 209 16. 068 101. 85 6 0. 1517 8. 209 16. 068 101. 85
2. 8.样品测定结果： 测定 3批样品， 结果见表 8。 2. 8. Sample measurement results: Three batches of samples were determined, and the results are shown in Table 8.
表 8、 样品测定结果  Table 8, sample measurement results
批号 5糖含量 （mg/粒） 总糖含量 （mg/粒）  Batch No. 5 Sugar content (mg/grain) Total sugar content (mg/grain)
070601 16. 6 90. 4  070601 16. 6 90. 4
070602 16. 2 93. 2  070602 16. 2 93. 2
070603 17. 0 89. 4  070603 17. 0 89. 4
3方法耐用性研究 3 method durability study
3. 1  3. 1
色谱柱： MERK Lichrosorb N¾柱 （5 μ m， 4. 5 X 250 匪 ) ；  Column: MERK Lichrosorb N3⁄4 column (5 μ m, 4. 5 X 250 匪);
流动相： 乙腈： 水： 三乙胺 （68: 32： 0. 1 )； 检测器： RID-10A示差检测器； Mobile phase: acetonitrile: water: triethylamine (68: 32: 0.1); Detector: RID-10A differential detector;
温 度: 柱温 40°C， 检测器温度 40°C ; 流 速： 1. 2mL/min; Temperature: column temperature 40 ° C, detector temperature 40 ° C; flow rate: 1. 2mL / min;
结 果： 见表 9、 10。 Results: See Tables 9, 10.
表 9、 耐用性结果 1  Table 9. Durability results 1
批号 5糖含量 (mg/粒) ~~总糖含量 (mg/粒）
Figure imgf000011_0001
表 10、 巴戟天寡糖胶囊各色谱峰的相对保留时间 糖名称 相对保留时间Batch No. 5 sugar content (mg / grain) ~ ~ total sugar content (mg / grain)
Figure imgf000011_0001
Table 10. Relative retention time of each chromatographic peak of Morinda oligosaccharide capsules
3糖 0.68 3 sugar 0.68
4糖 0.82  4 sugar 0.82
5糖 1.00  5 sugar 1.00
6糖 1.22 6 sugar 1.22
7糖 1.50 7 sugar 1.50
8糖 1.86  8 sugar 1.86
9糖 2.30 9 sugar 2.30
3.2 3.2
色谱柱： SEPAX Amethyst amino柱 （5μηι， 4.6x250 mm )； 流动相： 乙腈： 水： 三乙胺 （68: 32： 0.1 )； Column: SEPAX Amethyst amino column (5μηι, 4.6x250 mm); Mobile phase: Acetonitrile: Water: Triethylamine (68: 32: 0.1);
检测器： RID-10A示差检测器； Detector: RID-10A differential detector;
温 度: 柱温 40°C， 检测器温度 40°C ; 流 速： 1.2mL/min; Temperature: column temperature 40 ° C, detector temperature 40 ° C; flow rate: 1.2 mL / min;
结 果： 见表 11、 12。 Results: See Tables 11, 12.
表 11、 耐用性结果 2  Table 11. Durability results 2
批号 5糖含量 (mg/粒） 总糖含量 (mg/粒） Batch No. 5 Sugar content (mg/grain) Total sugar content (mg/grain)
070602 17.5 89.4 表 12、 巴戟天寡糖胶麵囊各色谱峰的相对保留时间 糖名称 相对保留时间070602 17.5 89.4 Table 12. Relative retention times of chromatographic peaks of Morinda oligosaccharide gelatin capsules Sugar name Relative retention time
3糖 0.633 sugar 0.63
4糖 0.794 sugar 0.79
5糖 1.005 sugar 1.00
6糖 1.266 sugar 1.26
7糖 1.607 sugar 1.60
8糖 2.048 sugar 2.04
9糖 2.61 4、 相对保留时间范围的确定： 对多批巴戟天寡糖颗粒剂及胶囊进行了测定， 菊淀粉型寡 糖 3~9聚体的保留时间相对较为固定， 菊淀粉型寡糖 3~9聚体相对于 5聚体的相对保留时间 也较为稳定， 见表 13。 9 sugar 2.61 4. Determination of relative retention time range: The batches of Morinda oligosaccharides granules and capsules were determined. The retention time of inulin-type oligosaccharide 3~9mers was relatively fixed, and the amylose-type oligosaccharides 3~9 The relative retention time of the polymer relative to the 5-mer is also relatively stable, see Table 13.
表 13、 相对保留时间范围  Table 13, Relative retention time range
糖名称 胶囊中相对保留时间 颗粒剂中相对保留时间 平均值 -5% +5% Sugar name Relative retention time in capsules Relative retention time in granules Average -5% +5%
3糖 0. 58 0. 57 0. 58 0. 55 0. 613 sugar 0. 58 0. 57 0. 58 0. 55 0. 61
4糖 0. 76 0. 76 0. 76 0. 72 0. 804 sugar 0. 76 0. 76 0. 76 0. 72 0. 80
5糖 1. 00 1. 00 1. 00 1 15 sugar 1. 00 1. 00 1. 00 1 1
6糖 1. 31 1. 32 1. 31 1. 25 1. 386 sugar 1. 31 1. 32 1. 31 1. 25 1. 38
7糖 1. 73 1. 75 1. 74 1. 65 1. 837 sugar 1. 73 1. 75 1. 74 1. 65 1. 83
8糖 2. 28 2. 33 2. 30 2. 19 2. 428 sugar 2. 28 2. 33 2. 30 2. 19 2. 42
9糖 3. 01 3. 09 3. 05 2. 90 3. 21 经过系统研究， 本发明采用菊淀粉性寡糖 5聚体为对照品， 以 3-9糖的峰面积之和计算 巴戟天寡糖含量的方法是切实可行的。 该方法准确、 灵敏、 专一、 空白辅料对测定无干扰， 能有效控制本品含量。 本发明的质量控制方法快速便捷， 重复性良好， 适合实验室的小样检 测以及工业化大生产的质量检测。 具体实施方案 9 Sugar 3. 01 3. 09 3. 05 2. 90 3. 21 After systematic study, the present invention uses the amyloid oligosaccharide 5-mer as a reference substance, and calculates the Bayu day by the sum of the peak areas of 3-9 sugars. The method of oligosaccharide content is practical. The method is accurate, sensitive, specific, and blank excipients have no interference to the determination, and can effectively control the content of the product. The quality control method of the invention is fast, convenient and reproducible, and is suitable for laboratory sample detection and quality inspection of industrialized large production. Specific implementation
以下结合实施例详细说明本发明， 但不限定本发明的实施范围。 实施例 1.巴戟天寡糖胶囊含量测定  The present invention will be described in detail below with reference to the examples, but without limiting the scope of the invention. Example 1. Determination of the content of Morinda oligosaccharide capsules
1仪器与试剂：  1 instruments and reagents:
岛津 20AT高效液相色谱系统； LCsolution色谱工作站；  Shimadzu 20AT high performance liquid chromatography system; LCsolution chromatography workstation;
试剂： 乙腈一色谱纯 （天津四友生物医学技术开发公司）， 7j—纯水， 其余均为分析纯； 对照品： 菊淀粉型寡糖 5聚体， 中国药品生物制品检定所提供， 纯度 99.99%。  Reagents: Acetonitrile-chromatographic pure (Tianjin Siyou Biomedical Technology Development Company), 7j-pure water, the rest are of analytical grade; Reference: Chrysanthemum-type oligosaccharide 5-mer, supplied by China National Institute for the Control of Pharmaceutical and Biological Products, purity 99.99 %.
2. 方法与结果：  2. Methods and results:
2. 1.色谱分析条件  2. 1. Chromatographic conditions
色谱柱： Inertsil NH2柱 （5μηι， 4.5x250 mm )； Column: Inertsil NH 2 column (5μηι, 4.5x250 mm);
流动相： 乙腈： 水： 三乙胺 （68: 32： 0.1 )；  Mobile phase: acetonitrile: water: triethylamine (68: 32: 0.1);
检测器： RID-10A示差检测器；  Detector: RID-10A differential detector;
温 度: 柱温 40°C， 检测器温度 40°C ; 流 速： 1.2mL/min;  Temperature: column temperature 40 ° C, detector temperature 40 ° C; flow rate: 1.2 mL / min;
在此条件下各组分达到基线分离。 测得菊淀粉型寡糖 5聚体峰理论板数不低于 2000。 对照品溶液的制备 取菊淀粉型寡糖 5聚体 （5糖） 对照品适量， 精密称定， 加水制成每 lml含 lmg的溶液， 即得。 Under these conditions, each component reached baseline separation. The number of theoretical plates of the amylose-type oligosaccharide 5-mer peak was determined to be not less than 2000. The preparation of the reference solution is taken from the appropriate amount of the amylose-type oligosaccharide 5-mer (5 sugar) reference substance, accurately weighed, and water is added to make each Lml contains 1mg of solution, that is.
供试品溶液的制备 取巴戟天寡糖胶囊样品 0.3g，精密称定，置 10ml量瓶中，加入水 10ml， 超声， 取上清液， 用水稀释至刻度， 经 0.45μηι微孔滤膜过滤， 取续滤液， 即得。  Preparation of the test solution Take 0.3g of Morinda oligosaccharide capsule sample, accurately weigh it, put it into a 10ml volumetric flask, add 10ml of water, sonicate, take the supernatant, dilute with water to the mark, pass 0.45μηι microporous membrane Filter and take the filtrate to obtain.
测定法 分别精密吸取对照品溶液与供试品溶液各 20μ1， 注入液相色谱仪， 测定， 按外标 法以峰面积分别计算菊淀粉型寡糖 3聚体 -9聚体的含量， 将上述各寡糖的含量进行加和， 即得 巴戟天寡糖有效成分的含量。  The measurement method accurately absorbs the reference solution and the test solution 20μ1, respectively, and injects into the liquid chromatograph, and determines the content of the amylose-type oligosaccharide 3-mer-9-mer by the external standard method. The content of each oligosaccharide is added, that is, the content of the active ingredient of the Morinda oligosaccharide is obtained.
巴戟天寡糖胶囊中每粒含菊淀粉型寡糖 5聚体不得低于 9.0mg， 含菊淀粉型寡糖 3~9聚体的 总量以菊淀粉型寡糖 5聚体计， 不得低于 75.0mg。 实施例 2. 巴戟天寡糖颗粒剂的含量测定  Each of the capsules containing the inulin-type oligosaccharide 5 mer is not less than 9.0 mg, and the total amount of the stearyl amylose-containing oligosaccharide 3-9 mer is determined by the amylose-type oligosaccharide 5-mer. Less than 75.0 mg. Example 2. Determination of the content of Morinda granules
1仪器与试剂：  1 instruments and reagents:
岛津 20AT高效液相色谱系统； LCsolution色谱工作站；  Shimadzu 20AT high performance liquid chromatography system; LCsolution chromatography workstation;
试剂： 同实施例 1 ;  Reagents: same as in Example 1;
对照品：菊淀粉型寡糖 5聚体，中国药品生物制品检定所提供，批号 200701，纯度 99.99%。 2. 方法与结果：  Reference substance: Chrysanthemum starch type oligosaccharide 5-mer, provided by China National Institute for the Control of Pharmaceutical and Biological Products, batch number 200701, purity 99.99%. 2. Methods and results:
2. 1.色谱分析条件  2. 1. Chromatographic conditions
色谱柱： Inertsil NH2柱 （5μηι， 4.5x250 mm )； Column: Inertsil NH 2 column (5μηι, 4.5x250 mm);
流动相： 乙腈： 水： 三乙胺 （70: 30： 0.05 )；  Mobile phase: acetonitrile: water: triethylamine (70: 30: 0.05);
检测器： RID-10A示差检测器；  Detector: RID-10A differential detector;
温 度: 柱温 40°C， 检测器温度 40°C ; 流 速： 1.0mL/min;  Temperature: column temperature 40 ° C, detector temperature 40 ° C; flow rate: 1.0 mL / min;
对照品溶液的制备 取菊淀粉型寡糖 5聚体 （5糖） 对照品适量， 精密称定， 加流动相制 成每 lml含 lmg的溶液， 即得。  Preparation of the reference solution The chrysanthemum-type oligosaccharide 5-mer (5-glycan) reference substance was accurately weighed, and the mobile phase was added to prepare a solution containing 1 mg per lml.
供试品溶液的制备： 取巴戟天寡糖颗粒剂样品 0.25g， 精密称定， 置 10ml量瓶， 加流动 相 10ml， 超声， 取上清液， 用流动相稀释至刻度， 经 0.45μηι微孔滤膜过滤， 取续滤液。  Preparation of test solution: Take 0.25g sample of Morinda granules granules, accurately weighed, set 10ml volumetric flask, add mobile phase 10ml, ultrasonic, take the supernatant, dilute to the mark with mobile phase, pass 0.45μηι The microporous membrane was filtered and the filtrate was taken.
测定法： 分别精密吸取对照品溶液与供试品溶液各 20μ1， 注入液相色谱仪， 按外标法以峰 面积分别计算菊淀粉型寡糖 3聚体 -9聚体的含量， 将上述各寡糖的含量进行加和， 即得巴戟天 寡糖有效成分的含量。  Determination method: respectively, accurately draw the reference solution and the test solution 20μ1, inject into the liquid chromatograph, calculate the content of the amylose-type oligosaccharide 3-mer-9-mer by the external standard method, respectively. The content of the oligosaccharide is added, that is, the content of the active ingredient of the Morinda oligosaccharide is obtained.
巴戟天寡糖颗粒剂 （1.5g/袋） 中每克含菊淀粉型寡糖 5聚体不得低于 6.0mg， 含菊淀粉型 寡糖 3~9聚体的总量以菊淀粉型寡糖 5聚体计， 不得低于 50.0mg。 实施例 3. 巴戟天寡糖片剂的含量测定  Bayer oligosaccharide granules (1.5g/bag) should not be less than 6.0mg per gram of inulin-containing oligosaccharide 5g, and the total amount of chrysanthemum-type oligosaccharide 3~9-mers The sugar 5 polymer meter shall not be less than 50.0 mg. Example 3. Determination of the content of Morinda oligosaccharide tablets
1仪器与试剂：  1 instruments and reagents:
岛津 20AT高效液相色谱系统； LCsolution色谱工作站； 试剂： 乙腈， 色谱纯 （天津四友生物医学技术开发公司）； 纯水， 其余试剂均为分析纯； 对照品： 菊淀粉型寡糖 5聚体， 中国药品生物制品检定所提供， 纯度 99.99%。 Shimadzu 20AT high performance liquid chromatography system; LCsolution chromatography workstation; Reagents: acetonitrile, chromatographic purity (Tianjin Siyou Biomedical Technology Development Company); pure water, the rest of the reagents are of analytical grade; Reference: Chrysanthemum-type oligosaccharide 5-mer, provided by China National Institute for the Control of Pharmaceutical and Biological Products, purity 99.99% .
2. 方法与结果：  2. Methods and results:
2. 1.色谱分析条件  2. 1. Chromatographic conditions
色谱柱： NH2柱 （5μηι， 4.5x250 mm )； Column: NH 2 column (5μηι, 4.5x250 mm);
流动相： 乙腈： 水： 三乙胺 （60: 40： 0.15 )；  Mobile phase: acetonitrile: water: triethylamine (60: 40: 0.15);
检测器： RID-10A示差检测器；  Detector: RID-10A differential detector;
温 度: 柱温 40°C， 检测器温度 40°C ; 流 速： 1.2mL/min;  Temperature: column temperature 40 ° C, detector temperature 40 ° C; flow rate: 1.2 mL / min;
对照品溶液的制备： 取菊淀粉型寡糖 5聚体 （5糖） 对照品适量， 精密称定， 加水制成每 lml含 lmg的溶液， 即得。  Preparation of the reference solution: Take the amylose-type oligosaccharide 5-mer (5 sugar). The appropriate amount of the reference substance, accurately weighed, add water to make a solution containing lmg per lml, that is.
供试品溶液的制备:取巴戟天寡糖片剂样品 0.4g，精密称定，置 10ml量瓶中，加入水 10ml， 超声， 取上清液， 用水稀释至刻度， 经 0.45μηι微孔滤膜过滤， 取续滤液， 即得。  Preparation of test solution: Take 0.4g of Morinda oligosaccharide tablet sample, accurately weigh it, put it into a 10ml volumetric flask, add 10ml of water, sonicate, take the supernatant, dilute with water to the mark, pass 0.45μηι microporous Filter the membrane and take the filtrate to obtain.
测定法 分别精密吸取对照品溶液与供试品溶液各 20μ1， 注入液相色谱仪， 按外标法以峰 面积分别计算菊淀粉型寡糖 3聚体 -9聚体的含量， 将上述各寡糖的含量进行加和， 即得巴戟天 寡糖有效成分的含量。  The measurement method accurately absorbs the reference solution and the test solution for each 20μ1, and injects into the liquid chromatograph, and calculates the content of the amylose-type oligosaccharide 3-mer-9-mer by the external standard method. The content of the sugar is added, that is, the content of the active ingredient of the Morinda oligosaccharide is obtained.
巴戟天寡糖片剂中每片 （0.4g/片） 含菊淀粉型寡糖 5聚体不得低于 9.0mg， 含菊淀粉型寡 糖 3~9聚体的总量以菊淀粉型寡糖 5聚体计， 不得低于 75.0mg。 实施例 4. 巴戟天寡糖冻干粉针含量测定  Each tablet (0.4g/tablet) of the Morinda oligosaccharide tablets should not be less than 9.0mg, and the total amount of the chrysanthemum-type oligosaccharide 3~9-mers should be The sugar 5 polymer meter shall not be less than 75.0 mg. Example 4. Determination of the content of freeze-dried powder of Morinda officinalis
1仪器与试剂： 同实施例 1。  1 Instruments and reagents: Same as Example 1.
2. 方法与结果：  2. Methods and results:
2. 1.色谱分析条件：  2. 1. Chromatographic conditions:
色谱柱： NH2柱 （5 μ ηι， 4.5 X 250 mm );  Column: NH2 column (5 μ ηι, 4.5 X 250 mm);
流动相： 乙腈： 水： 三乙胺 （40: 60： 0.01 )；  Mobile phase: acetonitrile: water: triethylamine (40: 60: 0.01);
检测器： 蒸发光散射检测器；  Detector: evaporative light scattering detector;
温 度: 柱温 35°C， 检测器温度 40°C ; 流 速： 1.0mL/min;  Temperature: column temperature 35 ° C, detector temperature 40 ° C; flow rate: 1.0 mL / min;
在此条件下各组分达到基线分离。 测得菊淀粉型寡糖 5聚体峰理论板数不低于 4000。 对照品溶液的制备 取菊淀粉型寡糖 5聚体 （5糖） 对照品适量， 精密称定， 加水制成每 lml含 lmg的溶液， 即得。  Under these conditions, each component reached baseline separation. The number of theoretical plates of the amylose-type oligosaccharide 5-mer peak was determined to be not less than 4,000. Preparation of reference solution Take the amylose-type oligosaccharide 5-mer (5 sugar). The appropriate amount of the reference substance, accurately weighed, add water to make a solution containing lmg per lml, that is.
供试品溶液的制备 取巴戟天寡糖冻干粉针样品 0.3g， 精密称定， 置 10ml量瓶中， 加入 流动相 10ml至刻度， 经 0.45μηι微孔滤膜过滤， 取续滤液， 即得。  Preparation of the test solution: Take 0.3g of the Morinda oligosaccharide freeze-dried powder sample, accurately weigh it, put it into a 10ml volumetric flask, add 10ml of mobile phase to the mark, filter through 0.45μηι microporous membrane, and take the filtrate. That is.
测定法 同实施例 1。 巴戟天寡糖冻干粉针中每克含菊淀粉型寡糖 5聚体不得低于 60.0mg， 含菊淀粉型寡糖 3~9 聚体的总量以菊淀粉型寡糖 5聚体计， 不得低于 500.0mg。 实施例 5. 巴戟天寡糖口服液的含量测定 The measurement method was the same as in Example 1. The gluten-free oligosaccharide freeze-dried powder needle should not be less than 60.0 mg per gram of inulin-containing oligosaccharide, and the total amount of inulin-containing oligosaccharide 3-9 mer is in the form of inulin-type oligosaccharide 5-mer. No less than 500.0mg. Example 5. Determination of the content of Morinda oligosaccharide oral solution
1仪器与试剂： 同实施例 1。  1 Instruments and reagents: Same as Example 1.
2. 方法与结果：  2. Methods and results:
2. 1.色谱分析条件：  2. 1. Chromatographic conditions:
色谱柱： NH2柱 （5 μ ηι， 4.5 X 250 mm );  Column: NH2 column (5 μ ηι, 4.5 X 250 mm);
流动相： 乙腈： 水： 三乙胺 （80: 20： 5 )；  Mobile phase: acetonitrile: water: triethylamine (80: 20: 5);
检测器： RID-10A示差检测器；  Detector: RID-10A differential detector;
温 度: 柱温 40°C， 检测器温度 40°C ; 流 速： 0.8mL/min;  Temperature: column temperature 40 ° C, detector temperature 40 ° C; flow rate: 0.8 mL / min;
对照品溶液的制备 取菊淀粉型寡糖 5聚体 （5糖） 对照品适量， 精密称定， 加流动相制 成每 lml含 lmg的溶液， 即得。  Preparation of the reference solution The chrysanthemum-type oligosaccharide 5-mer (5-glycan) reference substance was accurately weighed, and the mobile phase was added to prepare a solution containing 1 mg per lml.
供试品溶液的制备 取巴戟天寡糖口服液样品 10ml， 精密称定， 置 10ml量瓶中， 用水稀 释至刻度， 经 0.45μηι微孔滤膜过滤， 取续滤液， 即得。  Preparation of the test solution Take 10 ml of the sample of Morinda oligosaccharide oral solution, accurately weigh it, place it in a 10 ml volumetric flask, dilute to the mark with water, filter through a 0.45 μηι microporous membrane, and take the filtrate to obtain.
测定法 同实施例 1。  The measurement method was the same as in Example 1.
巴戟天寡糖口服液中每 10ml含菊淀粉型寡糖 5聚体不得低于 9.0mg，含菊淀粉型寡糖 3~9聚 体的总量以菊淀粉型寡糖 5聚体计， 不得低于 75.0mg。 实施例 6. 巴戟天寡糖糖浆剂的含量测定  The 5 mg of the inulin-containing oligosaccharide-containing oligosaccharide per 10 ml of the Morinda oligosaccharide oral solution shall not be less than 9.0 mg, and the total amount of the chrysanthemum-type oligosaccharide 3-9-mer is determined by the amylose-type oligosaccharide 5-mer. Not less than 75.0mg. Example 6. Determination of the content of Morinda oligosaccharide syrup
1仪器与试剂： 同实施例 1。  1 Instruments and reagents: Same as Example 1.
2. 方法与结果：  2. Methods and results:
2. 1.色谱分析条件：  2. 1. Chromatographic conditions:
色谱柱： NH2柱 （5 μ ηι， 4.5 X 250 mm );  Column: NH2 column (5 μ ηι, 4.5 X 250 mm);
流动相： 乙腈： 水 ( 68： 32)；  Mobile phase: acetonitrile: water (68: 32);
检测器： 蒸发光散射检测器；  Detector: evaporative light scattering detector;
温 度: 柱温 40°C， 检测器温度 40°C ; 流 速： 1.0mL/min;  Temperature: column temperature 40 ° C, detector temperature 40 ° C; flow rate: 1.0 mL / min;
对照品溶液的制备： 取菊淀粉型寡糖 5聚体 （5糖） 对照品适量， 精密称定， 加水制成每 lml含 lmg的溶液， 即得。  Preparation of the reference solution: Take the amylose-type oligosaccharide 5-mer (5 sugar). The appropriate amount of the reference substance, accurately weighed, add water to make a solution containing lmg per lml, that is.
供试品溶液的制备 取巴戟天寡糖糖浆剂样品 5ml， 精密称定， 置 10ml量瓶中， 用水稀 释至刻度， 经 0.45μηι微孔滤膜过滤， 取续滤液， 即得。  Preparation of test solution Take 5 ml of Morinda oligosaccharide syrup sample, accurately weigh it, place it in a 10 ml volumetric flask, dilute with water to the mark, filter through 0.45 μηι microporous membrane, and take the filtrate to obtain.
测定法： 同实施例 1。  Assay: Same as Example 1.
巴戟天寡糖糖浆剂中每 ml含菊淀粉型寡糖 5聚体不得低于 0.9mg，含菊淀粉型寡糖 3~9聚体 的总量以菊淀粉型寡糖 5聚体计， 不得低于 7.5mg。 实施例 7. 巴戟天寡糖片剂的含量测定 Bayan oligosaccharide syrup containing 5 mg of inulin-containing oligosaccharide per ml, containing amylose-type oligosaccharide 3~9-mer The total amount is not less than 7.5 mg based on the amylose-type oligosaccharide 5-mer. Example 7. Determination of the content of Morinda oligosaccharide tablets
1仪器与试剂： 同实施例 1。  1 Instruments and reagents: Same as Example 1.
2. 方法与结果：  2. Methods and results:
2. 1.色谱分析条件：  2. 1. Chromatographic conditions:
色谱柱： C18柱 （5 μ ηι， 4.5 X 250 mm );  Column: C18 column (5 μ ηι, 4.5 X 250 mm);
流动相： 乙腈： 7j : 三乙胺 （75 : 25： 0.1 )；  Mobile phase: acetonitrile: 7j: triethylamine (75: 25: 0.1);
检测器： RID-10A示差检测器；  Detector: RID-10A differential detector;
温 度: 柱温 40°C， 检测器温度 40°C ; 流 速： 1.0mL/min;  Temperature: column temperature 40 ° C, detector temperature 40 ° C; flow rate: 1.0 mL / min;
对照品溶液的制备： 取菊淀粉型寡糖 5聚体 （5糖） 对照品适量， 精密称定， 加流动相制 成每 lml含 lmg的溶液， 即得。  Preparation of the reference solution: Take the amylose-type oligosaccharide 5-mer (5 sugar). The appropriate amount of the reference substance, accurately weighed, and add the mobile phase to prepare a solution containing lmg per lml.
供试品溶液的制备： 取巴戟天寡糖片剂样品 0.4g， 精密称定， 置 10ml量瓶中， 加流动相 10ml, 超声， 取上清液， 用流动相稀释至刻度， 经 0.45μηι微孔滤膜过滤， 取续滤液， 即得。  Preparation of the test solution: Take 0.4g of Morinda oligosaccharide tablet sample, accurately weigh it, put it in a 10ml volumetric flask, add 10ml of mobile phase, sonicate, take the supernatant, dilute to the mark with mobile phase, pass 0.45 Filter through μηι microporous membrane and take the filtrate to obtain.
测定法 同实施例 1。  The measurement method was the same as in Example 1.
巴戟天寡糖片剂中每片含菊淀粉型寡糖 5聚体不得低于 9.0mg， 含菊淀粉型寡糖 3~9聚体的 总量以菊淀粉型寡糖 5聚体计， 不得低于 75.0mg。 实施例 8. 巴戟天寡糖胶囊的含量测定  Each of the Morinda oligosaccharide tablets contains not less than 9.0 mg of the amylose-type oligosaccharide, and the total amount of the chrysanthemum-type oligosaccharide 3-9-mer is calculated by the amylose-type oligosaccharide 5-mer. Not less than 75.0mg. Example 8. Determination of the content of Morinda oligosaccharide capsules
1仪器与试剂： 同实施例 1。  1 Instruments and reagents: Same as Example 1.
2. 方法与结果：  2. Methods and results:
2. 1.色谱分析条件：  2. 1. Chromatographic conditions:
色谱柱： NH2柱 （5 μ ηι， 4.5 X 250 mm );  Column: NH2 column (5 μ ηι, 4.5 X 250 mm);
流动相： 乙腈： 水： 三乙胺 （55 : 45： 0.05 )；  Mobile phase: acetonitrile: water: triethylamine (55: 45: 0.05);
检测器： RID-10A示差检测器；  Detector: RID-10A differential detector;
温 度: 柱温 40°C， 检测器温度 40°C ; 流 速： 1.2mL/min;  Temperature: column temperature 40 ° C, detector temperature 40 ° C; flow rate: 1.2 mL / min;
在此条件下各组分达到基线分离。 测得菊淀粉型寡糖 5聚体峰理论板数不低于 2500。 对照品溶液的制备 取菊淀粉型寡糖 5聚体 （5糖） 对照品适量， 精密称定， 加流动相制 成每 lml含 lmg的溶液， 即得。  Under these conditions, each component reached baseline separation. The number of theoretical plates of the amylose-type oligosaccharide 5-mer peak was not less than 2,500. Preparation of the reference solution The chrysanthemum-type oligosaccharide 5-mer (5-glycan) reference substance was accurately weighed, and the mobile phase was added to prepare a solution containing 1 mg per lml.
供试品溶液的制备 取巴戟天寡糖胶囊样品 0.3g， 精密称定， 置 10ml量瓶中， 加入流动 相 10ml， 超声， 取上清液， 用水稀释至刻度， 经 0.45μηι微孔滤膜过滤， 取续滤液， 即得。  Preparation of the test solution Take 0.3g of Morinda oligosaccharide capsule sample, accurately weighed, put it into a 10ml volumetric flask, add 10ml of mobile phase, sonicate, take the supernatant, dilute with water to the mark, filter through 0.45μηι microporous The membrane is filtered, and the filtrate is taken off.
测定法 同实施例 1。  The measurement method was the same as in Example 1.
巴戟天寡糖胶囊中每粒含菊淀粉型寡糖 5聚体不得低于 9.0mg， 含菊淀粉型寡糖 3~9聚体的 总量以菊淀粉型寡糖 5聚体计， 不得低于 75mg。 实施例 9.巴戟天寡糖原料药含量测定 Each of the capsules containing the inulin-type oligosaccharide 5 mer can be no less than 9.0 mg, containing the amylose-type oligosaccharide 3~9-mer. The total amount is not less than 75 mg based on the amylose-type oligosaccharide 5-mer. Example 9. Determination of the content of raw materials of Morinda officinalis
1仪器与试剂： 同实施例 1。  1 Instruments and reagents: Same as Example 1.
2. 方法与结果：  2. Methods and results:
2. 1.色谱分析条件： 同实施例 1。  2. 1. Chromatographic conditions: same as in Example 1.
对照品溶液的制备： 取菊淀粉型寡糖 5聚体 （5糖） 对照品适量， 精密称定， 加水制成每 lml含 lmg的溶液， 即得。  Preparation of the reference solution: Take the amylose-type oligosaccharide 5-mer (5 sugar). The appropriate amount of the reference substance, accurately weighed, add water to make a solution containing lmg per lml, that is.
供试品溶液的制备： 取巴戟天寡糖原料药， 研匀， 取约 150mg， 精密称定， 置具塞锥形 瓶中， 精密加入水 10ml， 超声 10分钟， 用 0.45um微孔滤膜滤过.， 取续滤液， 即得。  Preparation of the test solution: Take the raw material of Morinda oligosaccharide, stir it well, take about 150mg, accurately weigh it, place it in a conical flask, add 10ml of water precisely, sonicate for 10 minutes, filter with 0.45um microporous The membrane is filtered. The filtrate is taken and obtained.
测定法 同实施例 1。  The measurement method was the same as in Example 1.
巴戟天寡糖原料药按干燥品计算， 含菊淀粉型寡糖 5聚体不得少于 6. 0%， 含菊淀粉型寡糖 3~9聚体的总量以菊淀粉型寡糖 5聚体计， 不得少于 50. 0%。 实施例 10.巴戟天寡糖原料药的含量测定  The Morinda oligosaccharide bulk drug is calculated as a dry product, and the amylose-type oligosaccharide 5-mer is not less than 6.0%, and the total amount of the chrysanthemum-type oligosaccharide 3~9-mer is in the form of the chrysanthemum-type oligosaccharide 5 0%。 The polymer meter, not less than 50.0%. Example 10. Determination of the content of the raw material of Morinda oligosaccharide
1仪器与试剂： 同实施例 1。  1 Instruments and reagents: Same as Example 1.
2. 方法与结果：  2. Methods and results:
2. 1.色谱分析条件：  2. 1. Chromatographic conditions:
色谱柱： NH2柱 （5 μ ηι， 4.5 X 250 mm );  Column: NH2 column (5 μ ηι, 4.5 X 250 mm);
流动相： 乙腈： 水 ( 70： 30)；  Mobile phase: acetonitrile: water (70: 30);
检测器： 蒸发光散射检测器；  Detector: evaporative light scattering detector;
温 度: 柱温 30°C， 检测器温度 30°C ; 流 速： 1.0mL/min;  Temperature: column temperature 30 ° C, detector temperature 30 ° C; flow rate: 1.0 mL / min;
对照品溶液的制备 同实施例 1。  The preparation of the reference solution was the same as in Example 1.
供试品溶液的制备 取巴戟天寡糖液体原料药 lml， 置具塞锥形瓶中， 精密加入流动相 10ml， 用 0. 45um微孔滤膜滤过.取续滤液， 即得。  Preparation of the test solution Take 1 ml of the raw material medicine of Morinda oligosaccharide liquid, place it in a stoppered conical flask, precisely add 10 ml of the mobile phase, filter it with a 0.44 μm microporous membrane, and take the filtrate to obtain.
测定法 同实施例 1。  The measurement method was the same as in Example 1.
巴戟天寡糖原料药含菊淀粉型寡糖 5聚体不得少于 6.0%， 含菊淀粉型寡糖 3~9聚体的总量 以菊淀粉型寡糖 5聚体计， 不得少于 50.0%。 实施例 11. 天然植物中提取得到的含有巴戟天寡糖的提取物的含量测定  The amount of the amylose-type oligosaccharide 5-mer of the Morinda oligosaccharide raw material is not less than 6.0%, and the total amount of the 3-9-mer of the amylose-type oligosaccharide is determined by the amylose-type oligosaccharide 5-mer. 50.0%. Example 11. Determination of extracts containing Morinda offigo oligosaccharides extracted from natural plants
1仪器与试剂： 同实施例 1.  1 instruments and reagents: the same embodiment
2. 方法与结果：  2. Methods and results:
2. 1.色谱分析条件： 同实施例 1。  2. 1. Chromatographic conditions: same as in Example 1.
对照品溶液的制备 取菊淀粉型寡糖 5聚体 （5糖） 对照品适量， 精密称定， 加水制成每 lml含 lmg的溶液， 即得。 The preparation of the reference solution is taken from the appropriate amount of the amylose-type oligosaccharide 5-mer (5 sugar) reference substance, accurately weighed, and water is added to make each Lml contains 1mg of solution, that is.
供试品溶液的制备 取天然植物中提取得到的含有巴戟天寡糖的提取物 10g， 用水溶解， 使成浓度为 lg/ml的溶液， 上活性碳柱层析， 先用水洗脱至还原糖检出 （硫酸-苯酚法） 呈阴 性， 再用大约 6倍柱体积的 30%乙醇洗脱。 收集 30%乙醇洗脱部分， 经浓缩干燥得到巴戟天 寡糖（样品）。 取样品约 150mg， 精密称定， 置 10ml量瓶中， 用水稀释至刻度， 经 0.45μηι微 孔滤膜过滤， 取续滤液， 即得。  Preparation of the test solution 10 g of the extract containing Morinda officinose extracted from natural plants, dissolved in water to a concentration of lg / ml, on activated carbon column chromatography, first eluted with water to reduce The sugar was detected (sulfate-phenol method) negative and eluted with approximately 6 column volumes of 30% ethanol. The 30% ethanol fraction was collected and concentrated to obtain Morinda oligosaccharides (sample). Take about 150mg of sample, accurately weigh it, put it into a 10ml volumetric flask, dilute it to the mark with water, filter through 0.45μηι microporous membrane, and take the filtrate to obtain.
测定法 同实施例 1。  The measurement method was the same as in Example 1.
天然植物中提取得到的含有巴戟天寡糖的提取物按干燥品计算， 含菊淀粉型寡糖 5聚体 (C30H52O26) 不得少于 0.12%， 含巴戟天寡糖 （即菊淀粉型寡糖 3聚体 -9聚体的总量） 以菊淀 粉型寡糖 5聚体 （C3。H52026) 计， 不得少于 1.0%。 实施例 12. 天然植物中提取到的含有巴戟天寡糖的液体提取物含量测定 The extract containing Morinda officinalis oligosaccharides extracted from natural plants is calculated as a dry product, and the amylose-containing oligosaccharide 5-mer (C 30 H 52 O 26 ) is not less than 0.12%, containing Morinda officinalis ( That is, the total amount of the chrysanthemum-type oligosaccharide 3-mer-9-mer is not less than 1.0% in terms of the chrysanthemum-type oligosaccharide 5-mer (C 3 .H 52 0 26 ). Example 12. Determination of liquid extract containing Morinda officinalis extracted from natural plants
1仪器与试剂： 同实施例 1。  1 Instruments and reagents: Same as Example 1.
2. 方法与结果：  2. Methods and results:
2. 1.色谱分析条件  2. 1. Chromatographic conditions
色谱柱： cl8柱 （5 μ ηι， 4.5 X 250 mm );  Column: cl8 column (5 μ ηι, 4.5 X 250 mm);
流动相： 乙腈： 水 (70： 30)；  Mobile phase: acetonitrile: water (70: 30);
检测器： RID-10A示差检测器；  Detector: RID-10A differential detector;
温 度: 柱温 30°C， 检测器温度 30°C ; 流 速： 1.0mL/min;  Temperature: column temperature 30 ° C, detector temperature 30 ° C; flow rate: 1.0 mL / min;
对照品溶液的制备： 同实施例 1。  Preparation of the reference solution: Same as in Example 1.
供试品溶液的制备： 取天然植物中提取得到的含有巴戟天寡糖的提取物 10ml， 上活性碳 柱层析， 先用水洗脱至还原糖检出 （硫酸-苯酚法） 呈阴性， 再用大约 4倍柱体积的 30%乙醇 洗脱。 收集 30%乙醇洗脱部分， 经浓缩干燥得到巴戟天寡糖（样品）。 取样品约 150mg， 精密 称定， 置 10ml量瓶中， 用水稀释至刻度， 经 0.45μηι微孔滤膜过滤， 取续滤液， 即得。  Preparation of the test solution: 10 ml of the extract containing Morinda officinose extracted from natural plants, subjected to activated carbon column chromatography, first eluted with water to reduce sugar (sulfuric acid-phenol method) was negative, It was then eluted with about 4 column volumes of 30% ethanol. The fraction eluted with 30% ethanol was collected and concentrated to obtain Morinda oligosaccharides (sample). Take about 150mg of sample, accurately weigh it, put it into a 10ml volumetric flask, dilute it to the mark with water, filter through 0.45μηι microporous membrane, and take the filtrate to obtain.
测定法： 同实施例 1。  Assay: Same as Example 1.
天然植物中提取得到的含有巴戟天寡糖的提取物按干燥品计算， 含菊淀粉型寡糖 5聚体 (C30H52O26)不得少于 6%， 含巴戟天寡糖（即菊淀粉型寡糖 3聚体 -9聚体的总量） 以菊淀粉型 寡糖 5聚体 （C3。H52026) 计， 不得少于 50%。 The extract containing Morinda officinalis oligosaccharides extracted from natural plants is calculated as dry product, and the amylose-containing oligosaccharide 5-mer (C 30 H 52 O 26 ) is not less than 6%, containing Morinda officinalis ( That is, the total amount of the chrysanthemum-type oligosaccharide 3-mer-9-mer is not less than 50%, based on the amylose-type oligosaccharide 5-mer (C 3 .H 52 0 26 ).

Claims

权 利 要 求 Rights request
1.一种巴戟天寡糖的含量测定方法， 该巴戟天寡糖至少含有菊淀粉型寡糖 5聚体， 并 且存在于含有巴戟天寡糖的原料药或由天然植物提取得到的含有巴戟天寡糖的提取物或 含有巴戟天寡糖的制剂中， 该含量测定方法包括-A method for determining the content of Morinda offigo oligosaccharide, which comprises at least a chitosan-type oligosaccharide 5-mer, and is present in a raw material containing Morinda oligosaccharide or extracted from a natural plant. In the preparation containing the Morinda oligosaccharide or the preparation containing the Morinda oligosaccharide, the content determination method includes -
( 1 ) 采用高效液相色谱法进行测定， 以含有乙腈的水溶液为流动相， 采用示差检测 器或蒸发光散射检测器检测； (1) Determination by high performance liquid chromatography, using an aqueous solution containing acetonitrile as a mobile phase, using a differential detector or an evaporative light scattering detector;
(2) 对照品溶液的制备， 称取菊淀粉型寡糖 5聚体作为对照品， 加水或步骤 （1 ) 中 所述的流动相制成对照品溶液；  (2) preparing a reference solution, weighing the amylose-type oligosaccharide 5-mer as a control, adding water or the mobile phase described in the step (1) to prepare a reference solution;
(3 ) 供试品溶液的制备， 取含有巴戟天寡糖的制剂或含有巴戟天寡糖原料药或由天 然植物提取得到的含有巴戟天寡糖的提取物，力 M-100重量倍数的水或者流动相使其混合或 溶解， 取上清液， 滤过， 取续滤液， 即得供试品溶液；  (3) Preparation of the test solution, taking a preparation containing Morinda oligosaccharide or an extract containing Morinda oligosaccharide raw material or extracted from natural plants, containing Morinda oligosaccharide, force M-100 weight Multiple water or mobile phase is mixed or dissolved, and the supernatant is taken, filtered, and the filtrate is taken to obtain a test solution;
(4) 测定， 分别吸取对照品溶液与供试品溶液， 注入液相色谱仪， 测定；  (4) Determination, respectively, draw the reference solution and the test solution, inject into the liquid chromatograph, and measure;
(5 ) 计算， 依照供试品图谱中各个色谱峰与菊淀粉型寡糖 5聚体对照品相对保留时 间， 按外标法以峰面积计算该巴戟天寡糖中所含有的菊淀粉型寡糖 3〜9聚体中任一寡糖 聚体或其混合物的含量， 再将菊淀粉型寡糖 3〜9聚体的含量进行加和， 即得所述的巴戟 天总寡糖的含量。  (5) Calculate, according to the relative retention time of each chromatographic peak in the test sample and the amylose-type oligosaccharide 5-mer reference substance, calculate the inulin type contained in the Morinda officinalis oligosaccharide by the external standard method The content of any oligosaccharide polymer or a mixture thereof in the oligosaccharide 3~9mer, and the content of the chrysanthemum-type oligosaccharide 3~9-mer is further added, thereby obtaining the total oligosaccharide of the Morinda officinalis content.
2. 权利要求 1所述的含量测定方法，其中，所述的巴戟天寡糖还含有菊淀粉型寡糖 3〜 4、 6〜9聚体中任一寡糖或其混合物。  The content determination method according to claim 1, wherein the Morinda oligosaccharide further comprises any one of the oligosaccharides of the inulin type oligosaccharide 3 to 4, 6 to 9 or a mixture thereof.
3. 权利要求 1所述的含量测定方法， 其中， 所述的步骤 （1 ) 的流动相含有 40-80% 体积的乙腈。  The content determination method according to claim 1, wherein the mobile phase of the step (1) contains 40 to 80% by volume of acetonitrile.
4. 权利要求 2或 3所述的含量测定方法， 其中， 所述的步骤 （1 ) 的流动相中还包括 不超过该流动相中体积的 0.5 %的三乙胺。  The content determination method according to claim 2 or 3, wherein the mobile phase of the step (1) further comprises not more than 0.5% by volume of triethylamine in the mobile phase.
5. 权利要求 1所述的含量测定方法， 其中， 所述的步骤 （3 ) 包括： 取含有巴戟天寡 糖的固体制剂或含有巴戟天寡糖的固体原料药或由天然植物提取得到的含有巴戟天寡糖 的固体提取物， 加水或者流动相使其溶解或混合， 取上清液， 滤过， 取续滤液， 即得供试 品溶液。  The content determination method according to claim 1, wherein the step (3) comprises: taking a solid preparation containing Morinda oligosaccharide or a solid raw material containing Morinda oligosaccharide or extracting from a natural plant. The solid extract containing Morinda oligosaccharide is dissolved or mixed by adding water or a mobile phase, and the supernatant is taken, filtered, and the filtrate is taken to obtain a test solution.
6. 权利要求 1所述的含量测定方法， 其中， 所述的步骤 （3 ) 包括： 取含有巴戟天寡 糖的液体制剂或含有巴戟天寡糖的液体原料药或由天然植物提取得到的含有巴戟天寡糖 的液体提取物， 加水或者流动相稀释， 取上清液， 滤过， 取续滤液， 即得供试品溶液。 The content determination method according to claim 1, wherein the step (3) comprises: taking a liquid preparation containing Morinda oligosaccharide or a liquid raw material containing Morinda oligosaccharide or extracting from a natural plant. The liquid extract containing Morinda oligosaccharide is diluted with water or mobile phase, and the supernatant is taken, filtered, and the filtrate is taken to obtain a test solution.
7. 权利要求 5所述的含量测定方法， 其中， 所述的步骤 （3 ) 包括： The content determination method according to claim 5, wherein the step (3) comprises:
每 0.2-0.5g固体制剂或固体原料药或固体提取物，于具塞锥形瓶中加入水和 /或流动相 10ml, 超声， 取上清液， 微孔滤膜滤过， 取续滤液， 即得供试品溶液。  Add 0.2 ml of water and/or mobile phase to each of 0.2-0.5 g of the solid preparation or the solid drug substance or the solid extract. Ultrasonic, take the supernatant, filter through the microporous membrane, and take the filtrate. That is, the test solution is obtained.
8. 权利要求 6所述的含量测定方法， 其中， 所述的步骤 （3 ) 包括： 每 0.1-10ml的液 体制剂或液体原料药或液体提取物， 于具塞锥形瓶中加入水和 /或流动相至 10ml， 用微孔 滤膜滤过， 取续滤液， 即得供试品溶液。  The content determination method according to claim 6, wherein the step (3) comprises: adding water and/or to a conical flask for every 0.1-10 ml of the liquid preparation or the liquid medicine or the liquid extract. Or the mobile phase to 10ml, filtered through a microporous membrane, and the filtrate is taken to obtain the test solution.
9. 权利要求 5所述的含量测定方法， 其中， 所述的步骤（3 )包括： 每 0.3g含有巴戟 天寡糖的固体制剂加入水 10ml，即得供试品溶液；该固体制剂为胶囊剂的内容物、粉针剂、 片剂或颗粒剂。  The method for determining the content according to claim 5, wherein the step (3) comprises: adding 10 ml of water per 0.3 g of the solid preparation containing Morinda oligosaccharide to obtain a test solution; The contents of the capsule, powder injection, tablet or granule.
10. 权利要求 1所述的含量测定方法， 其中， 该方法步骤（1 ) 中所述的色谱条件包括 采用氨基柱或 C18反相柱。  The content determination method according to claim 1, wherein the chromatographic conditions described in the step (1) of the method comprise using an amino column or a C18 reverse phase column.
PCT/CN2008/072344 2008-06-26 2008-09-12 Method for determining the contents of oligosaccharides in morinda officinalis WO2009155755A1 (en)

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CN114550843A (en) * 2022-01-21 2022-05-27 梁军 Model for predicting monosaccharide composition and content in traditional Chinese medicine polysaccharide and construction method and application thereof
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CN115487206A (en) * 2022-10-08 2022-12-20 河南农业大学 Method for relieving poor development of tibial cartilage of broiler chicken by morinda officinalis polysaccharide and application of morinda officinalis polysaccharide

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