Construction method and application of characteristic maps of Poria cocos, red Poria cocos and Poria cocos peel

Abstract

The invention discloses a method for constructing a characteristic spectrum of Poria cocos, red Poria cocos and Poria cocos peel, comprising the following steps: preparing a reference solution, wherein the reference solution includes a uridine reference, an adenosine reference and a guanosine reference; taking Poria cocos medicinal materials, red Poria cocos medicinal materials or Poria cocos peel medicinal materials to prepare a test solution; taking a preset amount of the test solution and the reference solution respectively, injecting them into a liquid chromatograph for determination, wherein the liquid chromatograph uses octadecylsilane bonded silica gel as a filler, methanol as a mobile phase A, and potassium dihydrogen phosphate solution as a mobile phase B for gradient elution to obtain a characteristic spectrum. The invention has good reproducibility, is accurate and reliable, and can quickly and accurately identify medicinal materials from different parts of Poria cocos based on water-soluble components.

Description

Technical Field

[0001] The present invention relates to the technical field of quality analysis and detection of traditional Chinese medicines, and in particular to a method for constructing a characteristic spectrum of Poria cocos, red Poria cocos and Poria cocos peel and an application thereof. Background Art

[0002] Poria cocos is the dried sclerotium of the Polyporaceae fungus Poria cocos (Schw.) Wolf. It is mostly parasitic on the underground roots of Masson pine or Pinus densiflora or on pine wood and pine branches buried in the soil. It is mainly distributed in Yunnan, Hubei, Hunan, Anhui, Guizhou and other provinces. Because it needs to remove the outer skin of the sclerotium and the light red part near the outer skin of the sclerotium for clinical use, and retain the white part of the sclerotium, Poria cocos is also called "white Poria cocos". Poria cocos is a traditional Chinese medicine in my country. It is flat in nature, sweet and light in taste, and enters the heart, lung, spleen, and kidney meridians; it has the effects of diuresis, invigorating the spleen and nourishing the middle, and calming the mind. It is the raw material of many prescriptions and Chinese patent medicines. There is a saying that "nine out of ten medicines are Poria cocos".

[0003] The outer skin of the sclerotium of Poria cocos is called Poria cocos peel, and the light red part of the sclerotium near the outer skin is called "red Poria cocos". Poria cocos was first recorded as a medicine in "Shennong's Herbal Classic", which was listed as a top-grade product, saying that "taking it for a long time can calm the soul and nourish the spirit, and prolong life without hunger". Regarding the use of Poria cocos peel as medicine, "Sichuan Chinese Medicine Records" records that Poria cocos peel "is mild in nature, sweet and light in taste, and non-toxic. It enters the kidney and bladder meridians". "Compendium of Materia Medica" records that Poria cocos peel "treats edema and swelling of the skin, opens the water channels, and opens the pores". "Medical Collection" records that Poria cocos peel "moves water in the skin". Regarding the medicinal value of red Poria cocos, "New Correction of Jinling's Compendium of Materia Medica" records that red Poria cocos: "it can relieve dampness and heat in the heart, small intestine and bladder, and promote the flow of water". It can be seen that although Poria cocos peel, red Poria cocos and Poria cocos are derived from Poria cocos sclerotium and belong to the same category, they have different medicinal parts and different effects, and cannot be mixed clinically. Poria skin is the dried outer skin of Poria sclerotium, which has the effect of promoting diuresis and reducing swelling; red Poria is the light red part near the outer skin, which has the effects of promoting diuresis, promoting dampness and heat, and benefiting the heart and moistening the lungs; Poria is the white part after peeling and cutting off the red Poria, which has the effects of promoting diuresis and eliminating dampness, invigorating the spleen and calming the mind. It can be seen that Poria is good at invigorating the spleen and eliminating dampness, red Poria is good at promoting diuresis, and Poria skin is good at promoting diuresis and reducing swelling.

[0004] The 2020 edition of the Chinese Pharmacopoeia only includes Poria cocos and Poria cocos peel. Research on different medicinal parts of Poria cocos is limited to one or two medicinal parts. A comprehensive comparative study of different medicinal parts of Poria cocos (Poria cocos, red Poria cocos, and Poria cocos peel) has not yet been conducted. In addition, current research on the chemical composition of Poria cocos and its different medicinal parts is mostly focused on the analysis of triterpenoid components, and there is a lack of research on highly polar water-soluble components. Traditional Chinese medicine is mainly used in the form of decoctions. The water-soluble components in the decoction are one of the important material bases for the efficacy of Chinese medicine. At present, there is no research on the differences in water-soluble components in different medicinal parts of Poria cocos.

[0005] At present, the 2020 edition of the Chinese Pharmacopoeia only uses appearance, chemistry and thin-layer chromatography to identify and control the quality of Poria cocos and Poria cocos peel medicinal materials. It is difficult to accurately analyze the medicinal ingredients of Poria cocos, and thus it is difficult to make an objective and comprehensive evaluation of the quality of Poria cocos slices. In recent years, research on various aspects of Poria cocos has gradually increased, and its chemical composition, pharmacological effects and application development have attracted much attention, but the differences in the chemical composition of different medicinal parts are still not clear enough. For the accuracy and safety of clinical medication, it is necessary to distinguish Poria cocos from different parts.

[0006] Moreover, at present, the processing of Poria cocos is mainly manual, and the processing technology is relatively backward. The quality of Poria cocos medicinal materials varies greatly. There are dozens of processed commodity specifications without unified standards. The clinical use of red Poria cocos and Poria cocos peel is not large, and has not been extensively developed and utilized. Most of them are treated as waste, causing great waste. The previous editions of the Chinese Pharmacopoeia collectively refer to the light reddish brown and white parts of Poria cocos as Poria cocos, but the clinical use of traditional Chinese medicine clearly distinguishes red Poria cocos from Poria cocos (white Poria cocos). In the classic prescriptions that have emerged in recent years, the Poria cocos used in several prescriptions is clearly marked as red Poria cocos. As a relatively mature Chinese medicine quality control method in the field of modern Chinese medicine, the characteristic spectrum research has the advantages of high integrity and strong characteristics, and can be used to characterize the integrity, complexity and composition changes of Chinese medicine chemical components. Therefore, this is to urgently invent a water-soluble characteristic spectrum method of Poria cocos medicinal materials, which can quickly and accurately identify medicinal materials in different parts of Poria cocos, and provide a scientific basis for the formulation of Poria cocos medicinal material quality and commodity specification grades. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a method for constructing characteristic maps of Poria cocos, red Poria cocos and Poria cocos peel. The method has good reproducibility, accuracy and reliability, and can quickly and accurately identify medicinal materials from different parts of Poria cocos based on the water-soluble components in Poria cocos medicinal materials, red Poria cocos medicinal materials and Poria cocos peel medicinal materials.

[0008] In order to solve the above technical problems, the present invention provides a method for constructing characteristic maps of Poria cocos, red Poria cocos and Poria cocos peel, comprising the following steps:

[0009] preparing a reference solution, wherein the reference solution comprises a uridine reference, an adenosine reference and a guanosine reference;

[0010] Take Poria cocos, red Poria cocos or Poria cocos peel to prepare a test solution;

[0011] Preset amounts of the test solution and the reference solution were taken respectively and injected into a liquid chromatograph for determination. The liquid chromatograph used octadecylsilane bonded silica gel as a filler, methanol as a mobile phase A, and potassium dihydrogen phosphate solution as a mobile phase B for gradient elution to obtain a characteristic spectrum.

[0012] In one embodiment, the gradient elution is performed according to the following procedure:

[0013] 0-10min, mobile phase A is 0%, mobile phase B is 100%;

[0014] 10min-15min, mobile phase A from 0→4%, mobile phase B from 100%→96%;

[0015] 15min~28min, mobile phase A from 4→6%, mobile phase B from 96%→94%;

[0016] 28min~40min, mobile phase A from 6%→30%, mobile phase B from 94%→70%;

[0017] 40min~43min, mobile phase A changes from 30%→50%, and mobile phase B changes from 70%→50%.

[0018] In one embodiment, the concentration of the potassium dihydrogen phosphate solution is 2.5 mmol / L to 3.5 mmol / L.

[0019] In one embodiment, the preset amount of the test solution or the reference solution is 9 μL to 11 μL;

[0020] The liquid chromatograph uses octadecylsilane bonded silica gel as a filler, with a column length of 250 mm, an inner diameter of 4.6 mm, a particle size of 5 μm, and a column temperature of 23° C. to 25° C.;

[0021] When performing gradient elution, the flow rate of the mobile phase is 1.1 mL / min to 1.3 mL / min;

[0022] The ultraviolet detection wavelength of the liquid chromatograph is 260nm-270nm.

[0023] In one embodiment, the test solution is prepared by the following method:

[0024] Take Poria cocos medicinal material, red Poria cocos medicinal material or Poria cocos peel medicinal material, grind it and then use methanol for heating reflux extraction to obtain the test solution.

[0025] In one embodiment, in the heating reflux extraction process, 8% to 12% methanol is added to the powder of Poria cocos, red Poria cocos or Poria cocos peel, and the mixture is heated and refluxed for 2.5 h to 3.5 h.

[0026] In one embodiment, the characteristic spectrum of Poria cocos medicinal material includes 7 common peaks, among which, taking the peak 3 uridine chromatographic peak as the reference peak S, the relative retention times of peaks 1 to 2, peaks 4 to 6, and peak 7 and peak S are consistent with: peak 1 is 0.31, peak 2 is 0.63, peak 4 is 1.20, peak 5 is 1.50, peak 6 is 1.81, and peak 7 is 2.55, and the relative standard deviation is within ±10%.

[0027] In one embodiment, the characteristic spectrum of the Poria peel medicinal material includes 8 common peaks, wherein the uridine chromatographic peak of peak 2 is used as the reference peak S1, and the relative retention times of peaks 1, 3 to 6 and S1 are consistent with: peak 1 is 0.95, peak 3 is 1.15, peak 4 is 1.43, peak 5 is 1.66, peak 6 is 1.79, and the relative standard deviation is within ±10%;

[0028] Taking the adenosine peak 7 as the reference peak S2, the relative retention time of peak 8 and peak S2 is consistent with that of peak 8 being 1.05, and the relative standard deviation is within ±10%.

[0029] In one embodiment, the characteristic spectrum of the red tuckahoe medicinal material includes 8 common peaks, wherein the peak 3 uridine chromatographic peak is used as the reference peak S1, and the relative retention times of peaks 1 to 2, peaks 4 to 5 and S1 peak are consistent with: peak 1 is 0.32, peak 2 is 0.96, peak 4 is 1.20, peak 5 is 1.50, and the relative standard deviation is within ±10%;

[0030] Taking the adenosine peak 6 as the reference peak S2, the relative retention times of peaks 7, 8 and S2 are consistent with: peak 7 is 1.04, peak 8 is 1.06, and the relative standard deviation is within ±10%.

[0031] The present invention also provides the application of the method for constructing the characteristic spectrum of Poria cocos, red Poria cocos and Poria cocos peel in (1) or (2):

[0032] (1) Identification and quality control of the medicinal parts of Poria cocos;

[0033] (2) Differentiation and identification of Poria cocos, red Poria cocos and Poria cocos peel.

[0034] The implementation of the present invention has the following beneficial effects:

[0035] The present invention establishes a method for constructing characteristic maps of Poria cocos, red Poria cocos and Poria cocos peel. The method is rapid, stable and highly specific. According to the water-soluble components in Poria cocos medicinal materials, red Poria cocos medicinal materials and Poria cocos peel medicinal materials, different parts of Poria cocos can be identified, providing a scientific new method for quality control of Poria cocos medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is the characteristic spectrum of Poria cocos with different extraction solvents;

[0037] Figure 2 This is the characteristic spectrum of Poria cocos extracted by different methods;

[0038] Figure 3 This is the characteristic spectrum of Poria cocos medicinal material at different extraction times;

[0039] Figure 4 It is the characteristic spectrum of Poria cocos medicinal material on different chromatographic columns;

[0040] Figure 5 This is the characteristic spectrum of Poria cocos medicinal material with different concentrations of mobile phase;

[0041] Figure 6 This is the characteristic spectrum of Poria cocos medicinal material in full wavelength scanning;

[0042] Figure 7 It is the characteristic spectrum of Poria cocos medicinal material at different detection wavelengths;

[0043] Figure 8 This is the characteristic spectrum of Poria cocos medicinal material at different column temperatures;

[0044] Fig. 9 It is the characteristic spectrum of Poria cocos medicinal material at different flow rates;

[0045] Fig.10 It is the result of investigation on the specificity of the characteristic spectrum of Poria cocos medicinal material;

[0046] Fig.11 It is an overlay of characteristic spectra of 24 batches of Poria cocos medicinal materials;

[0047] Fig.12 This is an overlay of characteristic spectra of 22 batches of Poria peel medicinal materials;

[0048] Fig.13 This is the superposition of characteristic spectra of three batches of red tuckahoe medicinal materials;

[0049] Fig.14 It is the superposition diagram of the test solution and the reference solution;

[0050] Fig.15 It is the UV images (190nm~400nm) of the test solution (left) and the uridine reference solution (right);

[0051] Fig.16 It is the UV images (190nm~400nm) of the test solution (left) and the adenine reference solution (right);

[0052] Fig.17 It is the UV images (190nm~400nm) of the test solution (left) and the guanosine reference solution (right);

[0053] Fig.18It is the UV images (190nm~400nm) of the test solution (left) and adenosine reference solution (right);

[0054] Fig.19 It is the UV images (190nm~400nm) of the test solution (left) and the protocatechuic aldehyde reference solution (right);

[0055] Fig. 20 This is the reference characteristic spectrum of Poria cocos, where Peak 3 (S): uridine; Peak 5: adenine; Peak 6: guanosine; Peak 7: adenosine;

[0056] Fig.21 This is the reference characteristic spectrum of Poria peel medicinal material, among which, peak 2 (S1): uridine; peak 4: adenine; peak 6: guanosine; peak 7 (S2): adenosine; peak 8: protocatechuic aldehyde;

[0057] Fig. 22 This is the reference characteristic spectrum of the red tuckahoe medicinal material, where peak 3 (S1): uridine; peak 5: adenine; peak 6 (S2): adenosine; peak 7: protocatechuic aldehyde;

[0058] Fig.23 It is a comparison of the characteristic spectra of Poria cocos medicinal materials, Poria cocos peel medicinal materials, and red Poria cocos medicinal materials. DETAILED DESCRIPTION

[0059] In order to make the purpose, technical solutions and advantages clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific implementation methods.

[0060] 1. Instruments and test drugs

[0061] Instruments: Waters high performance liquid chromatograph (e2695, Waters Corporation), Waters HSS T3 C18 chromatographic column (4.6 mm × 250 mm, 5 μm), Agilent ZORBAX SB-Aq chromatographic column (4.6 mm × 250 mm, 5 μm), YMC TriartC18 chromatographic column (4.6 mm × 250 mm, 5 μm), one-tenth balance (Mettler-Toledo, ME204E), one-millionth balance (Mettler-Toledo, XP26), electric constant temperature water bath (Shanghai Yiheng Technology Co., Ltd., HWS-28), CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), ultrapure water system (Merck Co., Ltd., Milli-QDirect).

[0062] Reagents: ethanol (analytical grade, Guangdong Guanghua Science and Technology Co., Ltd.), methanol (analytical grade, Guangdong Guanghua Science and Technology Co., Ltd.); methanol (chromatographic grade, Merck Co., Ltd.), potassium dihydrogen phosphate (Guangzhou Chemical Reagent Factory), and ultrapure water (self-made in the laboratory).

[0063] Test drugs: uridine (China Food and Drug Inspection Institute, batch number: 110887-202104, content: 99.6%); adenosine (China Food and Drug Inspection Institute, batch number: 110879-202204, content: 99.4%); 24 batches of Poria cocos medicinal materials, 22 batches of Poria cocos peel medicinal materials, and 3 batches of red Poria cocos medicinal materials. The origin and number are shown in Table 1.

[0064] Table 1 Sample information table

[0065]

[0066]

[0067] 2 Chromatographic conditions and test solution preparation

[0068] 2.1 Chromatographic conditions

[0069] Adopting YMC Triart C 18 The chromatographic column (4.6 mm × 250 mm, 5 μm) was used, with methanol as mobile phase A and 3 mmol / L potassium dihydrogen phosphate solution as mobile phase B, and gradient elution was performed according to the regulations in Table 2; the flow rate was 1.2 ml per minute; the column temperature was 25°C; and the detection wavelength was 260 nm.

[0070] Table 2 Gradient elution table

[0071]

[0072] 2.2 Preparation of reference solution Take appropriate amount of uridine reference substance, adenosine reference substance and guanosine reference substance, weigh accurately, add 10% methanol to make a solution containing 10 μg of each substance per 1 ml as the reference solution.

[0073] 2.3 Preparation of test solution Take about 2 g of the product powder (passed through No. 3 sieve), add 25 ml of 10% methanol, vortex for 1 minute, then add 25 ml of 10% methanol, weigh the weight, heat under reflux for 3 hours, cool, weigh again, make up the lost weight with 10% methanol, filter, take 25 ml of the filtrate, evaporate to dryness, make up the volume of the residue to 2 ml with 10% methanol, shake well, filter, and take the filtrate.

[0074] 2.4 Determination method: Accurately pipette 10 μL of reference solution and test solution respectively, inject into liquid chromatograph for determination.

[0075] 3 Investigation of the preparation method of test solution

[0076] 3.1 Investigation of extraction solvents About 2 g of the same batch of Poria powder (batch number: S12, passed through No. 3 sieve) were taken, accurately weighed, placed in a stoppered conical flask, and accurately added 25 ml of water, 10% methanol, 30% methanol, 50% methanol, 10% ethanol, 30% ethanol, and 50% ethanol, vortexed for 1 minute, and then 25 ml of the above corresponding solvents were added, weighed, heated under reflux for 3 hours, cooled, weighed again, and the lost weight was supplemented with the corresponding solvent, shaken, filtered, and 25 ml of the filtrate was taken, evaporated to dryness, and the residue was made up to 2 ml with the above solvents, shaken, filtered, and the filtrate was taken. The chromatographic conditions under "2.1" were determined. The results are shown in Figure 1 The results show that the above 7 extraction solvents have a greater impact on the peak shape of each characteristic peak. When 10% methanol is used as the extraction solvent, the overall response of each characteristic peak is higher, so 10% methanol is selected as the extraction solvent.

[0077] 3.2 Investigation of extraction methods About 2 g of the same batch of Poria powder (batch number: S12, passed through No. 3 sieve) was taken, accurately weighed, placed in a stoppered conical flask, accurately added with 25 ml of 10% methanol and vortexed for 1 minute, then added with 25 ml of 10% methanol, weighed, ultrasonically treated (power 300 W, frequency 60 kHz) for 3 hours and heated under reflux for 3 hours, cooled, weighed again, supplemented with 10% methanol to make up the loss, filtered, took 25 ml of the filtrate, evaporated, and the residue was made up to 2 ml with 10% methanol, shaken, filtered, and the filtrate was taken. The chromatographic conditions under "2.1" were used for determination. The results are shown in Figure 2 The results showed that there was no significant difference in the peak shape and separation effect of each characteristic peak between the two extraction methods, but the overall response of each characteristic peak in the heating reflux extraction was higher than that in the ultrasound, so the heating reflux extraction method was selected.

[0078] 3.3 Investigation of extraction time Take about 2 g of the same batch of Poria powder (batch number: S12, passed through No. 3 sieve), accurately weigh, put in a stoppered conical flask, accurately add 25 ml of 10% methanol, vortex for 1 minute, then add 25 ml of 10% methanol, weigh the weight, heat and reflux for 0.5 hour, 1 hour, 2 hours, 3 hours, and 5 hours, cool, weigh again, make up the loss with 10% methanol, filter, take 25 ml of the filtrate, evaporate to dryness, make up the volume to 2 ml with 10% methanol, shake well, filter, take the filtrate, and measure according to the chromatographic conditions under "2.1". The results are shown in Figure 3 The results showed that different extraction times had a great influence on the response values ​​of each characteristic peak. Combined with the overall peak response of each characteristic peak, heating reflux for 3 hours was selected.

[0079] 4 Optimization of chromatographic conditions

[0080] 4.1 Selection of chromatographic column Take about 2g of Poria cocos powder (batch number: S12, passed through No. 3 sieve), prepare the test solution according to the method under "2.3", and measure according to the following chromatographic conditions. Chromatographic conditions: 0.1% phosphoric acid as mobile phase A, methanol as mobile phase B, gradient elution according to the provisions in Table 3; flow rate is 1.0ml per minute, column temperature is 30℃; injection volume is 10μl.

[0081] The results show that when different brands of hydrophilic chromatographic columns are analyzed with pure water as the initial concentration, there are still many highly polar components that cannot be retained at the solvent peak. Figure 4 , but the YMC Triart C18 column presented more information and better separation, so this column was used for further research.

[0082] Table 3 Gradient elution table

[0083]

[0084] 4.2 Mobile phase optimization Take about 2 g of Poria cocos powder (batch number: S12, passed through No. 3 sieve) and prepare the test solution according to the method under "2.3". Refer to the chromatographic conditions under "2.1" for sample injection analysis and examine different buffer salt concentrations. The results are as follows Figure 5 As shown in the figure, different buffer salt concentrations have a great influence on the separation of each characteristic peak. When the concentration of potassium dihydrogen phosphate is 3mmol / L, the separation of each characteristic peak is better and the peak shape is more symmetrical. Therefore, this system is used for further investigation.

[0085] 4.3 Selection of detection wavelength Take about 2g of Poria cocos powder (batch number: S12, passed through No. 3 sieve), prepare the test solution according to the method in "2.3", refer to the chromatographic conditions in "2.1" for sample analysis, and record the absorption spectrum in the range of 190-400nm. The results are as follows Figure 6 and Figure 7 By comparing the chromatograms of different detection wavelengths, it was found that at a wavelength of 260nm, the peak response and information were richer, the interference was smaller, and the separation was better. Therefore, after comprehensive consideration, 260nm was selected as the detection wavelength.

[0086] 4.4 Selection of column temperature Take about 2g of Poria cocos powder (batch number: S12, passed through No. 3 sieve) and prepare the test solution according to the method in "2.3". Except for the column temperature, the other chromatographic conditions are the same as the injection analysis in "2.1". The results are as follows Figure 8 As shown in the figure, by comparing the chromatograms of three different column temperatures, when 23℃ and 25℃ are selected as the column temperature, the information of the chromatographic peaks is more complete and the separation is better. Taking into account the performance of different chromatographs, a column temperature of 25℃ is selected as the column temperature for establishing the characteristic spectrum of Poria water solubility.

[0087] 4.5 Selection of flow rate Take about 2 g of Poria cocos powder (batch number: S12, passed through No. 3 sieve) and prepare the test solution according to the method in "2.3". Except for the flow rate, the other chromatographic conditions are the same as the injection analysis in "2.1". The results are as follows Fig. 9 As shown in the figure, by comparing the chromatograms of three different flow rates, when the flow rate is 1.2 ml / min, the information of each chromatographic peak is complete, and the separation of each chromatographic peak is better. After comprehensive consideration, 1.2 ml / min is selected as the flow rate for establishing the water-soluble characteristic spectrum of Poria cocos.

[0088] 5 Methodological investigation

[0089] 5.1 Specificity investigation Take about 2g of Poria cocos powder (batch number: S12, passed through No. 3 sieve), prepare the test solution according to the method in "2.3", accurately pipette 10μl of the test solution, the reference solution in "2.2" and the blank solvent, and inject and analyze according to the chromatographic conditions in "2.1". The results are as follows Fig.10 As shown, the test sample chromatogram has the same chromatographic peak at the corresponding retention time as the reference sample chromatogram, and there is no interference from the blank solvent, indicating that the method has good specificity.

[0090] 5.2 Precision investigation Take about 2g of Poria cocos powder (batch number: S12, passed through No. 3 sieve), prepare the test solution according to the method under "2.3", and repeat the injection 6 times according to the chromatographic conditions under "2.1". Take the uridine chromatographic peak as the reference peak S, calculate the relative retention time and relative peak area of ​​each characteristic peak and the S peak, and calculate the RSD value. The results show that the relative retention time RSD value of the 7 common peaks is in the range of 0.12% to 0.49%, and the relative peak area RSD value is in the range of 0.76% to 3.02%, indicating that the instrument has good precision.

[0091] 5.3 Repeatability Study Take about 2g of Poria cocos powder (batch number: S12, sieve No. 3), prepare 6 test solutions according to the method under "2.3", and inject and measure according to the chromatographic conditions under "2.1". Taking the uridine chromatographic peak as the reference peak, calculate the relative retention time and relative peak area of ​​each characteristic peak and the S peak, and calculate the RSD value. The results show that the relative retention time RSD value of the 7 common peaks is in the range of 0.18% to 2.05%, and the relative peak area RSD value is in the range of 0.73% to 3.17%, indicating that the method has good repeatability.

[0092] 5.4 Stability Study Take about 2g of Poria cocos powder (batch number: S12, sieve No. 3), prepare the test solution according to the method under "2.3", and inject and measure at 0, 4, 6, 8, 10, 12, 20, and 24 hours according to the chromatographic conditions under "2.1". Take the uridine chromatographic peak as the reference peak S, calculate the relative retention time and relative peak area of ​​each characteristic peak and the S peak, and calculate the RSD value. The relative retention time RSD value of the 7 common peaks is in the range of 0.10% to 0.48%, and the relative peak area RSD value is in the range of 0.44% to 3.28%, indicating that the test solution is relatively stable within 24 hours.

[0093] 5.5 The intermediate precision study was conducted by different analysts in different laboratories and on instruments of different brands. Take about 2g of Poria cocos powder (batch number: S12, passed through No. 3 sieve), prepare 6 test solutions according to the method under "2.3", and inject and measure according to the chromatographic conditions under "2.1". Taking the uridine chromatographic peak as the reference peak S, calculate the relative retention time and relative peak area of ​​each characteristic peak and the S peak, and calculate the RSD value. The results show that the relative retention time RSD value of the 7 common peaks is in the range of 0.06% to 0.20%, and the relative peak area RSD value is in the range of 0.60% to 4.12%, indicating that the established method can be reproduced under different conditions such as different equipment and laboratories, and the intermediate precision of this method is good.

[0094] 6 Establishment of characteristic profiles of Poria cocos, Poria cocos peel and red Poria cocos

[0095] 6.1 Determination of characteristic spectra of Poria cocos, Poria cocos peel and red Poria cocos samples

[0096] Take 24 batches of Poria cocos, 22 batches of Poria cocos peel and 3 batches of red Poria cocos, prepare the test solution according to the method under "2.3", inject and measure according to the chromatographic conditions under "2.1", record the characteristic spectrum chromatogram, automatically integrate the fingerprint of each sample, export the integrated characteristic spectrum in CDF format, import the characteristic spectrum of 24 batches of Poria cocos, 22 batches of Poria cocos peel and 3 batches of red Poria cocos in CDF format into "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software (2012.130723 Edition)" for peak matching and multi-point correction, the results are as follows: Figure 11 to Figure 13As shown in the figure, 7 common characteristic peaks were identified in the characteristic spectra of 24 batches of Poria cocos medicinal materials. The relative retention time was used for positioning, and the chromatographic peak of peak 3 uridine was used as the reference peak S. The average relative retention times of peaks 1-2, peaks 4-6, peak 7 and S were calculated to be 0.31 (peak 1), 0.63 (peak 2), 1.20 (peak 4), 1.50 (peak 5), 1.81 (peak 6), and 2.55 (peak 7). The characteristic spectra of 22 batches of Poria cocos peel medicinal materials identified 8 common characteristic peaks. The relative retention time was used for positioning, and the chromatographic peak of peak 2 uridine was used as the reference peak S1 to calculate the relative retention times of peaks 1, peaks 3-6 and S1. The chromatographic peak of peak 7 adenosine was used as the reference peak S2, and the relative retention times of peaks 8 and S2 were calculated. The relative retention times of the three batches of red tuckahoe were 0.95 (peak 1), 1.15 (peak 3), 1.43 (peak 4), 1.66 (peak 5), 1.79 (peak 6), and 1.05 (peak 8). A total of eight common characteristic peaks were identified in the characteristic spectra of the three batches of red tuckahoe medicinal materials. The relative retention time positioning was adopted. The chromatographic peak of uridine at peak 3 was taken as the reference peak S1 to calculate the relative retention times of peaks 1-2, peaks 4-5 and S1. The chromatographic peak of adenosine at peak 6 was taken as the reference peak S2 to calculate the relative retention times of peaks 7-8 and S2. The average values ​​were 0.32 (peak 1), 0.96 (peak 2), 1.20 (peak 4), 1.50 (peak 5), 1.04 (peak 7), and 1.06 (peak 8).

[0097] 6.2 Identification of characteristic peaks

[0098] The main components of Poria cocos are polysaccharides and triterpenoids. In addition, it has been reported that Poria cocos also contains steroidal components, fatty acids, proteins, adenine, amino acids, and inorganic elements such as calcium, magnesium, iron, and potassium. In order to clarify the common peaks in the characteristic spectrum of Poria cocos, reference substances such as uridine, guanosine, adenosine, adenine, and protocatechuic acid were used for identification based on their chemical composition. The results showed that chromatographic peaks with consistent retention times and spectra with uridine, adenine, guanosine, adenosine, and protocatechuic acid were found in the characteristic spectrum. Figures 14 to 19 .

[0099] 6.3 Establishment of the common pattern of characteristic spectra of Poria cocos, Poria cocos peel and red Poria cocos

[0100] The CDF format of characteristic spectra of 24 batches of Poria cocos, 22 batches of Poria cocos peel and 3 batches of red Poria cocos were imported into the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation Software (2012.130723 Edition)" for multi-point correction and peak matching. The comparison spectra of Poria cocos, Poria cocos peel and red Poria cocos were generated by the average method. The results are shown in Figure 20 to Figure 22 shown.

[0101] According to the measurement results of the characteristic spectra of 24 batches of Poria cocos samples, the standard of the characteristic spectrum of Poria cocos medicinal material was determined as follows: the characteristic spectrum should show 7 characteristic peaks, the peak corresponding to the peak of the uridine reference substance is the S peak, and the relative retention time of peaks 1 to 2, peaks 4 to 6, peak 7 and S peak is calculated. The relative retention time should be within ±10% of the specified value, and the specified value is: 0.31 (peak 1), 0.63 (peak 2), 1.20 (peak 4), 1.50 (peak 5), 1.81 (peak 6), 2.55 (peak 7).

[0102] According to the measurement results of the characteristic spectrum of 22 batches of Poria peel samples, the characteristic spectrum standard of Poria peel medicinal material was determined as follows: the characteristic spectrum should show 8 characteristic peaks, the peak corresponding to the uridine reference peak is the S1 peak, and the relative retention time of peak 1, peaks 3 to 6 and S1 peak is calculated, and the relative retention time should be within ±10% of the specified value; the peak corresponding to the adenosine reference peak is the S2 peak, and the relative retention time of peak 8 and S2 peak is calculated, and the relative retention time should be within ±10% of the specified value, and the specified values ​​are: 0.95 (peak 1), 1.15 (peak 3), 1.43 (peak 4), 1.66 (peak 5), 1.79 (peak 6), and 1.05 (peak 8).

[0103] According to the measurement results of the characteristic spectra of three batches of red tuckahoe samples, the standard of the characteristic spectrum of red tuckahoe medicinal material was determined as follows: the characteristic spectrum should show 8 characteristic peaks, the peak corresponding to the uridine reference peak is the S1 peak, and the relative retention times of peaks 1-2, 4-5 and S1 are calculated, and their relative retention times should be within ±10% of the specified value; the peak corresponding to the adenosine reference peak is the S2 peak, and 0.32 (peak 1), 0.96 (peak 2), 1.20 (peak 4), 1.50 (peak 5), 1.04 (peak 7), and 1.06 (peak 8) are calculated.

[0104] 6.4 Differentiation and Identification of Poria, Poria Peel and Red Poria

[0105] like Fig.23 As shown in the figure, the characteristic spectra of Poria cocos, Poria cocos peel and red Poria cocos all have the same 4 common peaks. Compared with Poria cocos, Poria cocos peel and red Poria cocos both have an additional chromatographic peak A with higher response at the chromatographic retention time of 39 minutes. Compared with Poria cocos peel, red Poria cocos has an additional chromatographic peak B with higher response at the retention time of 40 minutes, which can be used to distinguish and identify Poria cocos, Poria cocos peel and red Poria cocos.

[0106] Table 4 Comparison of peak areas of protocatechuic aldehyde and adenosine in Poria cocos, Poria cocos peel and red Poria cocos

[0107]

[0108]

[0109] As shown in the results of Table 4, the peak area ratio of protocatechuic aldehyde to adenosine in Poria cocos medicinal materials ranges from 0.000 to 0.084, with an average of 0.01; the peak area ratio in Poria cocos peel medicinal materials ranges from 0.167 to 1.359, with an average of 0.54; the peak area ratio of red Poria cocos medicinal materials ranges from 5.903 to 7.996, with an average of 6.90. The peak area ratio of protocatechuic aldehyde to adenosine in red Poria cocos medicinal materials is much higher than that of Poria cocos medicinal materials and Poria cocos peel medicinal materials, and there is a chromatographic peak with a higher response at 40 minutes; the ratio of Poria cocos peel medicinal materials is also significantly higher than that of Poria cocos medicinal materials, that is, the peak area ratio of protocatechuic aldehyde / adenosine: red Poria cocos>Poria cocos peel>Poria cocos. Therefore, the water-soluble characteristic spectrum method established by the present invention can effectively distinguish Poria cocos medicinal materials, red Poria cocos medicinal materials and Poria cocos peel medicinal materials.

[0110] The above is a preferred embodiment of the invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the invention, and these improvements and modifications are also considered to be within the scope of protection.

Claims

1. A method for constructing characteristic maps of Poria cocos, red Poria cocos and Poria cocos peel, characterized in that: The following steps are involved: preparing a reference solution, wherein the reference solution comprises a uridine reference, an adenosine reference and a guanosine reference; Take Poria cocos, red Poria cocos or Poria cocos peel to prepare a test solution; Preset amounts of the test solution and the reference solution were respectively taken and injected into a liquid chromatograph for determination. The liquid chromatograph used methanol as mobile phase A and potassium dihydrogen phosphate solution as mobile phase B for gradient elution to obtain a characteristic spectrum; The gradient elution was carried out according to the following procedure: 0~10min, mobile phase A is 0%, mobile phase B is 100%; 10min~15min, mobile phase A from 0→4%, mobile phase B from 100%→96%; 15min~28min, mobile phase A from 4→6%, mobile phase B from 96%→94%; 28min~40min, mobile phase A from 6%→30%, mobile phase B from 94%→70%; 40min~43min, mobile phase A from 30%→50%, mobile phase B from 70%→50%; The concentration of the potassium dihydrogen phosphate solution is 2.5 mmol / L to 3.5 mmol / L; The test solution was prepared by the following method: Take Poria cocos, red Poria cocos or Poria cocos peel, grind them, and then perform heating reflux extraction with 8% to 12% methanol to obtain the test solution; The ultraviolet detection wavelength of the liquid chromatograph is 260nm~270nm; The liquid chromatograph adopts YMC Triart C 18 The chromatographic column has a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm.

2. The method for constructing the characteristic spectrum of Poria cocos, red Poria cocos and Poria cocos peel according to claim 1, characterized in that: The preset volume of the test solution or reference solution is 9 μL to 11 μL; The column temperature of the liquid chromatograph is 23°C to 25°C; When performing the gradient elution, the flow rate of the mobile phase is 1.1 mL / min to 1.3 mL / min.

3. The method for constructing the characteristic spectrum of Poria cocos, red Poria cocos and Poria cocos peel according to claim 1, characterized in that: In the heating reflux extraction process, 8% to 12% methanol is added to the powder of Poria cocos, red Poria cocos or Poria cocos peel, and the mixture is heated and refluxed for 2.5 h to 3.5 h.

4. The method for constructing the characteristic spectrum of Poria cocos, red Poria cocos and Poria cocos peel according to claim 1, characterized in that: The characteristic spectrum of Poria cocos medicinal material includes 7 common peaks. Among them, the uridine chromatographic peak of peak 3 is taken as the reference peak S, and the relative retention times of peaks 1 to 2, 4 to 6, and 7 with S peak are consistent with: peak 1 is 0.31, peak 2 is 0.63, peak 4 is 1.20, peak 5 is 1.50, peak 6 is 1.81, and peak 7 is 2.

55. The relative standard deviation is within ±10%.

5. The method for constructing the characteristic spectrum of Poria cocos, red Poria cocos and Poria cocos peel according to claim 1, characterized in that: The characteristic spectrum of Poria peel medicinal material includes 8 common peaks, among which: Taking the uridine chromatographic peak of peak 2 as the reference peak S1, the relative retention times of peaks 1, 3 to 6 and S1 are consistent with: peak 1 is 0.95, peak 3 is 1.15, peak 4 is 1.43, peak 5 is 1.66, peak 6 is 1.79, and the relative standard deviation is within ±10%; Taking the adenosine peak 7 as the reference peak S2, the relative retention time of peak 8 and S2 peaks is consistent with that of peak 8 at 1.05, and the relative standard deviation is within ±10%.

6. The method for constructing the characteristic spectrum of Poria cocos, red Poria cocos and Poria cocos peel according to claim 1, characterized in that: The characteristic spectrum of red tuckahoe medicinal material includes 8 common peaks, among which: Taking the peak 3 uridine chromatographic peak as the reference peak S1, the relative retention times of peaks 1 to 2, 4 to 5 and S1 are consistent with: peak 1 is 0.32, peak 2 is 0.96, peak 4 is 1.20, peak 5 is 1.50, and the relative standard deviation is within ±10%; Taking the adenosine peak 6 as the reference peak S2, the relative retention times of peaks 7, 8 and S2 are consistent with: peak 7 is 1.04, peak 8 is 1.06, and the relative standard deviation is within ±10%.

7. Use of the method for constructing characteristic profiles of Poria cocos, red Poria cocos and Poria cocos peel according to any one of claims 1 to 6 in (1): (1) Differentiation and identification of Poria cocos, red Poria cocos and Poria cocos peel.

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