Method for determining content of effective components in radix morindae officinalis oligosaccharide

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for determining the content of effective components in a radix morindae officinalis oligosaccharide capsule. The determination method comprises the following steps: preparing a test solution, preparing a reference solution, taking the test solution and the reference solution by adopting a hydrophilic interaction chromatography-ultra-high performance liquid chromatography-differential refraction detector technology, injecting a sample, and determining. The determination method has high separation degree and detection sensitivity on oligosaccharide substances, shortens the analysis time when analyzing the effective components in the radix morindae officinalis oligosaccharide, does not need derivatization pretreatment operation before determination, reduces the use of an organic solvent and the waste of raw materials, improves the detection efficiency, is simple and convenient to operate and stable in operation, and is suitable for industrial production. And the automatic control of the liquid chromatogram can be ensured.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for determining the content of effective ingredients in Morinda officinalis oligosaccharide capsules. Background Art

[0002] Morinda officinalis How., a precious Chinese herbal medicine, has been used as a traditional herbal medicine and nutritional supplement for more than 2,000 years. Its inherent effects of nourishing the liver and kidneys and strengthening the tendons and bones make it widely used in the treatment of various chronic diseases, such as impotence, osteoporosis, diabetes and Alzheimer's disease. Morinda officinalis oligosaccharides are a type of inulin-type oligosaccharides (3≤ polymerization degree≤9) extracted from the roots of Morinda officinalis. Its main component structure is characterized by linear fructose unit chains of different lengths (connected by β-(2→1)-D-fructosyl-fructose bonds) and terminated with a single glucose residue (connected by α-D-pyranose glucopyranosyl bonds).

[0003] It is reported that Morinda officinalis oligosaccharides can alleviate the depressive-like behavior of chronic unpredictable mild stimulation (CUMS) rats, repair intestinal epithelial damage, reduce the levels of corticosterone in plasma and urine, and restore intestinal dysbiosis; another important study found that Morinda officinalis oligosaccharides can increase the level of tryptophan hydroxylase in the intestinal microbial flora, promote the synthesis of 5-hydroxytryptophan (5-HTP) from tryptophan, inhibit the activity of 5-HTP decarboxylase, reduce the production of 5-hydroxytryptamine (5-HT), and further promote the accumulation of 5-HTP. After being absorbed by the blood, 5-HTP in the intestine indirectly increases the level of 5-HT in the brain through the blood-brain barrier, thereby exerting a significant antidepressant effect.

[0004] Morinda officinalis oligosaccharide capsules (MOOC) are well tolerated by healthy Chinese subjects. Clinical trials have confirmed that MOOC is safe within the prescribed therapeutic dose and its efficacy is not inferior to fluoxetine. A recent meta-analysis report, based on 7 studies with a total of 1,384 participants, fully confirmed that MOOC is as effective as conventional antidepressants in treating mild to moderate depressive disorders, providing a new direction for the selection of clinical antidepressants. At present, with the medical popularization and market development of MOOC, more stringent requirements have been put forward for its quality control and safety monitoring. However, there has been no relevant report on the determination of MOOC content so far. A mature and complete analytical detection method is urgently needed to optimize the existing quality evaluation system and provide scientific guidance for the industrial development, effectiveness evaluation and rational clinical application of MOOC.

[0005] As we all know, conventional detection methods for oligosaccharides mainly include: (1) chemical analysis, such as Fehling's method, potassium permanganate method, Lan Ainong method, potassium ferrocyanide method, etc.; (2) chromatographic analysis, such as liquid chromatography, gas chromatography, thin layer chromatography, etc.; (3) mass spectrometry; (4) capillary electrophoresis. For MOOC, its capsules also contain microcrystalline cellulose excipients and other sugar substances. If chemical analysis is used, they will show the same color reaction as the oligosaccharide components, which will inevitably interfere with the detection results; if gas chromatography is used, the low volatility of sugar substances and the complicated operation process will greatly affect the detection efficiency; if thin layer chromatography is used, the separation effect of sugars with a large degree of polymerization is difficult to achieve the expected effect; in addition, sugar compounds themselves are difficult to ionize, have no ultraviolet absorption and fluorescent groups, and the use of mass spectrometry and capillary electrophoresis will inevitably avoid complex derivatization pretreatment processes. Therefore, liquid chromatography, which is convenient to operate, fast to analyze, and highly specific, has become the preferred method for quantitative detection of oligosaccharides.

[0006] When using liquid chromatography, the most common operation is to first effectively separate the main components in the mixture, and then find the standard reference substances of each component in the test substance, and use this as a reference to establish a standard curve, so as to calculate the actual content of each component. For MOOC, the main components, oligosaccharide monomers with a degree of polymerization of 3 to 9, have similar physical and chemical properties, which brings challenges to the initial separation and detection technology. In addition, the standard reference substances of each oligosaccharide in MOOC are not easily available or convenient to prepare. Following the traditional content determination method will face the difficulties of high cost, cumbersome steps and rare standards. Therefore, the quality control of MOOC completed by convention will be idealistic and out of touch with reality. Summary of the invention

[0007] Therefore, the technical problem to be solved by the present invention is to overcome the defects of the prior art such as cumbersome methods for determining the content of active ingredients in Morinda officinalis oligosaccharides and difficulty in separation due to similar physicochemical properties of the active ingredients, thereby providing a method for determining the content of active ingredients in Morinda officinalis oligosaccharide capsules.

[0008] To this end, the present invention provides the following technical solutions.

[0009] The present invention provides a method for determining the content of effective ingredients in Morinda officinalis oligosaccharides, comprising the following steps:

[0010] Prepare test sample solution;

[0011] Prepare reference solution;

[0012] Determination: Hydrophilic interaction chromatography-ultra-performance liquid chromatography-differential refractive index detector technology was used to take the test solution and the reference solution, inject and determine; the chromatographic conditions were: Waters ACQUITY BEH Amide (50 mm × 2.1 mm; 1.7 μm) was used as the chromatographic column, 68% acetonitrile aqueous solution was used as the mobile phase, isocratic elution, flow rate was 0.3 ± 0.05 ml / min, column temperature was 30 ± 0.2 °C, detector temperature was 35 ± 0.2 °C, sample temperature was 15 ± 0.2 °C; running time: the running time of the reference solution was 5 min, and the running time of the test solution was 20 min;

[0013] The content of oligosaccharide 5-glycan in the test sample was determined by external standard method.

[0014] The total concentration of oligosaccharides 3 to 9 in the test sample is obtained by formula 1 and formula 2;

[0015]

[0016]

[0017] Among them, DP5 is oligosaccharide 5;

[0018] DP3-9 oligosaccharides refer to oligosaccharide trisaccharides, oligosaccharide tetrasaccharides, oligosaccharide pentasaccharides, oligosaccharide hexasaccharides, oligosaccharide septasaccharides, oligosaccharide nonasaccharides and oligosaccharide 9-glycans.

[0019] When the external standard method is used for determination, the concentration of oligosaccharide 5 in the reference solution is 0.125-6 mg / ml.

[0020] The dosage form of the Morinda officinalis test sample is capsule type.

[0021] The preparation steps of the test sample solution include: taking the test sample, adding a solvent, and preparing the test sample solution.

[0022] The content of DP5 in the test solution is 0.125-6 mg / ml.

[0023] The content of DP3-9 oligosaccharides in the test solution is 1.25-60 mg / ml.

[0024] The effective ingredients in the Morinda officinalis oligosaccharide include oligosaccharide 5-glycan.

[0025] The effective ingredients in the Morinda officinalis oligosaccharide also include at least one of oligosaccharide tri-glycan, oligosaccharide tetra-glycan, oligosaccharide hexa-glycan, oligosaccharide 7-glycan, oligosaccharide 8-glycan and oligosaccharide 9-glycan.

[0026] The technical solution of the present invention has the following advantages:

[0027] 1. The method for determining the content of effective ingredients in Morinda officinalis oligosaccharides provided by the present invention comprises the preparation of a test solution and a reference solution, adopts hydrophilic interaction chromatography-ultra-high performance liquid chromatography-differential refractive index detector technology, takes a test solution and a reference solution, injects and determines. The method has high separation and detection sensitivity for oligosaccharide substances, shortens the analysis time when analyzing the effective ingredients in Morinda officinalis oligosaccharides, does not require derivatization pretreatment operations before determination, reduces the use of organic solvents and the waste of raw materials, improves the detection efficiency, is easy to operate, runs stably, and can ensure the realization of automatic control of liquid chromatography. Further, the present invention overcomes the problem of difficulty in separating 7 oligosaccharide DP3-9 components with similar structures and similar physical and chemical properties in Morinda officinalis oligosaccharides in the prior art, and the separation of each component is good in a relatively short analysis time.

[0028] The determination method provided by the present invention has accurate and stable determination results for the total sugar content of DP5 and DP3-9, good reproducibility and strong applicability.

[0029] 2. The method for determining the content of active ingredients in Morinda officinalis oligosaccharides provided by the present invention "maps" the relative content of each active ingredient DP3-4 and DP6-9 in Morinda officinalis oligosaccharides with the content of a single reference substance, thereby smoothly realizing the quality assessment of the preparation and the optimization of the quality control system, reducing the use of compound standards, simplifying the operation steps, and saving the detection cost. The present invention uses DP5 as a reference, which accounts for the largest proportion in DP3-9, is a quality marker and a major active ingredient, has the same core structure as other oligosaccharides, and has a moderate retention time, peak order, peak height, and peak area, which can reduce the systematic error during content determination and calculation. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 This is the characteristic spectrum of Morinda officinalis oligosaccharides in Example 1 of the present invention. DETAILED DESCRIPTION

[0032] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0033] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0034] Drug testing:

[0035] The mass content of Morinda officinalis oligosaccharide 5-glycan in the Morinda officinalis oligosaccharide 5-glycan reference substance is 94.2%, which is corrected during weighing, and the corrected weight = weighed weight × content.

[0036] The labeled content of Morinda officinalis oligosaccharide capsules: the mass of each Morinda officinalis oligosaccharide capsule is 300 mg, the content of Morinda officinalis oligosaccharide in each Morinda officinalis oligosaccharide capsule is 150 mg, the content of Morinda officinalis oligosaccharide 5-glycan is 15.0 mg, the content of total Morinda officinalis oligosaccharide (DP3~DP9) is 82 mg, and the concentration of the prepared solution is calculated based on Morinda officinalis oligosaccharide.

[0037] Example 1

[0038] This embodiment provides a method for determining the content of active ingredients in Morinda officinalis oligosaccharides, comprising the following steps:

[0039] Test solution: Take Morinda officinalis oligosaccharide capsules, accurately weigh, add water, and prepare a test solution containing 3 mg of Morinda officinalis oligosaccharide per 1 ml of solution.

[0040] Morinda officinalis oligosaccharide 5-glycan reference solution: Take the Morinda officinalis oligosaccharide 5-glycan reference solution, place it in a centrifuge tube, add ultrapure water to dilute it, and obtain a Morinda officinalis oligosaccharide 5-glycan reference solution with a concentration of 10 mg / ml.

[0041] Determination: Hydrophilic interaction chromatography-ultra-performance liquid chromatography-differential refractive index detector technology was used to take the test solution and the reference solution, inject and determine; the chromatographic conditions were: Waters ACQUITY BEH Amide (50mm×2.1mm; 1.7μm) was used as the chromatographic column, 68% acetonitrile aqueous solution was used as the mobile phase, isocratic elution, the flow rate was 0.3ml / min, the column temperature was 30±0.2℃, the detector temperature was 35±0.2℃, and the sample temperature was 15±0.2℃; running time: the running time of the reference solution was 5min, and the running time of the test solution was 20min; the injection volume was 1μL.

[0042] The content of oligosaccharide 5-glycan in the test sample was determined by external standard method.

[0043] Figure 1The chromatographic peaks of the characteristic spectrum of Morinda officinalis oligosaccharides are determined in sequence according to the peak order and relative retention time, with the retention time of DP5 as the benchmark. The retention time of DP3-4 and DP6-9 becomes longer as the degree of polymerization increases. The DP5 chromatographic peak is determined first, and the two adjacent chromatographic peaks on the left are DP3 and DP4, and the four adjacent chromatographic peaks on the right are DP6-9. There is no impurity peak between the DP3-DP9 chromatographic peaks. Among them, taking DP5 as the reference peak, the specified values ​​of the relative retention time of DP3-4 and DP6-9 are 0.66, 0.81, 1.23, 1.54, 1.92, and 2.40, respectively, and the relative retention time is within ±10% of the specified value.

[0044] The total concentration of oligosaccharides 3 to 9 in the test sample was calculated according to formula 1 and formula 2:

[0045]

[0046]

[0047] Among them, DP5 is oligosaccharide 5 glycan; the peak area of ​​DP5 is measured by the above method, and the measured concentration of DP5 is measured by the external standard method; when the external standard method is used for determination, the concentration of oligosaccharide 5 glycan in the standard solution is 0.125-6 mg / mL.

[0048] DP3-9 oligosaccharides refer to oligosaccharide trisaccharides, oligosaccharide tetrasaccharides, oligosaccharide pentasaccharides, oligosaccharide hexasaccharides, oligosaccharide septasaccharides, oligosaccharide nonasaccharides and oligosaccharide 9-glycans.

[0049] Take Morinda Officinalis Oligosaccharide Capsules according to the method of Example 1 to prepare 5 test solutions, and place them in sample bottles for standby use, and test them under conditions 1-5, respectively. Conditions 1-5 are: Condition 1 is to test the test solution 0h after preparation; Condition 2 is to test the test solution after preparation, and place it in the dark at 2-8°C for 1 day; Condition 3 is to test the test solution after preparation, and place it in the dark at 2-8°C for 4 days; Condition 4 is to test the test solution after preparation, and place it in the dark at 2-8°C for 10 days; Condition 5 is to test the test solution after preparation, first place it in the dark at 2-8°C for 11 days, and then place it in the dark at room temperature for 8h before testing, numbered 1-5, and the test solution was sampled three times for each condition for testing, from different positions of the sample bottle, namely, sampling from the bottom of the sample bottle, sampling from the middle of the sample bottle, and sampling from the top of the sample bottle, and measured according to Example 1. The test results are shown in the table below.

[0050] Table 1 Retention time of total sugar DP3-9 contained in Morinda officinalis oligosaccharide preparation

[0051]

[0052]

[0053] Table 2 Relative retention time of total sugars DP3-9 contained in Morinda officinalis oligosaccharide preparation

[0054]

[0055] According to the above results, the prescribed values ​​of the relative retention times of DP3 to DP4 and DP6 to DP9 are 0.66, 0.81, 1.23, 1.54, 1.92 and 2.40, respectively, and the relative retention times are within ±10% of the prescribed values.

[0056] Experimental Example 1

[0057] (1) Investigation of stability

[0058] Test solution 1: Prepare a test solution 1 with a concentration of 3 mg / ml according to Example 1;

[0059] Test solution 2: Prepare a test solution 2 with a concentration of 20 mg / ml according to the method of Example 1;

[0060] Each test solution was sampled three times, with the sampling positions being the bottom of the sample bottle, the middle of the sample bottle, and the top of the sample bottle. The average of the three measurement results was taken as the theoretical concentration of DP5 and DP3-9. The test results are shown in the table below.

[0061] Table 3 Theoretical concentration of DP5 concentration and DP3-9 total sugar in test solution 1-2

[0062]

[0063] The stability of the test solution 1 and the test solution 2 was investigated under different conditions, and the test conditions were as follows: condition 1 was to test the test solution 0h after preparation; condition 2 was to test the test solution after preparation and to place it in the dark at 2-8°C for 1 day; condition 3 was to test the test solution after preparation and to place it in the dark at 2-8°C for 4 days; condition 4 was to test the test solution after preparation and to place it in the dark at 2-8°C for 10 days; condition 5 was to test the test solution after preparation and to place it in the dark at 2-8°C for 11 days, and then to place it in the dark at room temperature for 8h; condition 6 was to test the test solution after preparation and to place it in the dark at room temperature for 4h. Each test solution was sampled three times, and the sampling positions were sampling at the bottom of the sample bottle, sampling at the middle of the sample bottle, and sampling at the top of the sample bottle, respectively, and the results were shown in the table below according to Example 1.

[0064] Table 4 Results of DP5 content determination and stability analysis in test solution 1

[0065]

[0066]

[0067] Table 5 Results of DP5 content determination and stability analysis in test solution 2

[0068]

[0069]

[0070] Table 6 Determination of the content of DP3-9 total sugars in the test solution 1 and the results of stability analysis

[0071]

[0072] Table 7 Determination of total sugar content and stability analysis results of DP3-9 total sugar in test solution 2

[0073]

[0074]

[0075] The above results show that: the recovery rates R(%) of the measured concentrations of the test solution 1 and the test solution 2 and the theoretical concentrations obtained in Table 3 are all within 100±15%, and the precision RSD≤10%; under different conditions, the recovery rates R(%) of the measured concentrations of DP5 and DP3-9 in the test solution 1 and the test solution 2 and their theoretical concentrations are all within 100±15%, and the deviations are all within ±10% when compared with the concentrations measured at 0h. Therefore, the method of the present invention can accurately, reliably and stably determine the content of DP3-9 in Morinda officinalis oligosaccharides, and then effectively and quickly evaluate and control its production quality.

[0076] Experimental Example 2

[0077] (1) Investigation of standard solution concentration

[0078] Prepare DP5 linear standard solutions with concentrations of 0.125, 0.25, 0.5, 1, 2, 4 and 6 mg / mL, respectively, inject according to Example 1, record the peak area, represented by Y, and perform linear regression using the least squares method to establish a standard curve, r 2 is the correlation coefficient, the regression equation is Y=aX+b, and the experiment was repeated three times. The results show that the three measurements r 2 The values ​​were 0.999904, 0.999939 and 0.999906, all of which were greater than 0.990000, and the accuracy of each point on the standard curve was between 90% and 110%, indicating that DP5 had a good linear relationship in the concentration range of 0.125 to 6 mg / mL.

[0079] Table 8 Linear range verification results of Morinda officinalis oligosaccharide 5 glycan external standard method

[0080]

[0081] (2) Investigation of accuracy and precision

[0082] Morinda officinalis oligosaccharide 5 glycan was prepared into Morinda officinalis oligosaccharide 5 glycan reference solution with concentrations of 0.375, 1.5, and 5 mg / mL respectively according to the method of Example 1. Three parallel experiments were set for the test solution at each concentration, and the measured concentration of Morinda officinalis oligosaccharide 5 glycan was obtained according to Example 1. The above experiment was repeated twice, and the first experiment was recorded as the first batch, the second experiment was recorded as the second batch, and the third experiment was recorded as the third batch.

[0083] The recovery rate of the measured concentration of Morinda officinalis oligosaccharide 5 glycan relative to the theoretical concentration was calculated to examine the average recovery rate of the same batch; the intra-batch RSD of the measured concentration relative to the theoretical concentration was calculated to examine the precision; the recovery rate of the measured concentration relative to the theoretical concentration was calculated to examine the average recovery rate between different batches, and the inter-batch RSD of the measured concentration relative to the theoretical concentration was calculated to examine the inter-batch precision. The results are shown in the table below. Among them, the theoretical concentration refers to the preparation concentration of the Morinda officinalis oligosaccharide 5 glycan reference solution.

[0084] The results showed that the intra-batch accuracy of different concentrations of Morinda officinalis oligosaccharide 5-glycan reference solution within the same batch ranged from 95.47% to 103.20%, and the intra-batch precision RSD ranged from 0.15% to 1.20%; the inter-batch accuracy of the same concentration between different batches ranged from 98.21% to 99.49%, and the inter-batch precision RSD ranged from 1.23% to 3.16%, indicating that the accuracy and precision of this method were good.

[0085] Table 9 Verification results of accuracy and precision of Morinda officinalis oligosaccharide 5 glycan external standard method

[0086]

[0087] (3) Verification of the minimum limit of quantification of Morinda officinalis oligosaccharide 5-glycan reference solution

[0088] According to Example 1, 3 portions of DP5 solutions with a concentration of 0.125 mg / mL were prepared for Morinda officinalis oligosaccharide 5 polysaccharide; the above experiment was repeated twice, and a total of 9 groups of experiments were obtained. The first experiment was recorded as the first batch, the second experiment was recorded as the second batch, and the third experiment was recorded as the third batch. The results of the same batch of measurements were used to evaluate the intra-batch accuracy and precision, and the results of the three different batches of measurements were used to evaluate the inter-batch accuracy and precision. The results are shown in the table below.

[0089] The results showed that the intra-batch accuracy ranged from 96.80% to 108.80%, and the intra-batch precision RSD was 0.47% to 1.49%; the inter-batch accuracy was 100.89%, and the inter-batch precision RSD was 4.78%.

[0090] Table 10 Verification results of the minimum limit of quantification of Morinda officinalis oligosaccharide 5 glycans by external standard method

[0091]

[0092] (4) Specificity inspection

[0093] According to the method of Example 1, the peak areas of the interference peaks of the mobile phase, blank auxiliary material solution and ultrapure water at the DP5 retention time were measured, and the results are shown in the following table. Wherein, the mobile phase refers to 68% acetonitrile aqueous solution, and the blank auxiliary material refers to microcrystalline cellulose.

[0094] The results show that the peak areas of the mobile phase, blank auxiliary material solution and ultrapure water are all less than 5% of the peak area of ​​the DP5 solution with a concentration of 0.125 mg / ml, indicating that the method has good specificity.

[0095] Table 11 Specificity verification results of Morinda officinalis oligosaccharide 5 glycans by external standard method

[0096]

[0097] (5) System suitability investigation

[0098] At the beginning, during and at the end of the system injection, 1 mg / mL DP5 standard solution was injected respectively, which was recorded as the first injection, the second injection, and the third injection. The Empower workstation calculated the theoretical plate number n and tailing factor of the chromatographic column and evaluated the system drift degree. The results are shown in the table below. The above experiment was repeated twice, the first experiment was recorded as the first batch, the second experiment was recorded as the second batch, and the third experiment was recorded as the third batch.

[0099] The results showed that the RSD% of the retention time of the DP5 chromatographic peaks of the three batches were 0.03%, 0.03% and 0.11%, respectively, and the RSD% of the peak areas were 0.52%, 0.35% and 0.17%, respectively. The tailing factors were all less than 2.0, and the theoretical plate numbers were all greater than 2000, indicating that the method had good system adaptability.

[0100] Table 12 Results of the system suitability investigation of Morinda officinalis oligosaccharide 5 glycan external standard method

[0101]

[0102] (6) Verification of the maximum quantitative limit of Morinda officinalis oligosaccharide 5-glycan reference solution

[0103] After the injection of the 6 mg / ml DP5 standard solution, the mobile phase blank solution (68% acetonitrile aqueous solution) was injected to detect the residual amount of the 6 mg / ml DP5 standard solution. The 6 mg / ml DP5 standard solution was injected three times, and the blank solution was used to detect the residual amount. The results are shown in the table below.

[0104] The results show that the peak area of ​​the blank solution at the chromatographic peak of different batches of DP5 standard solutions does not exceed 20% of the peak area of ​​Morinda officinalis oligosaccharide 5 glycan.

[0105] Table 13 Morinda officinalis oligosaccharide 5-glycan residue detection by external standard method

[0106]

[0107] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the scope of protection of the invention.

Claims

1. A method for determining the content of active ingredients in Morinda officinalis oligosaccharides, characterized in that: The following steps are involved: Prepare test sample solution; Prepare reference solution; Determination: Hydrophilic interaction chromatography-ultra-performance liquid chromatography-differential refractive index detector technology was used to take the test solution and the reference solution, inject and determine; the chromatographic conditions were: Waters ACQUITY BEH Amide (50 mm × 2.1 mm; 1.7 μm) was used as the chromatographic column, 68% acetonitrile aqueous solution was used as the mobile phase, isocratic elution, flow rate was 0.3 ± 0.05 ml / min, column temperature was 30 ± 0.2 °C, detector temperature was 35 ± 0.2 °C, sample temperature was 15 ± 0.2 °C; running time: the running time of the reference solution was 5 min, and the running time of the test solution was 20 min; The content of oligosaccharide 5-glycan in the test sample was determined by external standard method.

2. The measuring method according to claim 1, characterized in that The total concentration of oligosaccharides 3 to 9 in the test sample is obtained by formula 1 and formula 2; Among them, DP5 is oligosaccharide 5; DP3-9 oligosaccharides refer to oligosaccharide trisaccharides, oligosaccharide tetrasaccharides, oligosaccharide pentasaccharides, oligosaccharide hexasaccharides, oligosaccharide septasaccharides, oligosaccharide nonasaccharides and oligosaccharide 9-glycans.

3. The measuring method according to claim 1 or 2, characterized in that When the external standard method is used for determination, the concentration of oligosaccharide 5 in the reference solution is 0.125-6 mg / ml.

4. The assay method according to any one of claims 1 to 3, characterized in that The dosage form of the Morinda officinalis test sample is capsule type.

5. The assay method according to any one of claims 1 to 4, characterized in that The preparation steps of the test sample solution include: taking the test sample, adding a solvent, and preparing the test sample solution.

6. The measuring method according to claim 5, characterized in that The content of DP5 in the test solution is 0.125-6 mg / ml.

7. The measuring method according to claim 5, characterized in that The content of DP3-9 oligosaccharides in the test solution is 1.25-60 mg / ml.

8. The measuring method according to claim 1, characterized in that The effective ingredients in the Morinda officinalis oligosaccharide include oligosaccharide 5-glycan.

9. The measuring method according to claim 1 or 8, characterized in that The effective ingredients in the Morinda officinalis oligosaccharide also include at least one of oligosaccharide tri-glycan, oligosaccharide tetra-glycan, oligosaccharide hexa-glycan, oligosaccharide 7-glycan, oligosaccharide 8-glycan and oligosaccharide 9-glycan.

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