CN110954642A - Detection method of grifola quality and construction method of fingerprint spectrum thereof - Google Patents
Detection method of grifola quality and construction method of fingerprint spectrum thereof Download PDFInfo
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Abstract
The invention discloses a construction method of a grifola fingerprint, a detection method of grifola quality, a method for detecting monosaccharide composition of grifola polysaccharide and a separation method of grifola polysaccharide extract. The detection method of the grifola quality of the invention applies the grifola fingerprint spectrum which can reflect the monosaccharide composition characteristics of the grifola polysaccharide, and can effectively detect and analyze the quality of the grifola decoction and the formula granules thereof. The method has the advantages of simple operation, stability, good reproducibility, high detection efficiency and strong practicability.
Description
Technical Field
The invention relates to the field of quality analysis, in particular to a grifola quality detection method and a fingerprint spectrum construction method thereof.
Background
Polyporus umbellatus is dried sclerotia of Polyporus umbellatus (Pers.) Fr. of Polyporaceae, has sweet, light and neutral taste, has effects of promoting diuresis and eliminating dampness, and can be used for treating dysuresia, edema, diarrhea, stranguria with turbid urine, leucorrhea, etc., and also has many applications as medicinal and edible product in health food.
The main chemical components of the polyporus umbellatus are polysaccharide and steroid components, and modern researches believe that: polyporus polysaccharide has effects of resisting tumor and improving immunity, and its immunostimulation effect is similar to TLR4Activation of a signal path; ergosterol and ergosterone are the main active ingredients of diuresis. In 2015, ergosterol is only used as a content determination item (the content standard is less than one per thousand) under the item of grifola in the section of Chinese pharmacopoeia of the edition of 2015, and most of traditional Chinese medicine formulas adopt a water decoction method, so that ergosterol can hardly be detected in a water decoction of grifola and formula particles thereof, and the inherent quality of the grifola is difficult to effectively evaluate. The existing analytical method for polysaccharide is mostly measured by phenol-concentrated sulfuric acid color-rendering spectrophotometry, and the method has poor specificity and is difficult to be used as an effective quality control method.
Wuyou et al, applying a monosaccharide PMP derivatization and a capillary electrophoresis method to detect monosaccharide composition of the polyporus umbellatus polysaccharide (Wuyou et al, analyzing the monosaccharide composition in the polyporus umbellatus polysaccharide by a high-efficiency capillary electrophoresis method, Nature science of Heilongjiang university, 2013,30(2): 220-224), only 4 monosaccharides are identified, which are not enough to characterize the characteristics of the polyporus umbellatus polysaccharide; compared with a capillary electrophoresis method, the high performance liquid chromatography is more generally applied and has stronger practicability.
In the existing research, in order to extract more chemical components of the traditional Chinese medicine as much as possible, an organic solvent extraction mode is often adopted for quality control; the use of water-soluble ingredients as quality control has been less studied. The total polysaccharide content of the polysaccharides is generally measured by a phenol-sulfuric acid method, and the characteristics cannot be characterized (for example, see content analysis of wild polyporus polysaccharide and ergosterol in different producing areas, plum blossom, and the like, Chinese wild plant resources, 2014,33 (4): 11-16).
Disclosure of Invention
The invention aims to solve the technical problem that an effective detection method aiming at the grifola quality is lacked in the prior art, and provides a grifola fingerprint spectrum construction method, a grifola quality detection method, a method for detecting monosaccharide composition of grifola polysaccharide and a grifola polysaccharide extract separation method. The detection method of the grifola quality of the invention applies the grifola fingerprint spectrum which can reflect the monosaccharide composition characteristics of the grifola polysaccharide, and can effectively detect and analyze the quality of the grifola decoction and the formula granules thereof. The method has the advantages of simple operation, stability, good reproducibility, high detection efficiency and strong practicability.
The invention collects more than twenty batches of polyporus umbellatus medicinal materials in different producing areas, researches monosaccharide compositions of water-soluble polysaccharide of the polyporus umbellatus medicinal materials one by adopting a water decoction method, improves the detection sensitivity of HPLC (high performance liquid chromatography) by derivatization of the monosaccharide, and grops a large number of experiments to find out the mobile phase proportion of the HPLC, thereby better realizing analysis of the monosaccharide compositions of the polyporus umbellatus.
In order to solve the technical problems, the invention aims to provide a construction method of a grifola fingerprint, which comprises the following steps: (1) detecting a sample solution of the polyporus umbellatus by using high performance liquid chromatography, and (2) generating a fingerprint spectrum according to a detection result;
the chromatographic column of the high performance liquid chromatography is a reverse phase column;
the high performance liquid chromatography is carried out isocratic elution by using acetonitrile-phosphate buffer solution with the volume ratio of mobile phase of 15:85 to 20: 80;
the preparation of the test solution comprises the following steps: decocting Polyporus umbellatus water-soluble crude polysaccharide with water to obtain decoction; hydrolyzing the obtained crude polysaccharide with acid, performing derivatization treatment with 1-phenyl-3-methyl-5-pyrazolone, and taking a water layer to fix the volume after extraction as a test solution.
Preferably, the reverse phase column is a silica gel bonded phase chromatographic column, preferably an octadecylsilane bonded chromatographic column or an octylsilane bonded silica gel chromatographic column silica gel column.
Preferably, the pH value of the phosphate buffer solution is 6.5-7.0, and preferably 6.80.
Preferably, the concentration of the phosphate buffer solution is 30-50 mmol/L, preferably 50 mmol/L.
Preferably, the phosphate buffer solution is KH2PO4NaOH buffer solution.
Preferably, the flow rate of the mobile phase is 0.8-1.2 ml/min, preferably 1 ml/min.
Preferably, the temperature of the reverse phase column is 25-30 ℃, preferably 30 ℃.
Preferably, the volume ratio of the mobile phase is 18:82 or 17.5: 82.5.
Preferably, the detection wavelength of the high performance liquid chromatography is 240-250 nm, and preferably 250 nm.
Preferably, the sample injection volume of the high performance liquid chromatography is 10-20 μ L, and preferably 10 μ L.
Preferably, the length of the octadecylsilane chemically bonded chromatographic column is 150-250 mm, and preferably 250 mm; and/or the inner diameter of the octadecylsilane chemically bonded chromatographic column is 2.1-4.6 mm, preferably 4.6 mm; and/or the particle size of the filler of the octadecylsilane chemically bonded chromatographic column is 3-5 μm, preferably 5 μm; more preferably, the octadecylsilane bonded chromatography column is Agilent extended C18, 4.6 × 250mm, 5 μm.
Preferably, the water decoction method comprises the steps of: (1) decocting the polyporus umbellatus with water to obtain a sample, and (2) carrying out post-treatment on the sample obtained in the step (1);
the post-treatment is carried out by one of the following methods: concentrating or dissolving the sample in water, mixing with ethanol, precipitating, and freeze drying to obtain water-soluble crude Polyporus polysaccharide, and drying with microporous membrane but not ultrafiltration membrane to obtain crude Polyporus polysaccharide;
more preferably, the volume of the ethanol accounts for 80-90% of the total volume of the mixed solution.
Preferably, the acid hydrolysis is a step of heating after mixing an acidic solution and the obtained crude polysaccharide, the heating mode is preferably a water bath, and the heating temperature is preferably 90-100 ℃, and more preferably 90 ℃; the heating time is preferably 4-6 h, and more preferably 6 h; the acidic solution is preferably hydrochloric acid, trifluoroacetic acid or sulfuric acid.
Preferably, the derivatization treatment comprises the steps of mixing the obtained mixture with a sodium hydroxide solution and a methanol solution of 1-phenyl-3-methyl-5-pyrazolone, and then heating, wherein the volume ratio of the methanol solution of 1-phenyl-3-methyl-5-pyrazolone to the sodium hydroxide solution is preferably greater than or equal to 1, the heating mode is preferably water bath, and the heating temperature is preferably 60-70 ℃, and more preferably 70 ℃; the heating time is preferably 30-40 min, and more preferably 30 min.
Preferably, the extractant for extraction is chloroform.
In order to solve the technical problems, the invention aims to provide a method for detecting the quality of polyporus umbellatus, the method is to generate a fingerprint of a polyporus umbellatus sample to be detected according to the construction method of claim 1, the obtained fingerprint has 8 characteristic peaks, 7 characteristic peaks are mannose, ribose, rhamnose, glucose, galactose, xylose and fucose, and the polyporus umbellatus sample to be detected is a qualified product.
Preferably, relative retention times of characteristic peaks of mannose, ribose, rhamnose, glucose, galactose, xylose and fucose are respectively 0.50 +/-5% min, 0.64 +/-5% min, 0.69 +/-5% min, 1.00 +/-5% min, 1.14 +/-5% min, 1.21 +/-5% min and 1.44 +/-5% min by taking the characteristic peak of glucose as a control.
Preferably, the polyporus umbellatus sample to be detected is a polyporus umbellatus decoction or a polyporus umbellatus formula particle.
In order to solve the technical problems, the invention aims to provide a method for detecting monosaccharide composition of polyporus polysaccharide, which is a test solution for detecting polyporus by using high performance liquid chromatography;
the chromatographic column of the high performance liquid chromatography is a reverse phase column;
the high performance liquid chromatography is carried out isocratic elution by using acetonitrile-phosphate buffer solution with the volume ratio of mobile phase of 15:85 to 20: 80;
the preparation of the test solution comprises the following steps: decocting Polyporus umbellatus water-soluble crude polysaccharide with water to obtain decoction; hydrolyzing the obtained crude polysaccharide with acid, performing derivatization treatment with 1-phenyl-3-methyl-5-pyrazolone, and taking a water layer to fix the volume after extraction as a test solution.
Preferably, the reverse phase column is a silica gel bonded phase chromatographic column, preferably an octadecylsilane bonded chromatographic column or an octylsilane bonded silica gel chromatographic column silica gel column.
Preferably, the pH value of the phosphate buffer solution is 6.5-7.0, and preferably 6.80.
Preferably, the concentration of the phosphate buffer solution is 30-50 mmol/L, preferably 50 mmol/L.
Preferably, the phosphate buffer solution is KH2PO4NaOH buffer solution.
Preferably, the flow rate of the mobile phase is 0.8-1.2 ml/min, preferably 1 ml/min.
Preferably, the temperature of the reverse phase column is 25-30 ℃, preferably 30 ℃.
Preferably, the detection wavelength of the high performance liquid chromatography is 240-250 nm, and preferably 250 nm.
Preferably, the volume ratio of the mobile phase is 18:82 or 17.5: 82.5.
Preferably, the sample injection volume of the high performance liquid chromatography is 10-20 μ L, and preferably 10 μ L.
Preferably, the length of the octadecylsilane chemically bonded chromatographic column is 150-250 mm, and preferably 250 mm; and/or the inner diameter of the octadecylsilane chemically bonded chromatographic column is 2.1-4.6 mm, preferably 4.6 mm; and/or the particle size of the filler of the octadecylsilane chemically bonded chromatographic column is 3-5 μm, preferably 5 μm;
more preferably, the octadecylsilane bonded chromatography column is Agilent extended C18, 4.6 × 250mm, 5 μm.
Preferably, the water decoction method comprises the steps of: (1) decocting the polyporus umbellatus with water to obtain a sample, and (2) carrying out post-treatment on the sample obtained in the step (1);
the post-treatment is carried out by one of the following methods: concentrating or dissolving the sample in water, mixing with ethanol, precipitating, and freeze drying to obtain water-soluble crude Polyporus polysaccharide, and drying with microporous membrane but not ultrafiltration membrane to obtain crude Polyporus polysaccharide;
more preferably, the volume of the ethanol accounts for 80-90% of the total volume of the mixed solution.
Preferably, the acid hydrolysis is a step of heating after mixing an acidic solution and the obtained crude polysaccharide, the heating mode is preferably a water bath, and the heating temperature is preferably 90-100 ℃, and more preferably 90 ℃; the heating time is preferably 4-6 h, and more preferably 6 h; the acidic solution is preferably hydrochloric acid, trifluoroacetic acid or sulfuric acid.
Preferably, the derivatization treatment comprises the steps of mixing the obtained mixture with a sodium hydroxide solution and a methanol solution of 1-phenyl-3-methyl-5-pyrazolone, and then heating, wherein the volume ratio of the methanol solution of 1-phenyl-3-methyl-5-pyrazolone to the sodium hydroxide solution is preferably greater than or equal to 1, the heating mode is preferably water bath, and the heating temperature is preferably 60-70 ℃, and more preferably 70 ℃; the heating time is preferably 30-40 min, and more preferably 30 min.
Preferably, the extractant for extraction is chloroform.
In order to solve the technical problems, the invention aims to provide a separation method of polyporus polysaccharide extract, which comprises the steps of carrying out acid hydrolysis on polyporus water-soluble crude polysaccharide obtained by a water decoction method, and then carrying out column chromatography separation; the column chromatography is performed isocratic elution using acetonitrile-phosphate buffer solution in a volume ratio of 15:85 to 20: 80.
Preferably, the chromatographic column used for the column chromatography is a reverse phase column.
Preferably, the pH value of the phosphate buffer solution is 6.5-7.0, and preferably 6.80.
Preferably, the concentration of the phosphate buffer solution is 30-50 mmol/L, preferably 50 mmol/L.
Preferably, the phosphate buffer solution is KH2PO4NaOH buffer solution.
Preferably, the volume ratio of the mobile phase is 18:82 or 17.5: 82.5.
Preferably, the water decoction method comprises the steps of: (1) decocting the polyporus umbellatus with water to obtain a sample, and (2) carrying out post-treatment on the sample obtained in the step (1);
the post-treatment is carried out by one of the following methods: concentrating or dissolving the sample in water, mixing with ethanol, precipitating, and freeze drying to obtain water-soluble crude Polyporus polysaccharide, and drying with microporous membrane but not ultrafiltration membrane to obtain crude Polyporus polysaccharide;
more preferably, the volume of the ethanol accounts for 80-90% of the total volume of the mixed solution.
The polyporus umbellatus is a traditional Chinese medicinal material polyporus umbellatus in the field, for example, genuine medicinal materials purchased from various places in China.
The methanol solution of the 1-phenyl-3-methyl-5-pyrazolone (PMP) is conventional in the field, and mainly the 1-phenyl-3-methyl-5-pyrazolone (PMP) is dissolved in the methanol solution. The concentration is conventional in the art, e.g., 0.5 mol/L.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows: the detection method of the grifola quality of the invention applies the grifola fingerprint spectrum which can reflect the monosaccharide composition characteristics of the grifola polysaccharide, and can effectively detect and analyze the quality of the grifola decoction and the formula granules thereof. The construction method of the grifola fingerprint, the detection method of the grifola quality, the method for detecting the monosaccharide composition of the grifola polysaccharide and the separation method of the grifola polysaccharide extract have the advantages of simple operation, stability, good reproducibility, high detection efficiency and strong practicability.
Drawings
FIG. 1A and FIG. 1B are HPLC chromatogram of monosaccharide composition of water-soluble polysaccharide of Polyporus umbellatus; FIG. 1A is a chromatogram under the conditions of example 5, and FIG. 1B is a chromatogram under the conditions of example 6; in the chromatograms of the two, peak 1 is mannose, peak 3 is ribose, peak 4 is rhamnose, peak 5 is glucose, peak 6 is galactose, peak 7 is xylose, and peak 8 is fucose.
FIG. 2 is an HPLC chromatogram of different monosaccharide references and their mixed references; wherein peak 1 is mannose (Man), peak 3 is ribose (Rib), peak 4 is rhamnose (Rha), peak 5 is glucose (Glc), peak 6 is galactose (Gal), peak 7 is xylose (Xyl), and peak 8 is fucose (Fuc); the numbers here correspond to the peaks numbered 1, 3, 4, 5, 6, 7, 8 of the umbellate pore fungus sample, and since no peak 2 is not identified in the umbellate pore fungus sample, no peak 2 is present in the monosaccharide reference profile.
FIG. 3 shows HPLC finger prints of monosaccharide composition of 20 batches of water-soluble Polyporus umbellatus polysaccharide in different production places.
FIG. 4 is a fingerprint of 20 batches of Polyporus umbellatus constructed in different producing areas, wherein S1-S20 correspond to batch numbers ZL-001-ZL-020 of Chinese medicinal materials in Table 1, respectively.
FIG. 5 is an HPLC chromatogram under the conditions of comparative example 1; wherein peak 1 is mannose, peak 2 is ribose, peak 3 is glucose, peak 4 is galactose, and peak 5 is fucose.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Example 1: preparation of water-soluble polyporus polysaccharide
Taking 50g of polyporus umbellatus, adding 450ml of water to soak for about 30-60 min, decocting for 30min, and filtering; adding 350ml water into the residue, decocting for 25min, filtering, mixing the two filtrates, concentrating under reduced pressure to 50ml, slowly stirring, adding 200ml anhydrous ethanol, standing overnight at 4 deg.C in refrigerator, centrifuging, precipitating, and drying to obtain Polyporus water-soluble crude polysaccharide.
Example 2: preparation of water-soluble polyporus polysaccharide
Taking 50g of polyporus umbellatus, adding 450ml of water to soak for about 30-60 min, decocting for 30min, and filtering; decocting the residue with 350ml water for 25min, filtering, mixing the filtrates, filtering with 0.5 μm microporous membrane, collecting the part retained by 10nm ultrafiltration membrane, and drying to obtain water-soluble crude polysaccharide of Polyporus umbellatus.
Example 3: preparation of test solution for analyzing monosaccharide composition fingerprint of water-soluble polyporus umbellatus polysaccharide
Weighing the prepared water-soluble crude polyporus umbellatus polysaccharide about 20mg, adding 5ml of 4mol/L HCl solution, and performing water bath at 90 ℃ for 6 h; taking out, adding appropriate amount of methanol, concentrating under reduced pressure, repeating the operation for several times to remove hydrochloric acid, adding water into the residue to constant volume to 1ml to obtain monosaccharide hydrolysate for use; absorbing 100 mu L of monosaccharide hydrolysate, adding 50 mu L of 0.3mol/L NaOH solution, adding 60 mu L of 0.5 mol/L1-phenyl-3-methyl-5-pyrazolone (PMP) methanol solution, carrying out water bath at 70 ℃ for 30min, taking out, cooling to room temperature, and adding 50 mu L of 0.3mol/L HCl solution for neutralization. Adding appropriate amount of chloroform, extracting for several times, and collecting the water layer to a constant volume of 1ml to obtain a sample solution.
Example 4: preparation of reference solutions of different monosaccharide references
Preparation of control solutions of different monosaccharide references: respectively weighing D-galactose (D-Gal; batch No. 840215; Shanghai reagent II plant); d-mannose (D-Man, batch No. F20120107, chemical reagents of national drug group, Ltd.); l-rhamnose (L-Rha, batch No. 839801, Shanghai reagent II plant); d-xylose (D-Xyl, Lot 820115, Shanghai reagent II); anhydrous glucose (Glc, lot 20070115, national drug group chemical agents ltd); d-ribose (D-Rib, batch No. 1451793, SIGMA); 10mg of each of D-fucose (D-Fuc, 050M1909, SIGMA) was dissolved in water to prepare a monosaccharide stock solution of 1 mg/ml. Precisely transferring 100 μ L of each monosaccharide stock solution, adding 50 μ L of 0.3mol/L NaOH solution, adding 60 μ L of 0.5mol/L PMP methanol solution, water-bathing at 70 deg.C for 30min, taking out, cooling to room temperature, and adding 50 μ L of 0.3mol/L HCl solution for neutralization. Adding appropriate amount of chloroform, extracting for several times, and collecting water layer to desired volume of 1ml to obtain monosaccharide derivatization reference substance solution (with concentration of about 0.1 mg/ml).
Preparation of mixed reference solution: transferring 10mg of each monosaccharide, adding water to a constant volume of 10ml to prepare a mixed reference substance storage solution of 1mg/ml, precisely transferring 100 mu L of the mixed reference substance storage solution, adding 50 mu L of 0.3mol/L NaOH solution, adding 60 mu L of 0.5mol/L PMP methanol solution, carrying out water bath at 70 ℃ for 30min, taking out, cooling to room temperature, adding 50 mu L of 0.3mol/L HCl solution, and neutralizing. Adding appropriate amount of chloroform, extracting for several times, and collecting the water layer to a constant volume of 1ml to obtain mixed reference solution (with concentration of about 0.015 mg/ml).
The HPLC chromatographic conditions for the different monosaccharide references and their mixed references are shown in example 5, namely: an ExtendC18 chromatographic column (5 μm, 4.6X 250mm, Agilent) was taken, acetonitrile-50 mmol/L phosphate buffer water (pH 6.80) (17.5:82.5) was used as a mobile phase, the flow rate was 1.0ml/min, the detection wavelength was 250nm, and the column temperature: 30 ℃; the injection volume was 10. mu.l. The results are shown in FIG. 2.
Example 5: polyporus umbellatus water-soluble polysaccharide monosaccharide composition fingerprint analysis liquid chromatography system 1
An Extend C18 chromatographic column (5 μm, 4.6X 250mm, Agilent) was used as a mobile phase with acetonitrile-50 mmol/L (concentration of phosphate buffer before mixing) phosphate buffer solution (pH 6.80) (17.5:82.5, V/V), wherein the phosphate buffer solution is KH2PO4NaOH buffer, flow rate 1.0ml/min, detection wavelength 250nm, column temperature: 30 ℃; the injection volume was 10. mu.l.
A test solution for fingerprint analysis of monosaccharide composition of water-soluble polysaccharide of Polyporus umbellatus purchased from decoction piece Co., Ltd, product number 1803002, in Anhui Shitian, was prepared as shown in examples 1 and 3. The HPLC results are shown in FIG. 1A. The figure shows that 8 characteristic peaks can be well separated.
Example 6: liquid chromatography system for analyzing monosaccharide composition fingerprint of water-soluble polyporus umbellatus polysaccharide 2
Taking an Extend C18 chromatographic column (5 μm, 4.6 × 250mm, Agilent), and taking acetonitrile-50 mmol/L (concentration of phosphate buffer solution before mixing) phosphate buffer solution water (pH 6.80) (18:82, V/V) as a mobile phase, wherein the phosphate buffer solution is KH2PO4NaOH buffer, flow rate 1.0ml/min, detection wavelength 250nm, column temperature: 30 ℃; the injection volume was 10. mu.l.
A test solution for fingerprint analysis of monosaccharide composition of water-soluble polysaccharide of Polyporus umbellatus, purchased from Shanghai Lei Shang pharmaceutical industry, West district Co., Ltd., product number 170315-1, was prepared as shown in examples 1 and 3. The HPLC results are shown in FIG. 1B. The figure shows that 8 characteristic peaks can be well separated.
Example 7: fingerprint establishment and identification of common characteristic peaks for composition of 20 batches of polyporus umbellatus water-soluble polysaccharide monosaccharides
Taking 20 batches of polyporus umbellatus samples, preparing a test solution for analyzing the monosaccharide composition fingerprint of the polyporus umbellatus water-soluble polysaccharide according to the preparation methods of the embodiment 1 and the embodiment 3, detecting the prepared test solution according to the condition of the embodiment 5, and obtaining the result of HPLC fingerprint as shown in figure 3. The chromatograms of the obtained 20 batches of Polyporus umbellatus samples chromatogram were introduced into traditional Chinese medicine chromatogram fingerprint similarity evaluation system (2004A) in AIA format for similarity evaluation, and the obtained Polyporus umbellatus control fingerprint is shown in FIG. 4. Determining 8 characteristic peaks, comparing with the monosaccharide reference substance in FIG. 2, and identifying that 7 peaks are: mannose, ribose, rhamnose, glucose, galactose, xylose, fucose. The retention times of the 8 common peaks, relative retention times against glucose (peak 5 in fig. 4), peak area averages are summarized as follows:
the peak 1 is mannose, the average retention time RT is 10.463min, the relative retention time is 0.50min, the average peak area is 10.793mAU, and the RSD% value is 44.67%
The peak 3 is ribose, the average retention time RT is 13.512min, the relative retention time is 0.64min, the average peak area is 2.758mAU, and the RSD% value is 55.12%
The No. 5 peak is glucose, the average retention time RT is 21.028min, the relative retention time is 1.00min, the average peak area is 41.707mAU, and the RSD% value is 43.34%
The No. 6 peak is galactose, the average retention time RT is 23.874min, the relative retention time is 1.14min, the average peak area is 31.873mAU, and the RSD% value is 39.19%
PMP remained as the derivatization reagent at RT of 9min and the solvent peak before 5 min.
Similarity evaluation HPLC chromatogram was comprehensively evaluated by using Chinese medicine fingerprint similarity evaluation software (2004 version A) recommended by the Committee of Chinese pharmacopoeia, and the similarity analysis results of monosaccharide fingerprints of 20 batches of Polyporus umbellatus samples are shown in Table 1. The similarity of 19 batches was greater than 0.9, except that the ZL-004 similarity was 0.85. The results show that the analysis method can better characterize the characteristics of the polyporus umbellatus and can be used as a reference for evaluating the quality of the polyporus umbellatus.
Similarity analysis results of 120 grifola polysaccharide samples in attached table
Comparative example
Comparative example 1: polyporus umbellatus water-soluble polysaccharide monosaccharide composition fingerprint analysis liquid chromatography system 3
Get extended C18Chromatography column (5 μm, 4.6X 250mm, Agilent), flow rate 1.0ml/min, detection wavelength 250nm, column temperature 30 deg.C; the sample injection volume is 10 mu l; mobile phase: acetonitrile (B) -0.1% phosphoric acid (a); elution gradient: 0-10 min: 15% -15% of B; 15-30% of B for 10-50 min. A mixed control solution of 5 selected monosaccharides (i.e., mannose, ribose, glucose, galactose, and fucose) was prepared according to examples 1 and 3, and detected under the HPLC conditions described in this example, and the chromatogram obtained was shown in FIG. 5. The figure shows that: neither peak 2 nor peak 3 nor peak 4 nor peak 5 achieved a good separation.
Claims (10)
1. The construction method of the agaric fingerprint is characterized by comprising the following steps: (1) detecting a sample solution of the polyporus umbellatus by using high performance liquid chromatography, and (2) generating a fingerprint spectrum according to a detection result;
the chromatographic column of the high performance liquid chromatography is a reverse phase column;
the high performance liquid chromatography is carried out isocratic elution by using acetonitrile-phosphate buffer solution with the volume ratio of mobile phase of 15:85 to 20: 80;
the preparation of the test solution comprises the following steps: decocting Polyporus umbellatus water-soluble crude polysaccharide with water to obtain decoction; hydrolyzing the obtained crude polysaccharide with acid, performing derivatization treatment with 1-phenyl-3-methyl-5-pyrazolone, and taking a water layer to fix the volume after extraction as a test solution.
2. The method of claim 1, wherein the reverse phase column is a silica gel bonded phase chromatography column, preferably an octadecylsilane bonded phase chromatography column or an octylsilane bonded silica gel chromatography column;
and/or the pH value of the phosphate buffer solution is 6.5-7.0, preferably 6.80;
and/or the concentration of the phosphate buffer solution is 30-50 mmol/L, preferably 50 mmol/L;
and/or, the phosphate buffer solution is KH2PO4-NaOH buffer;
and/or the flow rate of the mobile phase is 0.8-1.2 ml/min, preferably 1 ml/min;
and/or the temperature of the reverse phase column is 25-30 ℃, preferably 30 ℃;
and/or the volume ratio of the mobile phase is 18:82 or 17.5: 82.5;
and/or the detection wavelength of the high performance liquid chromatography is 240-250 nm, preferably 250 nm;
and/or the sample injection volume of the high performance liquid chromatography is 10-20 mu L, preferably 10 mu L.
3. The construction method according to claim 1, wherein the water decoction method comprises the steps of: (1) decocting the polyporus umbellatus with water to obtain a sample, and (2) carrying out post-treatment on the sample obtained in the step (1);
the post-treatment is carried out by one of the following methods: concentrating or dissolving the sample in water, mixing with ethanol, precipitating, and freeze drying to obtain water-soluble crude Polyporus polysaccharide, and drying with microporous membrane but not ultrafiltration membrane to obtain crude Polyporus polysaccharide;
preferably, the ethanol is used in an amount of 80-90% of the total volume of the mixed solution.
4. The construction method according to claim 1, wherein the acid hydrolysis is a step of heating after mixing an acidic solution and the obtained crude polysaccharide, the heating is preferably performed in a water bath, and the heating temperature is preferably 90-100 ℃, and more preferably 90 ℃; the heating time is preferably 4-6 h, and more preferably 6 h; the acidic solution is preferably hydrochloric acid, trifluoroacetic acid or sulfuric acid;
and/or the derivatization treatment comprises the steps of mixing the derivative with a sodium hydroxide solution and a methanol solution of 1-phenyl-3-methyl-5-pyrazolone, and then heating, wherein the volume ratio of the methanol solution of 1-phenyl-3-methyl-5-pyrazolone to the sodium hydroxide solution is preferably more than or equal to 1, the heating mode is preferably water bath, and the heating temperature is preferably 60-70 ℃, and more preferably 70 ℃; the heating time is preferably 30-40 min, and more preferably 30 min;
and/or the extractant for extraction is chloroform.
5. A detection method for polyporus umbellatus quality is characterized in that a fingerprint of a polyporus umbellatus sample to be detected is generated according to the construction method of claim 1, the obtained fingerprint has 8 characteristic peaks, 7 of the characteristic peaks are mannose, ribose, rhamnose, glucose, galactose, xylose and fucose, and the polyporus umbellatus sample to be detected is a qualified product;
preferably, the polyporus umbellatus sample to be detected is a polyporus umbellatus decoction or a polyporus umbellatus formula particle.
6. A method for detecting monosaccharide composition of Polyporus umbellatus polysaccharide is characterized by detecting sample solution of Polyporus umbellatus by high performance liquid chromatography;
the chromatographic column of the high performance liquid chromatography is a reverse phase column;
the high performance liquid chromatography is carried out isocratic elution by using acetonitrile-phosphate buffer solution with the volume ratio of mobile phase of 15:85 to 20: 80;
the preparation of the test solution comprises the following steps: decocting Polyporus umbellatus water-soluble crude polysaccharide with water to obtain decoction; hydrolyzing the obtained crude polysaccharide with acid, performing derivatization treatment with 1-phenyl-3-methyl-5-pyrazolone, and taking a water layer to fix the volume after extraction as a test solution.
7. The process according to claim 6, wherein the reverse phase column is a silica gel bonded phase chromatography column, preferably an octadecylsilane bonded chromatography column or an octylsilane bonded silica gel chromatography column silica gel column;
and/or the pH value of the phosphate buffer solution is 6.5-7.0, preferably 6.80;
and/or the concentration of the phosphate buffer solution is 30-50 mmol/L, preferably 50 mmol/L;
and/or, the phosphate buffer solution is KH2PO4-NaOH buffer;
and/or the flow rate of the mobile phase is 0.8-1.2 ml/min, preferably 1 ml/min;
and/or the temperature of the reverse phase column is 25-30 ℃, preferably 30 ℃;
and/or the volume ratio of the mobile phase is 18:82 or 17.5: 82.5;
and/or the detection wavelength of the high performance liquid chromatography is 240-250 nm, preferably 250 nm;
and/or the sample injection volume of the high performance liquid chromatography is 10-20 mu L, preferably 10 mu L.
8. The method of claim 6, wherein the water decoction process comprises the steps of: (1) decocting the polyporus umbellatus with water to obtain a sample, and (2) carrying out post-treatment on the sample obtained in the step (1);
the post-treatment is carried out by one of the following methods: concentrating or dissolving the sample in water, mixing with ethanol, precipitating, and freeze drying to obtain water-soluble crude Polyporus polysaccharide, and drying with microporous membrane but not ultrafiltration membrane to obtain crude Polyporus polysaccharide;
preferably, the volume of the ethanol accounts for 80-90% of the total volume of the mixed solution.
9. The method according to claim 6, wherein the acid hydrolysis is a step of heating after mixing an acidic solution and the obtained crude polysaccharide, the heating is preferably performed in a water bath, and the heating temperature is preferably 90-100 ℃, and more preferably 90 ℃; the heating time is preferably 4-6 h, and more preferably 6 h; the acidic solution is preferably hydrochloric acid, trifluoroacetic acid or sulfuric acid;
and/or the derivatization treatment comprises the steps of mixing the derivative with a sodium hydroxide solution and a methanol solution of 1-phenyl-3-methyl-5-pyrazolone, and then heating, wherein the volume ratio of the methanol solution of 1-phenyl-3-methyl-5-pyrazolone to the sodium hydroxide solution is preferably more than or equal to 1, the heating mode is preferably water bath, and the heating temperature is preferably 60-70 ℃, and more preferably 70 ℃; the heating time is preferably 30-40 min, and more preferably 30 min;
and/or the extractant for extraction is chloroform.
10. A separation method of polyporus polysaccharide extract is characterized in that the method comprises the steps of carrying out acid hydrolysis on polyporus water-soluble crude polysaccharide obtained by a water decoction method, and then carrying out column chromatography separation; the column chromatography is performed isocratic elution using acetonitrile-phosphate buffer solution in a volume ratio of 15:85 to 20: 80.
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