Bighead atractylodes rhizome main homogeneous polysaccharide BP1 standard substance as well as preparation method, detection method and application thereof

Abstract

The invention provides a atractylodes macrocephala main homogeneous polysaccharide BP1 standard substance and a preparation method, a detection method and application thereof, the weight-average molecular weight of the atractylodes macrocephala main homogeneous polysaccharide BP1 standard substance is 3850 + / -300, the atractylodes macrocephala main homogeneous polysaccharide BP1 standard substance is prepared by taking a traditional Chinese medicine atractylodes macrocephala as a raw material, performing water heating extraction, filtering, concentrating, performing alcohol precipitation, deproteinizing, performing cellulose column chromatography crude separation, and eluting and decoloring, and the atractylodes macrocephala main homogeneous polysaccharide BP1 standard substance is obtained. And further separating and purifying through gel column chromatography. The main homogeneous polysaccharide BP1 in the bighead atractylodes rhizome water extract is prepared through extraction, separation and purification, is high in purity and good in stability, can be prepared through repeated separation and purification, and can be subjected to qualitative identification and purity characterization on the molecular weight, monosaccharide composition and retention time; and the polysaccharide is used as a standard substance to establish a high performance liquid phase molecule exclusion chromatography-evaporative light scattering (HPSEC-ELSD) direct quantitative analysis method so as to carry out quantitative determination and quality control on the polysaccharide in the bighead atractylodes rhizome medicinal material.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine polysaccharide analysis, and in particular relates to a main homogeneous polysaccharide BP1 standard product of Atractylodes macrocephala and a preparation method, a detection method and an application thereof. Background Art

[0002] Atractylodes is the dried rhizome of Atractylodes macrocephala Koidz., a plant of the Asteraceae family. It has the effects of strengthening the spleen and replenishing qi, drying dampness and promoting diuresis, stopping sweating, and calming the fetus. The 2020 edition of the Chinese Pharmacopoeia contains two specifications of decoction pieces: raw Atractylodes and bran-fried Atractylodes. The quality control indicators of Atractylodes macrocephala for Atractylodes macrocephala include the qualitative analysis of atractylone by thin layer chromatography, but the content of such fat-soluble components in the water extract of Atractylodes macrocephala is very small and is not suitable as an indicator component for quality control of its polar parts. It is known that the main material basis of the water extract of Atractylodes macrocephala is polysaccharides and oligosaccharides. Atractylodes macrocephala polysaccharides have pharmacological effects such as anti-tumor, immunomodulation, protection of the nervous system, protection of the gastrointestinal mucosa, regulation of intestinal flora, liver protection, and hypoglycemic effects, which are mostly consistent with their clinical application efficacy in traditional Chinese medicine. They are the main active components in the decoction of Atractylodes macrocephala, but the literature reports that the structural composition and molecular weight range of Atractylodes macrocephala polysaccharides are diverse. At present, direct quantitative determination quality control analysis technology for the main homogeneous polysaccharides in Atractylodes macrocephala has not been established.

[0003] Existing quality control technologies for Chinese medicine polysaccharides mostly use phenol-sulfuric acid, anthrone-sulfuric acid and other colorimetric methods to determine the total polysaccharide content extracted from Chinese medicinal materials, or use acid hydrolysis followed by high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) to directly determine the monosaccharide composition and content in the total polysaccharides of Chinese medicine, or use acid hydrolysis followed by PMP derivatization and high performance liquid chromatography-ultraviolet detector (HPLC-UV) to determine the monosaccharide composition and content in the total polysaccharides of Chinese medicine, or use derivatization techniques such as methylation and acetylation after total polysaccharide hydrolysis and use gas chromatography-mass spectrometry (GC-MS) for determination. This type of quantitative quality analysis technology for polysaccharides in Chinese medicinal materials that controls the quality of Chinese medicinal materials by indirectly determining the monosaccharides in the components is not highly specific, and cannot exclude the influence of monosaccharides and oligosaccharides in the total polysaccharides. It is impossible to control the specific active polysaccharides and their content in Chinese medicine, and most of them have poor operability and repeatability in specific implementation. Summary of the invention

[0004] In view of this, the present invention aims to propose a standard product of the main homogeneous polysaccharide BP1 of Atractylodes macrocephala and its preparation method, detection method and application. The main homogeneous polysaccharide BP1 in the heated water extract of Atractylodes macrocephala is prepared by repeatable separation and is used as a standard product for quantitative determination of Atractylodes macrocephala medicinal materials and their extracts, thereby providing a more objective indicator component for the quality control of the water-soluble parts of Atractylodes macrocephala.

[0005] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0006] In a first aspect, the present invention provides a method for detecting atractylodes macrocephala, the method comprising the following steps:

[0007] (1) A main homogeneous polysaccharide BP1 of Atractylodes macrocephala is used as a standard to prepare a standard solution. The main homogeneous polysaccharide BP1 of Atractylodes macrocephala is extracted from Atractylodes macrocephala by heating with water, followed by filtration, concentration, alcohol precipitation, protein removal, crude separation by cellulose column chromatography, elution and color removal, and then separation and purification by gel column chromatography;

[0008] (2) Prepare the test solution; weigh the decoction pieces or powder of Atractylodes macrocephala, decoct in water / heat under reflux for extraction, filter, concentrate, and dry the precipitated part with 80% ethanol to obtain a crude polysaccharide powder. Accurately weigh an appropriate amount of the powder and dissolve it in pure water to obtain the test solution.

[0009] (3) The standard solution and the test solution were subjected to high performance liquid chromatography, and the polysaccharide BP1 of the test sample was qualitatively identified based on the peak elution time of the standard and the test sample, and the content of the main homogeneous polysaccharide BP1 of Atractylodes macrocephala in the test sample was calculated by the external standard method.

[0010] Furthermore, the conditions for the HPLC detection are as follows: the chromatographic column is TSKgel G3000PW XL Chromatographic column; mobile phase: ultrapure water; flow rate 0.2-0.8 mL / min, for example, 0.2 mL / min, 0.3 mL / min, 0.4 mL / min, 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, column temperature 25-40°C, for example, 25°C, 28°C, 30°C, 32°C, 35°C, 38°C, 40°C; detector is ELSD detector; drift tube temperature 60-80°C, for example, 60°C, 65°C, 70°C, 75°C, 80°C, carrier gas flow rate Gain: 7, injection volume 8-14 μl, for example, 8 μl, 9 μl, 10 μl, 11 μl, 12 μl, 13 μl, 14 μl; analysis time 20-40 min, for example, 20 min, 25 min, 30 min, 35 min, 40 min.

[0011] Furthermore, the column flow rate was 0.5 mL / min, the column temperature was 27° C., the ELSD detector drift tube temperature was 70° C., the carrier gas flow rate Gain: 7, the injection volume was 10 μl, and the analysis time was 30 min.

[0012] Furthermore, the concentration of the standard solution is 0.5-4 mg / mL, for example, it can be 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL; preferably, the concentration of the test solution is 3-5 mg / mL, for example, it can be 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL.

[0013] Furthermore, the method for preparing the test solution includes the following steps: mixing the Atractylodes macrocephala decoction pieces or powder with water, performing heating reflux extraction or water decoction, filtering the water extract or decoction, precipitating with alcohol, and drying to obtain a crude polysaccharide dry powder, accurately weighing an appropriate amount of the dry powder, and diluting it to a certain concentration with ultrapure water to obtain the test solution.

[0014] Furthermore, the dosage ratio of the test sample to water is 1:10-200, for example, 1:10, 1:30, 1:50, 1:80, 1:100, 1:120, 1:150, 1:180, 1:200, the water extraction temperature is 100° C., and the water extraction time is 0.5-1.5 h.

[0015] Furthermore, the retention time of the main homogeneous polysaccharide BP1 of Atractylodes macrocephala was about 16.2 min, the linear range was 5-40 μg, the detection limit and quantification limit were 1.3 and 2.5 μg respectively, the RSD of precision, stability and repeatability tests were all <5%, and the recoveries of the three kinds of addition amounts were all in the range of 90-110%. The content of homogeneous polysaccharide BP1 of Atractylodes macrocephala in Atractylodes macrocephala from different origins was measured by the water heating reflux extraction preparation method of medicinal material powder, which was greater than 10%.

[0016] In a second aspect, the present invention provides a method for preparing a homogeneous polysaccharide BP1 of Atractylodes macrocephala, the preparation method comprising the following steps:

[0017] a. Heat the water of Atractylodes macrocephala to extract by reflux and filter to obtain a water extract;

[0018] b. After concentrating the water extract, add ethanol, stir until precipitation is complete, refrigerate overnight, filter, and dry to obtain crude polysaccharide after water extraction and alcohol precipitation of Atractylodes macrocephala; preferably, the final concentration of ethanol is about 80%;

[0019] c. preparing a crude polysaccharide aqueous solution of Atractylodes macrocephala, removing protein from the crude polysaccharide aqueous solution, concentrating and drying the aqueous solution to obtain the deproteinized crude polysaccharide;

[0020] d. preparing a crude polysaccharide aqueous solution after removing protein, subjecting it to cellulose column chromatography, eluting and removing color, concentrating and drying to obtain Atractylodes macrocephala polysaccharide;

[0021] e. Prepare an aqueous solution of Atractylodes macrocephala polysaccharide, perform gel column chromatography, elute until the absorbance of the eluate is close to 0, combine the eluate flow intervals with strong absorbance near normal distribution, concentrate and dry to obtain the main homogeneous polysaccharide BP1 of Atractylodes macrocephala.

[0022] Furthermore, in step a, the dosage ratio of Atractylodes macrocephala to water is 1:5-10, for example, it can be 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, and the heating reflux extraction temperature is 90-100°C, for example, it can be 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, 100°C; preferably, the number of extractions is more than 2 times, and the extraction time for each time is 0.5-2h.

[0023] Furthermore, the concentration of the crude Atractylodes polysaccharide aqueous solution in step c is 30-100 mg / mL; preferably, the reagent used for protein removal is a mixed solution of chloroform and n-butanol; further preferably, the usage ratio of the crude Atractylodes polysaccharide aqueous solution, chloroform and n-butanol is 20-30:3-8:1.

[0024] In a third aspect, the present invention provides a main homogeneous polysaccharide BP1 of Atractylodes macrocephala obtained by the preparation method described in the second aspect; preferably, the weight average molecular weight of the main homogeneous polysaccharide BP1 of Atractylodes macrocephala is 3850±300.

[0025] Furthermore, the monosaccharide composition of the main homogeneous polysaccharide BP1 of Atractylodes macrocephala includes glucosamine, mannose, glucose, galactose, arabinose and rhamnose.

[0026] Furthermore, the main homogeneous polysaccharide BP1 of Atractylodes macrocephala is obtained by using Atractylodes macrocephala as raw material, and then subjected to water heating extraction, filtration, concentration, alcohol precipitation, protein removal, crude separation by cellulose column chromatography, elution and color removal, and then separation and purification by gel column chromatography.

[0027] Furthermore, the Atractylodes macrocephala medicinal material is selected from Atractylodes macrocephala decoction pieces or medicinal material powder.

[0028] Furthermore, the purity of the homogeneous polysaccharide BP1 of Atractylodes macrocephala was greater than 98% by high performance liquid phase size exclusion chromatography-differential detection (HPSEC-RID) or high performance liquid phase size exclusion chromatography-evaporative light scattering detection (HPSEC-ELSD) analysis technology. The weight average molecular weight was calculated to be approximately 3850±300 by HPGPC-RID analysis, which meets the requirements for use as a standard for the detection of Atractylodes macrocephala medicinal materials.

[0029] In a fourth aspect, the present invention provides the use of the main homogeneous polysaccharide BP1 of Atractylodes macrocephala obtained by the detection method of the first aspect or the preparation method of the second aspect or the main homogeneous polysaccharide BP1 of Atractylodes macrocephala as described in the third aspect in the quality evaluation of Atractylodes macrocephala medicinal material.

[0030] Compared with the prior art, the homogeneous polysaccharide BP1 standard of Atractylodes macrocephala and its preparation method, detection method and application described in the present invention have the following advantages:

[0031] The invention prepares the main homogeneous polysaccharide BP1 in the water extract of Atractylodes macrocephala by extraction, separation and purification. The polysaccharide BP1 has high purity and good stability, can be separated and purified repeatedly, and can be qualitatively identified and characterized in purity by measuring its molecular weight and retention time. A high performance molecular exclusion chromatography-evaporative light scattering (HPSEC-ELSD) direct quantitative analysis method is established as a standard product to quantitatively measure and control the quality of the polysaccharide in the Atractylodes macrocephala medicinal material. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0033] Figure 1 This is a schematic diagram of the elution curve of the homogeneous polysaccharide BP1 of Atractylodes macrocephala described in Example 1 of the present invention on a Sephadex G100 column;

[0034] Figure 2 This is a schematic diagram of the HPGPC-RID chromatogram of the homogeneous polysaccharide BP1 of Atractylodes macrocephala described in Example 1 of the present invention;

[0035] Figure 3 This is a schematic diagram of the LC-MS analysis results of the main monosaccharide composition of the homogeneous polysaccharide BP1 of Atractylodes macrocephala described in Example 1 of the present invention, wherein the six peaks from left to right are glucosamine, mannose, rhamnose, glucose, galactose, and arabinose;

[0036] Figure 4 This is a schematic diagram of the LC-MS analysis results of the monosaccharide standard PMP derivative product described in Example 1 of the present invention, wherein 1 is glucosamine, 2 is mannose, 3 is rhamnose, 4 is glucose, 5 is galactose, and 6 is arabinose;

[0037] Figure 5 This is a schematic diagram of the elution curve of the homogeneous polysaccharide BP1 of Atractylodes macrocephala described in Example 2 of the present invention on a Sephadex G100 column;

[0038] Figure 6 This is a schematic diagram of the HPGPC-RID chromatogram of the homogeneous polysaccharide BP1 of Atractylodes macrocephala described in Example 2 of the present invention;

[0039] Figure 7 This is the HPSEC-ELSD quantitative analysis chromatogram described in Example 2 of the present invention (A: negative control, B: BP1 standard, C: representative Atractylodes macrocephala sample). DETAILED DESCRIPTION

[0040] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0041] The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments.

[0042] Example 1

[0043] 1. Purification and preparation of homogeneous polysaccharide BP1 from medicinal materials

[0044] Take 1kg of bran-fried Atractylodes macrocephala slices, add water at a solid-liquid ratio of 1:8, heat and reflux at 100℃ for 2 times, 1h each time, filter and combine the water extracts. After concentration, add ethanol to 80%, stir while adding until the polysaccharide is completely precipitated, put it in a 4℃ cold storage overnight and take it out, filter it and soak the alcohol precipitate residue in a small amount of anhydrous ethanol and centrifuge it, soak and wash the insoluble matter in a small amount of acetone and centrifuge it, and dry the obtained crude polysaccharide in a fume hood with nitrogen to prepare the crude polysaccharide part of Atractylodes macrocephala, weigh 380g, and store it in a -80℃ refrigerator for later use; take 50g of crude Atractylodes macrocephala polysaccharide and prepare a 50mg / mL aqueous solution. The Sevage method was used to remove protein. The solution was poured into a separatory funnel in a ratio of polysaccharide aqueous solution: chloroform: n-butanol (25:4:1), shaken thoroughly for 3 minutes, and then allowed to stand for stratification. The organic layer and the precipitate were discarded, and the protein removal was repeated 5 times. The supernatant was collected, concentrated using a rotary evaporator, re-dissolved in pure water, and freeze-dried to prepare a crude polysaccharide without protein. The crude Atractylodes macrocephala polysaccharide after protein removal was dissolved in a small amount of water, chromatographed on a pretreated DEAE-52 cellulose column (35×ID3cm), and depigmented by elution with ultrapure water at 1 mL / min. The elution was eluted until no sugar was detected in the effluent using the phenol-concentrated sulfuric acid method. The eluate was collected, concentrated using a rotary evaporator, and then freeze-dried. That is, the Atractylodes macrocephala polysaccharide was prepared; the Atractylodes macrocephala polysaccharide was dissolved in pure water, chromatographed on a Sephadex G-100 gel column (100×ID2cm), eluted with pure water, the flow rate was 1mL / min, and the eluate was collected into one tube for every 10mL, and followed and monitored by the universal phenol-concentrated sulfuric acid method until the absorbance of the eluate was zero. The polysaccharide elution curve (such as Figure 1 The BP1 enriched fractions were combined, concentrated, and freeze-dried to obtain pure BP1.

[0045] 2. Molecular weight determination, purity and monosaccharide composition analysis of homogeneous polysaccharide BP1 HPGPC-RID

[0046] Molecular weight determination and purity analysis: High performance liquid chromatograph: Agilent 1260 liquid chromatograph equipped with RID-1200 differential refractive index detector, Japan TOSOH TSK-GEL G4000PWxL molecular exclusion chromatography column: mobile phase: ultrapure water, flow rate: 0.5ml / min, detector temperature: 35℃, column temperature: 40℃ (analysis condition 1). Results The average retention time of the Atractylodes macrocephala polysaccharide BP1 separated and purified under the analysis conditions was 20.35min (e.g. Figure 2 The purity of the polysaccharide standard was calculated by the peak area normalization method to be 98.7%. The retention time of the polysaccharide standard was further determined (as shown in Table 1), and the retention time of the chromatographic peak was used to analyze the purity of the standard M. W Manual calculation of linear regression equation for logarithmic values w =-0.265t R +9.056, r=-0.997, the method calculated the average molecular weight of Atractylodes macrocephala polysaccharide BP1 to be 3580.

[0047] Table 1. Weight average molecular weight retention time of polysaccharide standards HPGPC-RID determination data

[0048]

[0049]

[0050] Analysis of BP1 monosaccharide composition: Weigh 4 mg of homogenized Atractylodes macrocephala polysaccharide BP1 accurately, put it in a 25 mL round-bottom flask, connect a three-way valve and a small balloon, add 4 mL of 4 mol / L trifluoroacetic acid, fill with N2 and seal the tube, heat in an oil bath at 110 ° C for 8 hours to hydrolyze the polysaccharide into monosaccharides. Place it at room temperature, evaporation concentrate, add methanol several times, concentrate until there is no trifluoroacetic acid smell, add 500 μL of 1% acetic acid water to dissolve it again. Accurately draw 50 μL of the polysaccharide hydrolyzate, mix it with 200 μL of ammonia solution and 200 μL of 0.2 mol / L PMP methanol solution in an EP tube. The mixture was reacted in a 70°C constant temperature water bath for 90 min. After cooling, 300 μL of 0.3 mol / L HCL solution was added to neutralize it, and the mixture was concentrated by rotary evaporation. 500 μL of 1% acetic acid water was added to the residue for redissolution, 500 μL of CHCl3, vortexed for 1 min, the CHCl3 organic phase was removed, and the extraction was repeated 3 times. Finally, the supernatant was transferred to a centrifuge tube, centrifuged at high speed for 10 min, and filtered through a 0.22 μm microporous filter. Accurately weigh 1.0 mg of each monosaccharide reference substance, dissolve it in 1 mL of ultrapure water, and prepare a 1 mg / mL stock solution. Accurately transfer 100 μL of each stock solution, dilute to 2.0 mL with ultrapure water, and obtain a monosaccharide standard solution with a mass concentration of 50 μg / mL. Ammonia water was diluted to a mass concentration of 10.0 μg / mL. The PMP derivatization method was the same as above.

[0051] Analytical conditions: AB5500 triple quadrupole LC / MS, positive ion MRM mode, CUR value: 35, CAD value: 6, TEM value 500℃, GS1: 45, GS2: 45, electrospray voltage: 5500V; chromatographic column: Hypersil GOLD C18 column (50mm×2.1mm, 1.9μm); mobile phase: aqueous solution containing 0.5mmol / L ammonium acetate and 0.05% acetic acid-acetonitrile (81:19, V / V); flow rate: 0.25mL / min; column temperature: 35℃; injection volume 3μL.

[0052] The analysis results are as follows Figure 3-4 As shown in Table 2, the main monosaccharide composition of Atractylodes macrocephala homogeneous polysaccharide BP1 includes glucosamine, mannose, glucose, galactose, arabinose, and rhamnose.

[0053] Table 2 Retention time and mass spectrometry detection parameters of PMP derivatives of monosaccharide standards

[0054]

[0055]

[0056] Example 2

[0057] 1. Preparation of homogeneous polysaccharide BP1 from Atractylodes macrocephala powder

[0058] The raw Atractylodes macrocephala was crushed, 150 g of the powder was taken, and water was added at a solid-liquid ratio of 1:25, and the mixture was heated and refluxed at 100°C for 2 times, each time for 1 hour, and the water extract was combined after filtration. After concentration, ethanol was added to 80%, and the mixture was stirred while adding until the polysaccharide was completely precipitated, and the mixture was placed in a 4°C explosion-proof refrigerator for overnight refrigeration, and then taken out. After filtration, a small amount of anhydrous ethanol was added to the alcohol precipitation part, and then centrifuged. The obtained crude polysaccharide was placed in a fume hood and dried with nitrogen to prepare the crude polysaccharide part of Atractylodes macrocephala, and stored in a -20°C refrigerator for later use; 20 g of crude Atractylodes macrocephala polysaccharide was taken and prepared into a 40 mg / mL aqueous solution. The solution was poured into a separatory funnel in a ratio of polysaccharide aqueous solution: chloroform: n-butanol (25:4:1), shaken thoroughly for 3 minutes, and then allowed to stand to separate the layers. The organic layer and the precipitate were discarded, and the protein removal was repeated 5 times. The supernatant was collected, concentrated using a rotary evaporator, re-dissolved in pure water, and freeze-dried to prepare a crude polysaccharide without protein. The crude Atractylodes macrocephala polysaccharide after protein removal was dissolved in a small amount of water, chromatographed on a pretreated DEAE-52 cellulose column (50×ID 5cm), and depigmented by elution with ultrapure water at 2.5mL / 1min. The elution was eluted until no sugar was detected in the effluent using the phenol-concentrated sulfuric acid method. The eluate was collected, concentrated using a rotary evaporator, and then freeze-dried. That is, the Atractylodes macrocephala polysaccharide is prepared; the Atractylodes macrocephala polysaccharide is dissolved in pure water, eluted with pure water, eluted at normal pressure, and the eluate is collected in one tube for every 10 mL, and followed and monitored by the phenol-concentrated sulfuric acid method until the absorbance of the eluate is zero. The eluate in the tube is diluted 10 times, and detected by the phenol-concentrated sulfuric acid method, with the detection tube number as the horizontal axis and the absorbance as the vertical axis, and the polysaccharide elution curve (such as Figure 5 The BP1 enriched fractions were combined, concentrated, and freeze-dried to obtain pure BP1.

[0059] 2. Molecular weight determination and purity analysis of homogeneous polysaccharide BP1 by HPGPC-RID

[0060] High performance liquid chromatograph: Agilent 1260 liquid chromatograph with RID-1200 differential refractometer, Japan TOSOH TSK-GEL G3000 PWxL (7μm, 7.8*300mm) molecular exclusion chromatography column: mobile phase: 0.1% NaCl, flow rate: 0.4mL / min, analysis time 40min, detector temperature: 35°C, column temperature: 25°C (analysis condition 2). Accurately weigh 1mg of Atractylodes macrocephala polysaccharide BP1 standard, add 1ml of ultrapure water, ultrasonicate for 10min, and prepare a 1mg / mL standard solution after complete dissolution for sampling and analysis. Under this elution condition, the retention time of BP1 is about 20.8min, and its purity is calculated by peak area normalization method to be 98.5% (such as Figure 6 shown).

[0061] Preparation of dextran standard solution: weigh 1 mg of standard solution with molecular weight of 4300, 5300, 6230, 7200, 9200, 17900, 41100, 45800, 603006, dissolve in 1 mL ultrapure water, and sample after high-speed centrifugation for 10 minutes. Inject 10 μL and collect the chromatogram. Draw the "logMw-tR" curve with the logarithm of the molecular weight of the standard dextran logMw as the ordinate and the retention time tR as the abscissa. Use BreezeGPC system software to perform regression fitting on the curve to obtain the dextran GPC calibration curve equation lgM w =-0.265t R +9.056, r=0.994, after the purified BP1 was substituted into the dextran curve, the mass average molecular weight of BP1 was calculated to be 3617, the number average molecular weight was 3324, the weight average molecular weight was 3887, and the polydispersity was 1.17.

[0062] 3. Determination of the content of Atractylodes macrocephala homogenate polysaccharide BP1 by HPSEC-ELSD

[0063] 3.1. Instruments and test drugs

[0064] 3.1.1 Instruments

[0065] Agilent 1260LC high performance liquid chromatograph, equipped with G4318A evaporative light scattering detector (Agilent Technologies, Inc.), MS105DU 1 / 100000 electronic balance (Mettler Toledo, Switzerland), 30B2 herbal medicine decoction pot (Chaozhou Kangyashun Electric), Microfuge 16 desktop microcentrifuge (Beckman Coulter, USA), RODI-220B1 water purifier (Rui Sijie Water Purification Technology Co., Ltd.).

[0066] 3.1.2 Medicinal materials and reagents

[0067] 13 batches of Atractylodes macrocephala extracts (7 batches of raw Atractylodes macrocephala, 6 batches of bran-fried Atractylodes macrocephala) were purchased from the pharmacy of Guangdong Provincial Hospital of Traditional Chinese Medicine, and 15 batches of powdered Atractylodes macrocephala extracts (13 batches of raw Atractylodes macrocephala, 2 batches of bran-fried Atractylodes macrocephala) were provided by Jiangyin Tianjiang Pharmaceutical (specific origin and batch information are shown in Tables 4 and 5). The main homogeneous polysaccharide BP1 of Atractylodes macrocephala was purified and prepared in the laboratory, and its weight average molecular weight was calculated by HPSEC-RID analysis and was about 3800, and the analytical purity was greater than 98%, meeting the quantitative purity requirements of the standard product.

[0068] 3.2. Methods and results

[0069] 3.2.1 Chromatographic conditions

[0070] TSKgel G3000PW XL(7μm, 7.8*300mm) chromatographic column; mobile phase: ultrapure water; flow rate 0.5mL / min, column temperature 27℃; ELSD detector drift tube temperature 70℃, carrier gas flow rate (Gain:7), injection volume 10μl; analysis time 30min (analysis condition 3).

[0071] 3.2.2 Solution preparation

[0072] 3.2.2.1 Preparation of test solution

[0073] Weigh 50g of 13 batches of Atractylodes macrocephala slices, soak in 10 times the volume of water for 30 minutes, and then boil in an automatic decoction pot for 1 hour. Weigh 50g of 15 batches of Atractylodes macrocephala, crush, pass through a 60-mesh sieve, mix, accurately weigh 0.1g of powder, and heat and reflux extract at 100℃ with 200 times the volume of water for 1 hour. Filter the medicinal slices and powder water extracts, freeze-dry, and calculate the yield. About 0.5 g of the lyophilized powder of each decoction was weighed, dissolved in 10 mL of ultrapure water, and 40 mL of pure ethanol was added. The mixture was mixed well, allowed to stand overnight at 4°C, and then centrifuged. The supernatant was removed, and the alcohol precipitate was dried with nitrogen gas. The yield was calculated as shown in Tables 4 and 5. About 20 mg of the lyophilized powder of each medicinal material decoction was weighed, dissolved in 0.4 mL of ultrapure water, and 1.6 mL of pure ethanol was added. The mixture was mixed well, allowed to stand overnight at 4°C, and then centrifuged. The supernatant was removed, and the alcohol precipitate was dried with nitrogen gas. The yield was calculated as shown in Tables 4 and 5. 4 mg of the water-extracted alcohol precipitate was accurately weighed, dissolved in 1 mL of ultrapure water, and centrifuged at 13,000 rpm. The supernatant was taken for sampling and determination.

[0074] 3.2.2.2 Preparation of reference solution

[0075] Accurately weigh 20 mg of homogenized Atractylodes macrocephala polysaccharide BP1, dissolve it in 2 mL of ultrapure water to prepare a 10 mg / mL stock solution, and dilute it with ultrapure water to prepare a series of standard solutions with concentrations of 4, 3.5, 3, 2.5, 2, 1.5, 1, and 0.5 mg / mL.

[0076] 3.2.3 Methodological validation

[0077] 3.2.3.1 Specificity Verification

[0078] The solution prepared under 2.2, the test sample (batch number: 212003252), the polysaccharide standard, and the negative control solution (pure water) were subjected to chromatographic detection. Figure 7 Under the analytical conditions, the retention time of the BP1 standard was about 16.2 min, with a single symmetrical peak shape and no interference from impurity peaks.

[0079] 3.2.3.2 Limit of detection and limit of quantification

[0080] Take the polysaccharide BP1 reference solution (1 mg / mL) under "3.2.2.2", dilute it step by step with ultrapure water to prepare a series of mass concentrations, inject and analyze according to the chromatographic conditions under "3.2.1", and record the peak height H of the chromatographic peak. When the signal-to-noise ratio is about 3:1, the detection limit concentration is 0.125 mg / mL (1.3 μg), and when the signal-to-noise ratio is about 10:1, the quantification limit concentration is 0.250 mg / mL (2.5 μg).

[0081] 3.2.3.3 Investigation of linear relationship

[0082] The reference solution under 3.2.2 was injected according to the conditions under 3.2.1, and linear regression was performed with the chromatographic peak area as the ordinate (y) and the injection volume as the abscissa (x). The linear regression equation of the two was y=342.8x-1268(R 2 =0.9933), indicating that BP1 has a good linear relationship in the mass range of 5-40 μg.

[0083] 3.2.3.4 Precision test

[0084] The reference solution (c = 1.5 mg / mL) under "3.2.2.2" was injected 6 times according to "3.2.1", and the peak area was measured and the retention time was calculated. The RSD of the BP1 peak area was 1.5%, and the RSD of the retention time was 4.1%, indicating that the instrument used for the determination had high precision.

[0085] 3.2.3.5 Stability test

[0086] Take a sample of Atractylodes macrocephala and prepare the test solution according to the method under "3.2.2.1". Samples were injected and tested at 0, 2, 4, 8, 12, and 24 hours after preparation. The RSD of the target peak area was 0.2%, indicating that the test solution was stable within 24 hours.

[0087] 3.2.3.6 Repeatability test

[0088] Weigh 6 samples of Atractylodes macrocephala powder from the same batch, process them according to the powder extraction method under "3.2.2.1", and then test them. The peak area is determined, and the RSD of the BP1 peak area is 0.6%, indicating that the method has good repeatability.

[0089] 3.2.3.7 Sample recovery test

[0090] Weigh 0.05g of sample powder of known content from 9 batches of the same batch, and treat it according to the powder extraction method under "3.2.2.1". Take the equivalent of 0.85mg BP1 solution, add 50%, 100%, and 150% of the content to the corresponding reference substance BP1, prepare 3 tests in parallel, and calculate the recovery rate. The results are shown in Table 3. The recovery rates of low, medium, and high additions are 106.4%, 97.0%, and 99.6%, respectively, and the RSDs are 0.2%, 0.6%, and 0.6%, respectively (n=3).

[0091] Table 3 Recovery of BP1 at low, medium and high levels (n=3)

[0092]

[0093] 3.2.3.8 Determination of Atractylodes macrocephala samples

[0094] A total of 28 batches of samples of Atractylodes macrocephala slices and powder were taken, and the test solution was prepared according to the method under "3.2.2.1". The quantitative determination results of Atractylodes macrocephala slices are shown in Table 4, and the quantitative determination results of Atractylodes macrocephala powder are shown in Table 5. The homogeneous polysaccharide BP1 extracted from Atractylodes macrocephala by the traditional decoction method in clinical water decoction varies from 1.6% to 12.2% in terms of medicinal material content, with a content difference of more than 5 times. After the medicinal material was crushed, 200 times the amount of hot water was refluxed and extracted, and the homogeneous polysaccharide BP1 extracted from Atractylodes macrocephala varied from 13.2% to 34.7% in terms of medicinal material content, with a content difference of about 2.5 times. This shows that the difference in polysaccharide BP1 content of Atractylodes macrocephala itself should be within 3 times, but the polysaccharide BP1 content of Atractylodes macrocephala slices from different origins varies more under the traditional decoction method, which may affect the clinical efficacy. Under the condition of sufficient extraction of medicinal material powder by heating and reflux, the content of homogeneous polysaccharide BP1 of different batches of medicinal materials does not differ by more than 3 times, indicating that this preparation method is more suitable for the quality evaluation of Atractylodes macrocephala. The content of BP1 measured in all analyzed batches of Atractylodes macrocephala powder is greater than 10%, and the maximum medicinal material content is as high as 34.7%, indicating that the content of this polysaccharide in Atractylodes macrocephala is very large, and it is suitable for the quality evaluation of Atractylodes macrocephala, extracts and related preparations.

[0095] Table 4 Information of 13 batches of Atractylodes macrocephala slices, water decoction extraction and alcohol precipitation yields, and BP1 content

[0096]

[0097] Table 5 Information of 15 batches of Atractylodes macrocephala powder, water reflux extraction yield and BP1 content

[0098]

[0099]

[0100] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work shall all fall within the scope of protection of the present invention.

Claims

1. A method for detecting Atractylodes macrocephala, characterized in that: The detection method comprises the following steps: (1) A main homogeneous polysaccharide BP1 of Atractylodes macrocephala is used as a standard to prepare a standard solution. The main homogeneous polysaccharide BP1 of Atractylodes macrocephala is extracted from Atractylodes macrocephala by heating with water, followed by filtration, concentration, alcohol precipitation, protein removal, crude separation by cellulose column chromatography, elution and color removal, and then separation and purification by gel column chromatography; (2) Prepare the test solution; (3) The standard solution and the test solution were subjected to high performance liquid exclusion chromatography-evaporative light scattering detection to obtain the chromatograms of the standard and test solutions, respectively. The test sample polysaccharide BP1 was qualitatively identified based on the peak elution time of the standard and test sample, and the content of the main homogeneous polysaccharide BP1 of Atractylodes macrocephala in the test sample was calculated by the external standard method.

2. The detection method according to claim 1, characterized in that: The conditions for the HPLC detection are as follows: the chromatographic column is TSKgel G3000PW XL Chromatographic column; mobile phase: ultrapure water; flow rate 0.2-0.8 mL / min, column temperature 25-40°C; detector is ELSD detector; drift tube temperature 60-80°C, carrier gas flow rate Gain: 7, injection volume 8-14 μl; analysis time 20-40 min.

3. The detection method according to claim 1, characterized in that: The concentration of the standard solution is 0.5-4 mg / mL; preferably, the concentration of the test solution is 3-5 mg / mL.

4. The detection method according to claim 1, characterized in that: The preparation method of the test solution comprises the following steps: mixing the decoction pieces or powder of Atractylodes macrocephala with water, performing heating reflux extraction or water decoction, filtering the water extract or decoction, precipitating with alcohol, and drying to obtain a crude polysaccharide dry powder, accurately weighing an appropriate amount of the dry powder, and diluting it with pure water to a certain concentration to obtain a test solution.

5. The detection method according to claim 4, characterized in that: The dosage ratio of Atractylodes macrocephala slices or powder to water is 1:10-200, the water extraction temperature is 100°C, and the extraction time is 0.5-1.5h.

6. A method for preparing a homogeneous polysaccharide BP1 from Atractylodes macrocephala, characterized in that: The preparation method comprises the following steps: a. Heat the water of Atractylodes macrocephala to extract by reflux and filter to obtain a water extract; b. After concentrating the water extract, add ethanol, stir until precipitation is complete, refrigerate overnight, filter, and dry to obtain crude polysaccharide after water extraction and alcohol precipitation of Atractylodes macrocephala; c. preparing a crude polysaccharide aqueous solution of Atractylodes macrocephala, removing protein from the crude polysaccharide aqueous solution, concentrating and drying the aqueous solution to obtain the deproteinized crude polysaccharide; d. preparing a crude polysaccharide aqueous solution after removing protein, subjecting it to cellulose column chromatography, eluting and removing color, concentrating and drying to obtain Atractylodes macrocephala polysaccharide; e. Prepare an aqueous solution of Atractylodes macrocephala polysaccharide, perform gel column chromatography and elution, combine the eluate with strong absorbance near normal distribution into interval segments, concentrate and dry to obtain the main homogeneous polysaccharide BP1 of Atractylodes macrocephala.

7. The preparation method according to claim 6, characterized in that: In step a, the dosage ratio of Atractylodes macrocephala to water is 1:5-10, and the heating reflux extraction temperature is 90-100° C.; preferably, the extraction times are more than 2 times, and the extraction time for each time is 0.5-2 hours.

8. The preparation method according to claim 6, characterized in that: The concentration of the aqueous solution of crude Atractylodes macrocephala polysaccharide in step c is 30-100 mg / mL; preferably, the protein is removed by the Sevage method using a mixed solution of chloroform and n-butanol in a volume ratio of 4:

1.

9. Atractylodes macrocephala main homogeneous polysaccharide BP1 obtained according to the preparation method according to any one of claims 6-8; preferably, the weight average molecular weight of the Atractylodes macrocephala main homogeneous polysaccharide BP1 is 3850±300.

10. Use of the detection method according to any one of claims 1 to 5, or the preparation method according to any one of claims 6 to 8, or the main homogeneous polysaccharide BP1 of Atractylodes macrocephala according to claim 9 in the quality evaluation of Atractylodes macrocephala medicinal material.

Images