WO2009155756A1

WO2009155756A1 - Method for determining the contents of oligosaccharides in morinda officinalis chinese medicine or extraction thereof

Info

Publication number: WO2009155756A1
Application number: PCT/CN2008/072346
Authority: WO
Inventors: 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
Priority date: 2008-06-26
Filing date: 2008-09-12
Publication date: 2009-12-30
Also published as: CN101334390B; HK1122101A1; WO2009155755A1; CN101334389B; CN101334390A; HK1122102A1; CN101334389A

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, canculating 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

Method for determining content of Morinda officinalis in Morinda citrifolia or its extract

The 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 to a Chinese herbal medicine of Morinda officinalis or Chinese herbal medicine of Bashu. A method for determining the high-performance liquid phase content of Morinda officinalis in an extract. Background technique

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 shortcomings of measuring the unified target one by one are inevitable, the operation is cumbersome, time-consuming and laborious, and the efficiency is low. The relative content standards of the components in the same chemical part are independent and irrelevant, so it will inevitably cause a certain degree. Deviation, the quality cannot be controlled more effectively and accurately.

For the extract of Morinda officinalis or Morinda citrifolia containing Babentian oligosaccharide, the active ingredient is an oligosaccharide substance, and the oligosaccharide substance is a mixture of 3-9 sugars, if chemical coloration is used The reaction shows the same reaction characteristics as other sugars (monosaccharides, disaccharides, polysaccharides). Therefore, the data proves that for the detection of Morinda oligosaccharides, the chromatographic identification of HPLC is more specific, and the results are more scientific. And rigorously, it is necessary to develop a high-performance liquid phase content determination method which can determine the total amount of the chemical part while identifying each component, giving the relative content of each component, minimizing the type of the reference substance, and simple operation. . Summary of the invention

At present, in the extracts of Morinda officinalis or Morinda citrifolia containing Morinda citrifolia, there is no standardized and mature method for quality monitoring, and the object of the present invention is to provide a scorpion Sugar The method for determining the content, using the method and the technical indicators in the method, can well monitor the quality of the Morinda citrifolia containing the Morinda citrifolia or the extract thereof, so that the quality of the traditional Chinese medicine is stable and controllable, and the curative effect is ensured. , can better serve the community.

The object of the present invention is to develop a quality control method for the Morinda citrifolia containing the Morinda oligosaccharide component or the extract thereof, which is simple, efficient and suitable for scientific research, marketing, market supervision, clinical application and The need for quality testing in industrial production.

The invention provides a method for determining the content of Morinda officinalis oligosaccharide, wherein the Morinda oligosaccharide comprises at least a chitosan-type oligosaccharide 5-mer (also referred to as 1F-fructofuranosyl-sucrose, English name lF- Fructofuranosyl nystose), and is present in the Chinese herbal medicine or its extract, which is determined by:

(1) Determination by high performance liquid chromatography; chromatographic conditions are carried out using an aqueous solution containing acetonitrile as a mobile phase, using a differential detector or an evaporative light scattering detector;

(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) Preparation of the test solution: Take the sample of Morinda oligosaccharide, add water or dissolve the mobile phase in step (1), take the supernatant, filter, and take the filtrate to obtain the test solution; The continuous filtrate is the filtrate of the intermediate stage collected during the filtration process;

The scorpion oligosaccharide sample is prepared by the following method, taking the medicinal material of Morinda citrifolia and extracting the extract by using water, the extract is an extract of Chinese herbal medicine of Morinda citrifolia, and the extract is subjected to activated carbon column chromatography. First, it was eluted with water to reduce sugar (sulfuric acid-phenol method), and then eluted with 20-50% ethanol. The ethanol eluted fraction was collected and concentrated to obtain the Morinda oligosaccharide sample.

(4) Determination: separately draw the reference solution and the test solution, inject into the liquid chromatograph, and measure;

(5) Calculate, according to the relative retention time of each chromatographic peak in the test sample and the amyloid 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 a content of a mixture of any oligosaccharide or any oligosaccharide of the inulin oligosaccharide 3mer-9 mer contained in the oligosaccharide, thereby determining each of the daisy starches in the sorghum oligosaccharide Relative content of oligosaccharide 3-mer-9 mer The content ratio of the inulin-type oligosaccharide 3-mer-9-mer is further added to obtain the content of the total oligosaccharide of the Morinda officinalis.

The above-mentioned Morinda oligosaccharide is an active ingredient having an antidepressant action, and includes a chrysanthemum-type oligosaccharide 3 saccharide to 9 saccharide, that is, the Morinda oligosaccharide according to the present invention contains at least a chamomile-type oligosaccharide 5-mer. Oligosaccharide (1F-fructofuranosyl nystose), or both amylose-type oligosaccharide 5-mer and chrysanthemum-type oligosaccharide trimer, chrysanthemum-type oligosaccharide 4-mer ( Ness sugar, nystose, chrysanthemum-type oligosaccharide 6-mer ((2→1) fruit furanosyl sucrose h _exasaCC h _a ride), chrysanthemum-type oligosaccharide 7-mer (hep tasaccharide), chrysanthemum-type oligosaccharide A mixture of a sugar 8-mer and a chrysanthemum-type oligosaccharide 9-mer, any combination or combination thereof.

In a preferred embodiment of the present invention, the column in the step (1) in the above method is preferably an amino column or a C18 reverse phase column; for example, an Inertsil NH ₂ column (5 μm, 4.6 x 250 mm), and the detector is used for differential detection. Or an evaporative light scattering detector, the mobile phase generally selecting an aqueous solution containing 40-80% by volume of acetonitrile; preferably a mixed solution of acetonitrile and water in a volume ratio of 3:1 to 1:1.

The above-mentioned Morinda oligosaccharide preferably contains, in addition to the inulin-type oligosaccharide 5-mer, any of the oligosaccharides of the inulin type oligosaccharides 3 to 4, 6 to 9 or a mixture thereof.

Preferably, the Morinda oligosaccharide of the present invention comprises a chrysanthemum-type oligosaccharide 5mer, a chrysanthemum-type oligosaccharide 3mer, a chrysanthemum-type oligosaccharide 4mer, a chrysanthemum-type oligosaccharide 6-mer, a chrysanthemum starch A mixture of a type oligosaccharide 7-mer, a chrysanthemum-type oligosaccharide 8-mer and a chrysanthemum-type oligosaccharide 9-mer. In general, the chrysanthemum-type oligosaccharide n-mer can be simply referred to as n-saccharide, and 11 is 3 to 9. For example, the inulin-type oligosaccharide 5-mer is a 5-sugar, and the inulin-type oligosaccharide 9-mer is 9 sugar.

The inventors have found that in order to improve the tailing phenomenon of the chromatogram, 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 contains triethylamine, triethyl ethane. The volume of amine in the mobile phase generally does not exceed 0.5%; typically from 0.01% to 0.5%.

The filtration step described in the preparation of the test solution of the present invention preferably employs a microporous membrane having a pore size of generally 0.3, 0.45, 0.5, 0.75 um, more preferably 0.45 um. 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 mixed solution of acetonitrile and water in a volume ratio of 68:32, and the mixed solution further contains no more than the mobile phase volume. 0.5% triethylamine; for example, a mixed solution of acetonitrile, water and triethylamine in a volume ratio of 68:32:0.1 can be used as a mobile phase, and the obtained spectrum and peak shape exhibit surprisingly perfect effects.

In the detection method of the present invention, the activated carbon column chromatography on the water extract used in the step (3) is preferably first eluted with water, and then eluted with about 20-50% ethanol, more preferably after elution with water. It was eluted with 30% ethanol and concentrated to obtain a preferred sample of Morinda officinalis. Then, take 50-200mg of Morinda oligosaccharide sample, accurately weigh it, put it in a 10ml volumetric flask, dissolve it with water or the mobile phase in step (1), dilute to the mark, preferably filter through microporous membrane, continue The filtrate is used to obtain a test solution.

The reference substance of the invention is selected from the amylose-type oligosaccharide 5-mer, and the inulin-type oligosaccharide 5-mer is selected as the control because the Morinda oligosaccharide is a mixture of 3~9-mer oligosaccharides under the determined HPLC chromatographic conditions. 5, sugar relative retention time is centered, peak peak height or peak area is moderate, to calibrate other 3-4, 6-9 sugar, the relative position difference between each sugar and 5 sugar peak is more uniform, 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 5 sugars enables the HPLC of other sugars (SP, 3, 4, 6, 7, 8 and 9 sugars) to have clear peak shapes, good resolution and high accuracy.

The test solution of the step (3) is preferably a solution obtained by adding water or a mobile phase of 10 ml per 50-200 mg of Morinda oligosaccharide sample, and during the preparation of the test solution, the solution is subjected to ultrasonication, dissolution. After filtering with a 0.45um microporous membrane, the filtrate is taken as the test solution;

If the test product is a raw material of Morinda oligosaccharide, the test solution is directly prepared; if it is a Chinese herbal medicine or a Chinese herbal medicine extract, the test product is obtained by the following method: For Chinese herbal medicines containing Morinda oligosaccharides, the extracts are extracted with water, and the extracts are subjected to activated carbon column chromatography, and eluted with water to the reducing sugars (sulfuric acid-phenol method), and then 20-50%. Elution with ethanol, preferably 30% ethanol. The ethanol eluted fraction was collected and concentrated to obtain the Morinda citrus granule sample; the Chinese herbal extract containing Morinda oligosaccharide was directly subjected to activated carbon column chromatography, and then treated as described above. In another embodiment of the present invention, the method for determining content includes:

(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) Preparation of the reference solution, weigh the inulin-type oligosaccharide 5-mer as a control, add water to make a reference solution containing 1 mg per 1 ml;

(3) Preparation of the test solution, 50-200 mg of the Morinda oligosaccharide sample obtained by extracting the extract containing Morinda officinalis and then using activated carbon column chromatography, and placing the plug In the conical flask, add 10 ml of water, ultrasonic at a frequency of 100 W for 10 minutes at 40 kHz, let stand, take the supernatant, filter with a 0.45 um microporous membrane, and take the filtrate to obtain the test solution;

(4) Determination, draw the reference solution and the test solution 20μ1, inject into the liquid chromatograph, and measure;

(5) Calculate, according to the relative retention time of each chromatographic peak in the test sample and the amyloid oligosaccharide 5-mer 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, Total oligosaccharide content.

The above continuous filtrate is the intermediate stage filtrate collected during the filtration process.

The structure of Morinda oligosaccharides is as follows:

Wherein η = 1 is 3 sugars; η = 2 is 4 sugars; η = 3 is 5 sugars; η = 4 is 6 sugars; η = 5 is 7 sugars; η = 6 is 8 sugars; η = 7 is 9 sugars; .

In the present invention, the inulin oligosaccharide 5-mer in the Morinda oligosaccharide is used as a reference substance, and the oligosaccharide content is determined by HPLC. The innovation is: based on the component of the inulin-type oligosaccharide 5-mer , the retention time of the component is set to 1, and the retention time of other polymer peaks is converted into relative retention In the meantime, the relative retention time is used to identify the presence of other oligosaccharides other than 5 sugars, and then the content of each component and the total oligosaccharide is calculated by the external standard method to obtain a relatively stable and accurate Morinda oligosaccharide. content. That is, a plurality of structurally similar compounds can be characterized by a reference substance, and the relative contents 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. 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 Starch-type oligosaccharide 7-mer peak; 1.83~2.50 inulin-type oligosaccharide 8-mer peak; 1.85~3.40 in the amylose-type oligosaccharide 9-mer peak, and then calculate the oligosaccharide by peak area according to the external standard method The content.

The selection of the chromatographic conditions of the present invention is based on a large amount of experimental data, and various indicators including peak shape, resolution, peak area, etc., and the optimal column, mobile phase, detector, and column temperature conditions are comprehensively evaluated. . details as follows:

HPLC analysis of the reference chrysanthemum amylose oligosaccharide 5mer (5 sugar):

A 5 saccharide control solution was prepared for 20 μl, and HPLC analysis was carried out 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.

HPLC analysis of Morinda officinalis:

Take the content measurement item, the sputum oligosaccharide test solution 20μ1, perform HPLC analysis under the above chromatographic conditions, and record the chromatogram. The peaks in the figure are from 1 to 7 chromatographic peaks from left to right, by HPLC-MS- MS and other spectroscopy methods were determined in order (from left to right): chrysanthemum-type oligosaccharide trimer (C ₁₈ H ₃₂ 0 ₁₆ , abbreviated as: 3 sugar), chrysanthemum-type oligosaccharide 4-mer (C ₂₄ H ₄₂ 0 ₂₁ , abbreviated as: 4 sugar), chrysanthemum-type oligosaccharide 5-mer (C ₃₀ H ₅₂ O ₂₆ , abbreviated as: 5 sugar), chrysanthemum-type oligosaccharide 6-mer (C ₃₆ H ₆₂ 0 ₃₁ , abbreviation: 6 Sugar), inulin-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 , abbreviated: 9 sugar). The retention time of the 5 sugar peaks was consistent with the retention time of the control. Retention time of 3~9 polymer peaks, map analysis As shown below, the data is shown in Table 1 (Resolution: Resolution, T. Plate# Theoretical Plate Number, Ret Time: Retention Time, Area: Area).

Table 1

Chromatographic identification results: The above results were statistically analyzed to obtain the maximum, minimum and average values of the relative retention times of the respective peaks of 3 to 9 sugars, as shown in Table 2. Table 2 Relative retention time statistics of each peak of Morinda officinalis

Relative retention time

Sugar Name ·

Maximum 倌 minimum 倌 average 倌

3 sugar 0.577 0.572 0.574

4 sugar 0.760 0.757 0.758

5 sugar 1.000 1.000 1.000

6 sugar 1.316 1.311 1.314

7 sugar 1.755 1.740 1.748

8 sugar 2.330 2.289 2.316

9 sugar 3.098 3.053 3.075 HPLC analysis of the solvent:

Take the pure water solvent, or take the mobile phase solution, according to the preparation method of the test solution, take 20μ1, 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 the chromatogram of the blank sample, the retention time corresponding to the chromatogram of the Morinda oligosaccharide 5 sugar reference, and the chromatogram of the sample containing the Morinda oligosaccharide. At the relative retention time of the corresponding oligosaccharides in the figure, no chromatographic peaks were observed. Indicates that there is no white solvent interference. Morinda oligosaccharide, a chrysanthemum amylose oligosaccharide trimer (C ₁₈ H ₃₂ 0 ₁₆ , abbreviated as: 3 sugar), a chrysanthemum-type oligosaccharide 4mer (C ₂₄ H ₄₂ 0 ₂₁ , abbreviated as: 4 sugar) , chrysanthemum-type oligosaccharide 5-mer (C ₃₀ H ₅₂ O ₂₆ , abbreviated as: 5 sugar), chrysanthemum-type oligosaccharide 6-mer (C ₃₆ H ₆₂ 0 ₃₁ , abbreviated as 6 sugar), inulin-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 , abbreviated as: 9 sugar). The invention establishes a HPLC content determination method of the Morinda offigo oligosaccharide. The experiment is described as follows:

1 instruments and reagents:

Shimadzu 20AT high performance liquid chromatography system;

RID-10A differential detector;

LCsolution chromatography workstation;

Reagents: acetonitrile-chromatographically pure (Tianjin Siyou Biomedical Technology Development Co., Ltd.), water is high-purity water, and the remaining reagents are of analytical grade;

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. Chromatographic conditions

Column: Inertsil NH ₂ column (5μπι, 4.5x250 mm);

Mobile phase: acetonitrile: water: triethylamine (68: 32: 0.1);

Detector: RID-10A differential detector;

Temperature: column temperature 40 ° C, detector temperature 40 ° C; flow rate: 1.2 mL / min;

Under these conditions, the components in the sample reached baseline separation. The number of plates of the amylose-type oligosaccharide 5-mer 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, 30μ1, injected into the liquid chromatograph, determined according to the above chromatographic conditions, The peak area integral value is the ordinate. The injection volume of the amyloid oligosaccharide 5-mer is 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 5mer was in 0.0119~ A good linear relationship in the range of 0.0711 mg. See Table 3. Table 3 Investigation of linear relationship

Sugar injection volume (mg) peak shift

0.0119 60532.5

0.0237 123563

0.0356 190649

0.0474 259123

0.0593 321999.5

0.0711 388606.5

2. 3 precision experiment: Precision extraction of the 5-mer reference solution (2.37mg/ml) ΙΟμΙ, continuous injection 6 times, calculate the average peak area and relative standard deviation, see Table 4. Indicates good precision. Table 4 Precision Results

Number of peak areas mean RSD (%)

1 123774

2 122453

3 123119

123267 0.4

4 123130

5 123760

6 123366

2. Blank test: Take pure water solvent, or mobile phase solution, according to the test sample treatment method, take 20μ1, perform HPLC analysis under the above chromatographic conditions, record the chromatogram, and the result shows no interference.

2. Investigation of the stability of the test solution: The same batch of test solution, after the sample preparation 0, 2, 4, 8, 10,

After 22 hours of measurement, the results showed that the test solution was substantially stable within 22 hours. The results are shown in Table 5. Table 5 Stability test results

Time (hours) 5 Sugar content (mg/g) Mean (mg/g) RSD (%) Total sugar content (mg/g) Mean (mg/g) RSD (%)

0 55.8 54.6 1.8 311.5 310.4 0.6

2 55.2 308.7

4 54.5 311.2 53.8 310.3

22 54.8 313.0

2. 6. Repeatability test: Take the same batch of samples, make 6 samples in parallel, and measure. The results are shown in Table 6. The results show that the repeatability is better. Table 6 Repeatability test results

5 Sugar content (mg/g) Mean (mg/g) RSD (%) Total sugar content (mg/g) Mean (mg/g) RSD (%)

1 54.3 308.4

2 56.0 319.6

3 53.2 305.1

54.1 1.9 310.9 1.8

4 53.5 308.3

5 54.3 315.4

6 53.3 308.6

2. 7. Recovery test: Using the sample recovery method, we accurately weigh 150mg of the same batch (5 sugar content 54.1mg/g) of known content, and accurately add 5 sugar reference substance (7.716mg), parallel system Six samples were measured 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. Recovery rate (%) = - out 5 total sugar - in the sample 5 sugar containing

x l00%

Add 5 sugar control amount

Table 7 Recovery test results Sample sampling Samples 5 Sugar Add 5 Sugar amount Measured 5 Sugar Recovery Average RSD (Quantity (g) Amount (mg) (mg) Total (mg) (%) (%) %)

1 0.1502 8.128 15.876 100.42

2 0.1505 8.144 15.802 99.24

3 0.1511 8.177 16.001 101.40

7.716 100.83 1.72

4 0.1506 8.150 15.765 98.69

5 0.1509 8.166 16.142 103.38

6 0.1517 8.209 16.068 101.85

2. 8. Sample measurement results: Three batches of samples were measured, and the results are shown in Table 8. Table 8, sample determination results batch number 5 sugar content (mg / g) total sugar content (mg / g)

0701-1 114.0 612.7

0701-2 112.0 622.5

0701-3 112.3 605.3

3. Method durability study

Column: MERK Lichrosorb NH ₂ column (5 μιη, 4.5 x 250 mm); mobile phase: acetonitrile: water: triethylamine (68: 32: 0.1);

Detector: RID-10A differential detector;

Temperature: column temperature 40 ° C, detector temperature 40 ° C; flow rate: 1.2 mL / min; Results: See Tables 9, 10. Table 9. Durability results 1

Batch No. 5 Sugar content (mg/g) Total sugar content (mg/g)

0701-1 111.6 618.9 Table 10. Relative retention times of each chromatographic peak of Morinda officinalis

Relative retention time

3 sugar 0.68

4 sugar 0.82

5 sugar 1.00

6 sugar 1.22

7 sugar 1.50

8 sugar 1.86

9 sugar 2.30

SEPAX Amethyst amino column (5μπι, 4.6x250 mm); mobile phase: acetonitrile: water: triethylamine (68: 32: 0.1);

Detector: RID-10A differential detector; Temperature: column temperature 40 ° C, detector temperature 40 ° C; flow rate: 1.2 mL / min; Results: See Tables 11, 12. Table 11, durability results 2

~ ^ 5 sugar content (mg/g) total sugar content (mg/g)

0701-2 112.9 625.1 Table 12, Relative retention times of peaks of Morinda oligosaccharides

Sugar name relative retention time

3 sugar 0.63

4 sugar 0.79

5 sugar 1.00

6 sugar 1.26

7 sugar 1.60

8 sugar 2.04

9 sugar 2.61

4. Determination of relative retention time range: Determination of multiple batches of Morinda officinalis oligosaccharides, the retention time of chrysanthemum-type oligosaccharide 3-9 mers was relatively fixed, and the ratio of chrysanthemum-type oligosaccharides 3~9-mers The relative retention time of the 5-mer is also relatively stable, see Table 13. Table 13, Relative retention time range

Sugar name Relative retention time -5% +5%

3 sugar 0.574 0.55 0.61

4 sugar 0.758 0.72 0.80

5 sugar 1.000 1 1

6 sugar 1.314 1.25 1.38

7 sugar 1.748 1.65 1.83

8 sugar 2.316 2.19 2.42

9 Sugar 3.075 2.90 3.21 After systematic study, the present invention uses a sucrose amylose oligosaccharide 5-mer as a reference substance, with a peak of 3-9 sugar The method of calculating the oligosaccharide content of Morinda officinalis 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 industrial large production. Specific implementation

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 citrifolia

1 instruments and reagents:

Shimadzu 20AT high performance liquid chromatography system; LCsolution chromatography workstation

Reagents: acetonitrile-chromatographic purity (Tianjin Siyou Biomedical Technology Development Company), water-high purity water, the remaining reagents are of analytical grade

2. Methods and results:

2. 1. Chromatographic conditions

Column: Inertsil NH ₂ column (5μπι, 4.5x250 mm);

Mobile phase: acetonitrile: water: triethylamine (68: 32: 0.1);

Detector: RID-10A differential detector;

Preparation of reference solution Take the appropriate amount of the amylose-type oligosaccharide 5-mer (5 sugar) reference substance, accurately weighed, add water to make a solution containing lmg per lml, that is.

Preparation of the test solution Take 20g of Morinda citrifolia. According to the cold leaching method of Appendix XA of Chinese Pharmacopoeia 2005 edition, the extract is extracted, evaporated to dryness, and the dried matter is dissolved in water to make a solution with a concentration of lg/ml. , on activated carbon column chromatography, first eluted with water to reduce sugar (sulfate-phenol method) was negative, and then used 6 column volumes of 30% ethanol were eluted. The 30% ethanol fraction was collected and concentrated to obtain Morinda oligosaccharides (sample). Take about 150mg of the sample, accurately weigh it, place it in a 10ml volumetric flask, dilute it to the mark with water, filter through a 0.45μπι microporous membrane, and take the filtrate to obtain.

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.

Morinda citrifolia is calculated as dry product, containing inulin-type oligosaccharide 5-mer (C _3Q H ₅₂ 0 ₂₆ ) not less than 2.0%, containing Morinda oligosaccharide (ie, chrysanthemum-type oligosaccharide trimer-9 Total amount of polymer)

(C ₃₀ H ₅₂ O ₂₆ ), not less than 10.0%.

Example 2: Extract of Morinda citrifolia

1 instruments and reagents:

Shimadzu 20AT high performance liquid chromatography system; LCsolution chromatography workstation;

Reagent: same as Example 1;

Reference substance: Chrysanthemum-type oligosaccharide 5-mer (provided by China National Institute for the Control of Pharmaceutical and Biological Products, purity 99.99%). 2. Methods and results:

2. 1. Chromatographic conditions

Column: NH ₂ column (5μπι, 4.5x250 mm);

Mobile phase: acetonitrile: water: triethylamine (68: 32: 0.1);

Detector: RID-10A differential detector;

Preparation of the reference solution The appropriate amount of the amylose-type oligosaccharide 5-mer reference substance was accurately weighed, and the mobile phase was added to make a solution containing 1 mg per lml.

Preparation of the test solution Take 5g of the extract of Morinda citrifolia, dissolve it in water, and make a solution with a concentration of lg/ml. Perform on activated carbon column chromatography and elute with water to reduce sugar (sulfuric acid-phenol method). Negative, It was eluted with 30% ethanol. A 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 to the mark with mobile phase, filter through 0.45μπι microporous membrane, and take the filtrate to obtain.

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 3mer-9mer according to 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.

The extract of Morinda officinalis L. is calculated as a dry product, and the amylose-type oligosaccharide 5-mer (C _3Q H ₅₂ 0 ₂₆ ) is not less than 3.0%, and contains the glutinous oligosaccharide (ie, the amylose-type oligosaccharide trimer). The total amount of -9-mer is not less than 20.0% based on the amylose-type oligosaccharide 5-mer (C _3Q H ₅₂ 0 ₂₆ ).

Example 3. Determination of the content of Morinda officinalis

1 instruments and reagents:

Reagent: same as Example 1;

2. 1. Chromatographic conditions

Column: NH ₂ column (5μπι, 4.5x250 mm);

Mobile phase: acetonitrile: water (40: 60);

Detector: RID-10A differential detector;

Temperature: column temperature 40 ° C, detector temperature 40 ° C; flow rate: 1.0 mL / min;

Under these conditions, the components in the sample reached baseline separation. The number of plates of the amylose-type oligosaccharide 5-mer was determined to be not less than 2,500.

Preparation of the reference solution: Take the appropriate amount of the amylose-type oligosaccharide 5-mer (5-saccharide) reference substance, accurately weighed, and add the mobile phase to make a solution containing lmg per lml.

Preparation of the test solution Take 20g of Morinda citrifolia. According to the cold leaching method of Appendix XA of Chinese Pharmacopoeia 2005 edition, the extract is extracted, evaporated to dryness, and the dried matter is dissolved in water to make a solution with a concentration of lg/ml. , On activated carbon column chromatography, first eluted with water to reduce sugar (sulfate-phenol method) was negative, and then used approximately

Elected with 8 column volumes of 20% ethanol. The 20% ethanol fraction was collected and concentrated to obtain Morinda oligosaccharide (sample). Take about 150mg of sample, accurately weigh it, place it in a 10ml volumetric flask, dilute to the mark with mobile phase, filter through 0.45μπι microporous membrane, and take the filtrate to obtain.

According to the dry product, the medicinal material of Morinda citrifolia contains not less than 2.0% of the amylose-type oligosaccharide 5-mer (C _3Q H ₅₂ 0 ₂₆ ), and contains the amylose-type oligosaccharide 5-mer (C _3Q). H ₅₂ 0 ₂₆ ), not less than 10.0%. Example 4 Determination of the content of Morinda citrifolia

1 instruments and reagents:

Reagent: same as Example 1;

2. 1. Chromatographic conditions

Column: C18 column (5μπι, 4.5x250 mm);

Mobile phase: acetonitrile: water: triethylamine (78: 22: 0.2);

Detector: evaporative light scattering detector;

Temperature: column temperature 35 ° C, detector temperature 35 ° C; flow rate: 1.2 mL / min;

The components in the sample reached baseline separation. The number of theoretical plates of the amyloid oligosaccharide 5-mer peak is not less than 3,000. Preparation of the reference solution: Take the appropriate amount of the amylose-type oligosaccharide 5-mer (5-glycan) reference substance, accurately weighed, add water to make a solution containing lmg per lml, that is.

Preparation of test solution: Take 20g of Morinda citrifolia, extract the extract by cold soaking method, evaporate it, dissolve it in water, make a solution with concentration of lg/ml, and perform chromatography on activated carbon column. It was first eluted with water to a reducing sugar (sulfuric acid-phenol method) and then eluted with about 4 column volumes of 50% ethanol. The 50% ethanol fraction was collected and concentrated to obtain Morinda oligosaccharides (sample). Take about 150mg of the sample, place it in a 10ml volumetric flask, dilute to the mark with water, filter through a 0.45μπι microporous membrane, and take the filtrate to obtain.

Separately draw the reference solution and the test solution for 20μ1, respectively, and inject into the liquid chromatograph. Determine the peak size of the amylose-type oligosaccharide trimer, the chrysanthemum-type oligosaccharide 4-mer, and the chrysanthemum according to the external standard method. The content of the starch-type oligosaccharide 5-mer, the inulin-type oligosaccharide 6-mer, the inulin-type oligosaccharide 7-mer, the inulin-type oligosaccharide 8-mer, and the inulin-type oligosaccharide 9-mer The content of the sugar is added, that is, the content of the active ingredient of the Morinda oligosaccharide is obtained.

According to the dry product, the medicinal material of Morinda citrifolia contains not less than 2.0% of the amylose-type oligosaccharide 5-mer (C _3Q H ₅₂ 0 ₂₆ ), and contains the amylose-type oligosaccharide 5-mer (C _{3 ()} H ₅₂ 0 ₂₆ ), not less than 10.0%.

Claims

Rights request

A method for determining the content of Morinda officinalis oligosaccharide, which comprises at least a chitosan-type oligosaccharide 5-mer, and is present in a Chinese herbal medicine or an extract thereof, the method for determining the content comprises :

(1) Determination by high performance liquid chromatography, the chromatographic conditions are carried out using an aqueous solution containing acetonitrile as a mobile phase, using a differential detector or an evaporative light scattering detector;

(2) preparing a reference solution, weighing the amylose-type oligosaccharide 5-mer as a control, adding water or the mobile phase described in step (1) to prepare a reference solution;

(3) Preparation of the test solution, taking the sample of Morinda oligosaccharide, adding water or dissolving the mobile phase in step (1), taking the supernatant, filtering, and taking the filtrate to obtain the test solution;

The scorpion oligosaccharide sample is prepared by the following method, taking the medicinal material of Morinda citrifolia, extracting the extract with water, the extract is an extract of the Chinese medicinal material of Morinda citrifolia, and the extract is subjected to activated carbon column chromatography. First, it was negatively eluted with water to reduce sugar, and then eluted with 20-50% ethanol. The ethanol eluted fraction was collected and concentrated to obtain the Morinda oligosaccharide sample.

(4) Determination, respectively, draw the reference solution and the test solution, inject into the liquid chromatograph, and measure;

(5) Calculate, according to the relative retention time of each chromatographic peak in the test sample and the amyloid 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.

The content determination method according to claim 1, wherein the chromatographic conditions in the step (1) comprise using an amino column or a C18 reverse phase column, and the mobile phase contains 40-80% by volume of acetonitrile.

The content determination method according to claim 1, wherein the mobile phase in the step (1) is a mixed solution of acetonitrile and water in a volume ratio of 3:1 to 1:1.

The content determination method according to claim 1, wherein the Morinda oligosaccharide further comprises any of the oligosaccharides of the inulin oligosaccharide 3 to 4, 6 to 9 or a mixture thereof.

The content determination method according to claim 1, wherein the Morinda oligosaccharide contains a daisy A mixture of a powdered oligosaccharide 5mer, a chrysanthemum-type oligosaccharide 3~4mer, and a chrysanthemum-type oligosaccharide 6-9 polymer.

The content determination method according to claim 1, wherein the mobile phase in the step (1) is a mixed solution of acetonitrile and water in a volume ratio of 68:32, and the mixed solution further contains no more than the flow. A phase volume of 0.5% triethylamine.

The content determination method according to claim 1, wherein the water extract of the step (3) in the method is subjected to activated carbon column chromatography, first eluted with water, then eluted with 30% ethanol, and concentrated and dried. The described Morinda oligosaccharide sample was obtained.

8. The method of determining content according to claim 1, wherein the step (3) of the method comprises:

Take 50-200mg of Morinda oligosaccharide sample, accurately weigh it, put it in a 10ml volumetric flask, dissolve it with water or the mobile phase in step (1), dilute to the mark, filter through microporous membrane, take the filtrate, and obtain Sample solution.