CN117783321A - Construction method and application of characteristic maps of poria cocos, red poria cocos and poria cocos peel - Google Patents

Abstract

The invention discloses a construction method of characteristic maps of poria cocos, red poria cocos and poria cocos peel, which comprises the following steps: preparing a reference substance solution, wherein the reference substance solution comprises a uridine reference substance, an adenosine reference substance and a guanosine reference substance; taking Poria, poria or Poria skin, and making into sample solution; and respectively taking a preset amount of sample solution and a preset amount of reference substance solution, and injecting the sample solution and the reference substance solution into a liquid chromatograph for measurement, wherein the liquid chromatograph uses octadecylsilane chemically bonded silica 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 map. The invention has good reproducibility, is accurate and reliable, and can rapidly and accurately identify the medicinal materials at different parts of the poria cocos according to the water-soluble components.

Description

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

Technical Field

The invention relates to the technical field of traditional Chinese medicine quality analysis and detection, in particular to a construction method and application of characteristic maps of poria cocos, red poria cocos and poria cocos skin.

Background

Poria is dry sclerotium of Polyporaceae fungus Poria, poria cocos (Schw.) Wolf, and is mainly parasitic on underground root of Pinus massoniana or underground pine or pine branch buried under soil, and is mainly distributed in Yunnan provinces such as Hubei, hunan, anhui, guizhou, etc. The white part of sclerotium is remained because the light red part of sclerotium is removed from the cortex and the near outer skin part of sclerotium in clinical application, so Poria is also called as Poria. Poria is a traditional Chinese medicine in China, which has flat nature, sweet and light taste, and enters heart, lung, spleen and kidney meridians; has the effects of promoting diuresis, removing dampness, strengthening spleen, tonifying middle-jiao, soothing nerves and calming heart, is a raw material of various prescriptions and Chinese patent medicines, and has a theory of ten medicines, nine poria cocos.

The outer skin of the sclerotium is the skin of Poria cocos, and the light red part of the sclerotium near the outer skin is called as "Poria cocos. Poria is used as a medicine, which is listed in Shen nong Ben Cao Jing (Shen nong's herbal), and is called as the superior product, which is "it is taken for a long time to calm spirit and nourish spirit, not hunger and prolong life". Regarding the use of Poria as a drug, the following "Sichuan traditional Chinese medicine Zhi" describes: poria cocos peel is neutral in nature, sweet and light in taste and nontoxic. Enter kidney and bladder meridians. The compendium of materia medica records poria peel, pachyma and distention due to edema, water and skin striae. "medical understanding" describes Poria "Fu Pi" Shen Shui (skin lotion). Regarding the medicinal value of Poria, the "new correction of the" Ben Cao gang mu "of Jinling book" describes Poria: the heart, small intestine and bladder damp-heat are used for inducing resuscitation and promoting diuresis. Therefore, the poria peel, the red poria and the poria are derived from the sclerotium of the poria and belong to the same body, but the medicinal parts are different, the efficacy and the effect are different, and the poria cannot be clinically mixed. The poria peel is the dried skin of the sclerotium of the poria cocos, and has the effects of inducing diuresis to alleviate edema; poria cocos is a pale red part near the exodermis, and has the effects of promoting diuresis, removing dampness and heat, tonifying heart and moistening lung; poria is a white part of peeled and cut red Poria, has effects of promoting diuresis, removing dampness, invigorating spleen and calming heart, and it can be seen that Poria is longer than Poria for strengthening spleen, removing dampness, red Poria is longer than Poria for promoting urination, and Poria skin is longer than Poria for promoting urination and detumescence.

The Chinese pharmacopoeia 2020 edition only records Poria and Poria cortex, and researches on different medicinal parts of Poria are limited to one medicinal part or two medicinal parts, and have not been conducted on different medicinal parts (Poria, poria and Poria cortex) of Poria. In addition, the current research on chemical components of poria cocos medicinal materials and different medicinal parts thereof is mostly focused on analysis of triterpene components, and the research on water-soluble components with large polarity is lacking. The traditional Chinese medicine takes decoction as a main administration form, water-soluble components in the decoction are one of important substance bases for the traditional Chinese medicine to exert the efficacy, and the research on the difference of the water-soluble components of different medicinal parts of poria cocos is not yet seen at present.

At present, the Chinese pharmacopoeia of 2020 edition only adopts appearance characters, chemistry and thin layer chromatography to identify and control the quality of poria cocos and poria cocos skin medicinal materials, and the accurate analysis of the poria cocos medicinal components is difficult, so that objective comprehensive evaluation of the quality of poria cocos decoction pieces is difficult. In recent years, various aspects of poria cocos have been studied increasingly, chemical components, pharmacological actions and application and development thereof have been paid attention to, but differences of chemical components of different medicinal parts are not clear yet. For the accurate safety of clinical medication, it is necessary to distinguish between tuckahoe in different locations.

In addition, the current poria cocos processing is mainly performed manually, the processing technology is relatively backward, the poria cocos medicinal materials have large quality differences, the processed commercial specifications are tens of, and the unified standard is not available. The clinical dosage of the red tuckahoe and tuckahoe skin is not large, a large amount of comprehensive development and utilization are not obtained, and most of the raw materials are treated as waste materials, so that great waste is caused. The light red brown and white parts of Poria are called Poria together in the "Chinese pharmacopoeia", but clinical application of Chinese medicine clearly distinguishes Poria from Poria (Poria), and Poria used in several prescriptions in the classical prescription in recent years is clearly noted as Poria. The characteristic spectrum research is used as a mature traditional Chinese medicine quality control method in the field of modern traditional Chinese medicines, has the advantages of high integrity, strong characteristics and the like, and can be used for representing the integrity, complexity and component variation difference of chemical components of the traditional Chinese medicines. Therefore, the invention provides a method for identifying the water-soluble characteristic spectrum of the poria cocos medicinal material, which can rapidly and accurately identify the medicinal materials at different parts of the poria cocos and provides scientific basis for the quality and commodity specification grade preparation of the poria cocos medicinal material.

Disclosure of Invention

The invention aims to solve the technical problem of providing a construction method of characteristic maps of poria cocos, red poria cocos and poria cocos skin, which is good in reproducibility, accurate and reliable, and can rapidly and accurately identify medicinal materials of different parts of poria cocos according to water-soluble components in the poria cocos medicinal materials, the red poria cocos medicinal materials and the poria cocos skin medicinal materials.

In order to solve the technical problems, the invention provides a construction method of characteristic maps of poria cocos, red poria cocos and poria cocos peel, which comprises the following steps:

preparing a reference substance solution, wherein the reference substance solution comprises a uridine reference substance, an adenosine reference substance and a guanosine reference substance;

taking Poria, poria or Poria skin, and making into sample solution;

and respectively taking a preset amount of sample solution and a preset amount of reference substance solution, and injecting the sample solution and the reference substance solution into a liquid chromatograph for measurement, wherein the liquid chromatograph uses octadecylsilane chemically bonded silica 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 map.

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

0 to 10min, the mobile phase A is 0, and the mobile phase B is 100%;

10 min-15 min, mobile phase A from 0 to 4%, mobile phase B from 100 to 96%;

15-28 min, mobile phase A from 4-6% and mobile phase B from 96-94%;

28-40 min, mobile phase A from 6% -30%, mobile phase B from 94% -70%;

40-43 min, mobile phase A from 30% -50% and mobile phase B from 70% -50%.

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

In one embodiment, the predetermined amount of the test solution or the control solution is 9. Mu.L to 11. Mu.L;

the liquid chromatograph takes octadecylsilane chemically bonded silica gel as a filler, the column length is 250mm, the inner diameter is 4.6mm, the particle size is 5 mu m, and the column temperature is 23-25 ℃;

when gradient elution is carried out, the flow speed of the mobile phase is 1.1 mL/min-1.3 mL/min;

the ultraviolet detection wavelength of the liquid chromatograph is 260 nm-270 nm.

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

grinding Poria, poria or Poria skin, and reflux extracting with methanol under heating to obtain the test solution.

In one embodiment, in the heating reflux extraction treatment, 8% -12% of methanol is added into the powder of the poria cocos medicinal material, the red poria cocos medicinal material or the poria cocos peel medicinal material, and the heating reflux is carried out for 2.5-3.5 hours.

In one embodiment, the characteristic spectrum of the poria cocos medicinal material comprises 7 common peaks, wherein the relative retention time of peak 1-peak 2, peak 4-peak 6, peak 7 and S peak accords with the peak 3 uridine chromatographic peak as a reference peak S: peak 1 was 0.31, peak 2 was 0.63, peak 4 was 1.20, peak 5 was 1.50, peak 6 was 1.81, peak 7 was 2.55, and the relative standard deviation was within.+ -. 10%.

In one embodiment, the characteristic spectrum of the poria peel medicinal material comprises 8 common peaks, wherein a peak 2 uridine chromatographic peak is taken as a reference peak S1, and the relative retention time of the peak 1, the peak 3-peak 6 and the peak S1 accords with that of the following peak: 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, the relative standard deviation is within + -10%;

the relative retention times of peak 8 and S2 peaks were in agreement with the peak 7 adenosine chromatographic peak as reference peak S2: peak 8 was 1.05, within ±10% of the relative standard deviation.

In one embodiment, the characteristic spectrum of the red poria cocos medicinal material comprises 8 common peaks, wherein the peak 3 uridine chromatographic peak is taken as a reference peak S1, and the relative retention time of the peak 1 to the peak 2, the peak 4 to the peak 5 and the peak S1 accords with the following conditions: peak 1 was 0.32, peak 2 was 0.96, peak 4 was 1.20, peak 5 was 1.50, and the relative standard deviation was within ±10%;

the relative retention times of peak 7, peak 8 and S2 peaks, with the peak 6 adenosine chromatographic peak as reference peak S2, were in agreement: peak 7 was 1.04 and peak 8 was 1.06, with a relative standard deviation within + -10%.

The invention also provides application of the construction method of the characteristic maps of the poria cocos, the red poria cocos and the poria cocos peel in (1) or (2):

(1) Identifying and controlling the quality of medicinal parts of Poria;

(2) Distinguishing and identifying poria cocos medicinal materials, red poria cocos medicinal materials and poria cocos skin medicinal materials.

The implementation of the invention has the following beneficial effects:

the invention establishes a method for constructing characteristic maps of poria cocos, red poria cocos and poria cocos peel, which is quick, stable and strong in specificity, realizes identification of different parts of poria cocos according to water-soluble components in poria cocos medicinal materials, red poria cocos medicinal materials and poria cocos peel medicinal materials, and provides a scientific new method for quality control of the poria cocos medicinal materials.

Drawings

FIG. 1 is a characteristic map of Poria cocos (Poria cocos) medicinal materials with different extraction solvents;

FIG. 2 is a characteristic map of Poria cocos medicinal materials with different extraction modes;

FIG. 3 is a characteristic map of Poria cocos at different extraction times;

FIG. 4 is a characteristic spectrum of Poria cocos medicinal materials of different chromatographic columns;

FIG. 5 is a characteristic map of Poria cocos medicinal materials with different concentration mobile phases;

FIG. 6 is a full-wavelength scan of a characteristic map of Poria cocos;

FIG. 7 is a characteristic spectrum of Poria cocos medicinal materials with different detection wavelengths;

FIG. 8 is a characteristic map of Poria cocos medicinal materials with different column temperatures;

FIG. 9 is a characteristic map of Poria cocos medicinal materials with different flow rates;

fig. 10 is a specific investigation result of the characteristic spectrum of the poria cocos medicinal material;

FIG. 11 is a graph showing the superposition of characteristic maps of 24 batches of Poria cocos medicinal materials;

FIG. 12 is a superimposed graph of the characteristic maps of 22 batches of poria peel medicinal material;

FIG. 13 is a superimposed graph of characteristic maps of 3 batches of Poria cocos wolf medicinal materials;

FIG. 14 is a diagram of the superposition of a test solution and a control solution;

FIG. 15 is a UV diagram (190 nm-400 nm) of a sample solution (left) and a uridine control solution (right);

FIG. 16 is a UV diagram (190 nm-400 nm) of a sample solution (left) and an adenine control solution (right);

FIG. 17 is a UV diagram (190 nm-400 nm) of a sample solution (left) and a guanosine control solution (right);

FIG. 18 is a UV diagram (190 nm-400 nm) of a test solution (left) and an adenosine control solution (right);

FIG. 19 is a UV diagram (190 nm-400 nm) of a sample solution (left) and a protocatechuic aldehyde control solution (right);

fig. 20 is a control profile of the poria cocos medicinal material, wherein peak 3 (S): uridine; peak 5: adenine; peak 6: guanosine; peak 7: adenosine;

fig. 21 is a comparative characteristic map of poria peel drug, wherein peak 2 (S1): uridine; peak 4: adenine; peak 6: guanosine; peak 7 (S2): adenosine; peak 8: protocatechuic aldehyde;

fig. 22 is a control feature map of red poria medicinal material, wherein peak 3 (S1): uridine; peak 5: adenine; peak 6 (S2): adenosine; peak 7: protocatechuic aldehyde;

fig. 23 is a comparison of characteristic patterns of Poria cocos, poria cocos skin and Poria cocos.

Detailed Description

The invention will be described in further detail with reference to the drawings and the detailed description, in order to make the objects, technical solutions and advantages more apparent.

1. Instrument and reagent

Instrument: waters high performance liquid chromatography (E2695, waters), waters HSS T3C 18 column (4.6 mm. Times.250 mm,5 μm), agilent ZORBAX SB-Aq column (4.6 mm. Times.250 mm,5 μm), YMC Triart C18 column (4.6 mm. Times.250 mm,5 μm), one-ten-thousandth balance (Metler-Toli Co., ME 204E), one-ten-thousandth balance (Metler-Toli Co., XP 26), an electrothermal thermostatic water bath (Shanghai-Henry Co., HWS-28), a digital controlled ultrasonic cleaner (Kunke ultrasonic instrument Co., ltd.), an ultrapure water system (Merck Co., milli-QDirect).

Reagent: ethanol (analytically pure, guangdong photowage technologies, inc.), methanol (analytically pure, guangdong photowage technologies, inc.); methanol (chromatographic grade, merck corporation), potassium dihydrogen phosphate (guangzhou chemical reagent factory), and water (laboratory homemade) were ultrapure water.

Reagent: uridine (China food and drug inspection institute, batch No. 110887-202104, content: 99.6%); adenosine (China food and drug inspection institute, batch No. 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 place of origin and the number are shown in Table 1.

Table 1 sample information table

2 chromatographic conditions and sample solution preparation

2.1 chromatographic conditions

By YMC Triart C ₁₈ Chromatographic column (4.6mm.times.250mm, 5 μm) with methanol as mobile phase A and 3mmol/L potassium dihydrogen phosphate solution as mobile phase B, and performing gradient elution according to the specifications in Table 2; the flow rate is 1.2ml per minute; the column temperature is 25 ℃; the detection wavelength was 260nm.

TABLE 2 gradient elution table

2.2 preparation of control solution A solution of 10. Mu.g of each 1ml of solution was prepared as control solution by adding 10% methanol to a proper amount of uridine control, adenosine control and guanosine control, precisely weighing.

2.3 preparation of sample solution about 2g of the sample powder (sieving with a third sieve), adding 25ml of 10% methanol, swirling for 1 min, adding 25ml of 10% methanol, weighing, heating and refluxing for 3 hours, cooling, weighing again, supplementing the weight of the sample solution with 10% methanol, filtering, collecting 25ml of subsequent filtrate, evaporating to dryness, fixing the volume of the residue to 2ml with 10% methanol, shaking, filtering, and collecting the subsequent filtrate.

2.4, respectively precisely sucking 10 mu L of the reference substance solution and 10 mu L of the sample solution, and injecting into a liquid chromatograph for measurement.

3 investigation of sample solution preparation method

3.1, taking about 2g of the same batch of poria cocos medicinal material powder (batch number: S12, screening by a third sieve) respectively, precisely weighing, placing into a conical bottle with a plug, precisely adding water, 10% methanol, 30% methanol, 50% methanol, 10% ethanol, 30% ethanol and 50% ethanol respectively, swirling for 1 min, respectively adding 25ml of the corresponding solvent, weighing, heating and refluxing for 3 hours, cooling, weighing again, supplementing the reduced weight by the corresponding solvent, shaking uniformly, filtering, taking 25ml of the subsequent filtrate, evaporating to dryness, fixing the volume of the residues to 2ml by the solvent respectively, shaking uniformly, filtering, taking the subsequent filtrate, and measuring according to chromatographic conditions under the item "2.1", wherein the result is shown in figure 1. The results showed that the 7 kinds of extraction solvents have a large influence on the peak pattern of each characteristic peak, and that when 10% methanol is used as the extraction solvent, the overall response of each characteristic peak is high, and therefore 10% methanol is selected as the extraction solvent.

3.2 examining the extraction mode, namely taking about 2g of the same batch of poria cocos medicinal material powder (batch number: S12, screening by a third sieve), precisely weighing, placing into a conical bottle with a plug, precisely adding 25ml of 10% methanol, swirling for 1 min, adding 25ml of 10% methanol, weighing, respectively carrying out ultrasonic treatment (power 300W, frequency 60 kHz) for 3 hours and heating and refluxing for 3 hours, cooling, weighing again, supplementing the weight loss by 10% methanol, filtering, taking 25ml of subsequent filtrate, evaporating, fixing the volume of residues to 2ml by 10% methanol, shaking uniformly, filtering, taking the subsequent filtrate, and measuring according to the chromatographic condition under the term of 2.1, wherein the result is shown in figure 2. The results show that the two extraction modes have no obvious difference in peak type and separation effect of each characteristic peak, but the overall response of each characteristic peak is higher than that of ultrasonic wave during heating reflux extraction, so that the extraction mode of heating reflux is selected.

3.3 investigation of extraction time, about 2g of the same batch of poria cocos medicinal material powder (batch number: S12, screening by a third sieve) is respectively taken, precisely weighed, placed in a conical bottle with a plug, precisely added with 25ml of 10% methanol, vortexed for 1 minute, then added with 25ml of 10% methanol, weighed by weight, respectively heated and refluxed for 0.5 hour, 1 hour, 2 hours, 3 hours and 5 hours, cooled, weighed by weight again, complemented by 10% methanol for weight loss, filtered, and the subsequent filtrate is taken, 25ml is evaporated, the residue is subjected to constant volume to 2ml by 10% methanol, shaken uniformly, filtered, and the subsequent filtrate is taken, and measured according to the chromatographic condition under the item "2.1", and the result is shown in fig. 3. The result shows that the response value of each characteristic peak is greatly influenced by different extraction time, and the heating reflux is selected for 3 hours by combining the whole peak response of each characteristic peak.

4 chromatographic condition optimization

4.1 selection of chromatographic column about 2g of Poria powder (lot number: S12, sieving with a third sieve) was used to prepare a test solution according to the method under item "2.3", and the measurement was carried out under the following chromatographic conditions. Chromatographic conditions: gradient elution was performed as specified in table 3 with 0.1% phosphoric acid as mobile phase a and methanol as mobile phase B; the flow rate is 1.0ml per minute, and the column temperature is 30 ℃; the sample loading was 10. Mu.l.

The results show that when the hydrophilic chromatographic column of different brands is analyzed by taking a pure water item as an initial concentration, more large polar components which cannot be retained still exist at the solvent peak, and the results are shown in fig. 4, but the YMC Triart C18 chromatographic column presents more information and has better separation degree, so that the chromatographic column is adopted for further research.

TABLE 3 gradient elution table

4.2 taking about 2g of poria cocos medicinal material powder (batch number: S12, sieving with a third sieve) by mobile phase optimization, preparing a sample solution according to a method under a '2.3' item, referring to chromatographic condition sample injection analysis under a '2.1' item, respectively observing different buffer salt concentrations, and as shown in a result of FIG. 5, the influence of the different buffer salt concentrations on the separation degree of each characteristic peak is large, and when the concentration of potassium dihydrogen phosphate is 3mmol/L, the separation degree of each characteristic peak is good and the peak type is symmetrical, so that the system is adopted for further investigation.

4.3 selecting detection wavelength, taking about 2g of poria cocos medicinal material powder (batch number: S12, sieving with a third sieve), preparing a sample solution according to the method under the item "2.3", analyzing by referring to chromatographic condition sample injection under the item "2.1", and recording an absorption spectrum within the range of 190-400 nm. As shown in fig. 6 and 7, by comparing chromatograms of different detection wavelengths, it is found that at a wavelength of 260nm, the peak response and the information amount are more abundant, the interference is small, and the separation degree is better, so that 260nm is selected as the detection wavelength in consideration.

4.4 selecting the column temperature, taking about 2g of poria cocos medicinal material powder (batch number: S12, sieving with a third sieve), preparing a sample solution according to the method under the item "2.3", and carrying out sample injection analysis under the conditions of other than the column temperature under the item "2.1". As shown in FIG. 8, by comparing the chromatograms of 3 different column temperatures, when 23 ℃ and 25 ℃ are selected as the column temperatures, the information of chromatographic peaks is more complete, the separation degree is better, and the column temperature of 25 ℃ is selected as the column temperature for establishing the tuckahoe water-soluble characteristic map in consideration of the performances of different chromatographs.

4.5 selecting Poria powder (batch number: S12, sieving with a third sieve) about 2g, preparing sample solution according to the method under item "2.3", and performing sample injection analysis under the same chromatographic conditions as under item "2.1" except for the flow rate. As shown in FIG. 9, by comparing the chromatograms of 3 different flow rates, when the flow rate is 1.2ml/min, the information of each chromatographic peak is complete, the separation degree of each chromatographic peak is better, and 1.2ml/min is comprehensively considered to be selected as the flow rate for establishing the tuckahoe water-soluble characteristic map.

5 methodology investigation

5.1 investigation of specificity about 2g of Poria powder (batch number: S12, sieving with a third sieve) is taken, a sample solution is prepared according to the method under item "2.3", 10 μl of each of the reference substance solution and the blank solvent under item "2.2" is precisely sucked up, and sample injection analysis is performed according to the chromatographic condition under item "2.1". The results are shown in FIG. 10, and the sample chromatogram has the same chromatographic peak at the retention time corresponding to the reference chromatogram, and the blank solvent has no interference, which indicates that the method has good specificity.

5.2 examining the precision, about 2g of tuckahoe medicinal powder (batch number: S12, sieving with a third sieve) is taken, a sample solution is prepared according to the method under the item "2.3", and sample injection is repeated for 6 times according to the chromatographic condition under the item "2.1". The relative retention time and the relative peak area of each characteristic peak and the S peak are calculated by taking the uridine chromatographic peak as a reference peak S, and the RSD value is calculated, and the result shows that the relative retention time RSD value of 7 common peaks is in the range of 0.12% -0.49%, and the relative peak area RSD value is in the range of 0.76% -3.02%, which indicates that the instrument precision is good.

5.3 repeatability investigation about 2g of Poria powder (batch number: S12, sieving with a third sieve) is taken, 6 parts of sample solution is prepared according to the method under the item "2.3", and sample injection measurement is carried out according to the chromatographic condition under the item "2.1". The relative retention time and relative peak area of each characteristic peak and the S peak are calculated by taking the uridine chromatographic peak as a reference peak, and the RSD value is calculated, and the result shows that the relative retention time RSD value of 7 common peaks is in the range of 0.18% -2.05%, and the relative peak area RSD value is in the range of 0.73% -3.17%, which indicates that the method has good repeatability.

5.4 stability examination about 2g of Poria powder (batch number: S12, sieving with a third sieve) was taken, a sample solution was prepared according to the method under "2.3", and samples were taken and assayed under the chromatographic conditions under "2.1" under 0,4,6,8, 10, 12, 20, 24 hours, respectively. The relative retention time and relative peak area of each characteristic peak and the S peak are calculated by taking the uridine chromatographic peak as a reference peak S, the RSD value is calculated, the relative retention time RSD value of 7 common peaks is in the range of 0.10-0.48%, and the relative peak area RSD value is in the range of 0.44-3.28%, which indicates that the sample solution is relatively stable within 24 hours.

5.5 intermediate precision investigation was performed by different analysts on different laboratories and different brands of instruments. About 2g of poria cocos medicinal material powder (batch number: S12, sieving with a third sieve) is taken, 6 parts of sample solution is prepared according to the method under the item "2.3", and sample injection measurement is carried out according to the chromatographic condition under the item "2.1". The relative retention time and relative peak area of each characteristic peak and the S peak are calculated by taking the uridine chromatographic peak as a reference peak S, and the RSD value is calculated, and the result shows that the relative retention time RSD value of 7 common peaks is in the range of 0.06% -0.20%, and the relative peak area RSD value is in the range of 0.60% -4.12%, which indicates that the established method can be reproduced under different conditions of different equipment, laboratories and the like, and the intermediate precision of the method is good.

6 establishing characteristic maps of poria cocos, poria cocos skin and poria cocos

6.1 determination of characteristic Properties of Poria, poria skin and Poria cocos samples

Taking 24 batches of poria cocos medicinal materials, 22 batches of poria cocos skin medicinal materials and 3 batches of red poria cocos medicinal materials, respectively preparing a sample solution according to a method under a '2.3' item, carrying out sample injection measurement according to a chromatographic condition under a '2.1' item, recording a characteristic spectrum chromatogram, carrying out automatic integration treatment on each sample fingerprint, deriving the integrated characteristic spectrum in a CDF format, respectively introducing the 24 batches of poria cocos medicinal material characteristic maps, 22 batches of poria cocos skin medicinal material characteristic maps and 3 batches of red poria cocos medicinal material characteristic spectrum CDF format into traditional Chinese medicine chromatographic fingerprint similarity evaluation software (2012.130723 edition), carrying out peak matching and multipoint correction, as shown in the results of fig. 11-13, identifying 7 common characteristic peaks in total for the 24 batches of poria cocos medicinal material characteristic maps, adopting relative retention time positioning, and calculating peak 1-2, peak 4-6, peak 7-S peak relative retention time average values of peak 1.31 (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 spectrum of 22 batches of poria peel medicinal materials is marked with 8 common characteristic peaks, relative retention time positioning is adopted, peak 2 uridine chromatographic peak is taken as reference peak S1, relative retention time of peak 1, peak 3-6 and S1 peak is calculated, peak 7 adenosine chromatographic peak is taken as reference peak S2, and relative retention time of peak 8 and S2 peak is calculated, wherein the average value is 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); the characteristic spectrum of 3 batches of red poria medicinal materials is marked with 8 common characteristic peaks, relative retention time positioning is adopted, peak 3 uridine chromatographic peaks are used as reference peaks S1, relative retention time of peaks 1-2, peaks 4-5 and S1 peaks is calculated, peak 6 adenosine chromatographic peaks are used as reference peaks S2, and relative retention time of peaks 7-8 and S2 peaks is calculated, wherein the average value is 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).

6.2 identification of characteristic peaks

In addition to the polysaccharides and triterpenes as main components, poria has been reported to contain steroid components, fatty acids, proteins, adenine, amino acids, and inorganic elements such as calcium, magnesium, iron, and potassium. In order to determine the common peaks in the characteristic spectrum of Poria cocos, reference substances such as uridine, guanosine, adenosine, adenine and protocatechuic acid are adopted for identification, and the chemical components are adopted. As a result, it was found that chromatographic peaks consistent with retention times and spectra of uridine, adenine, guanosine, adenosine, protocatechuic aldehyde were found in the characteristic spectra, see FIGS. 14 to 19.

6.3 establishment of a common mode of characteristic maps of Poria cocos, poria cocos skin and Poria cocos

Respectively introducing 24 batches of poria cocos medicinal material characteristic maps, 22 batches of poria cocos skin medicinal material characteristic maps and 3 batches of red poria cocos medicinal material characteristic maps CDF format into traditional Chinese medicine chromatographic fingerprint similarity evaluation software (2012.130723 edition), performing multi-point correction and peak matching, and respectively generating poria cocos, poria cocos skin and red poria cocos reference maps according to an average method, wherein the results are shown in figures 20-22.

According to the measurement results of characteristic patterns of 24 batches of poria cocos samples, determining the characteristic pattern standard of the poria cocos medicinal materials as follows: the characteristic spectrum should show 7 characteristic peaks, the peak corresponding to the uridine reference substance peak is S peak, the relative retention time of peak 1-2, peak 4-6, peak 7 and S peak is calculated, the relative retention time is within + -10% of the specified value, 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).

According to the measurement results of 22 batches of poria peel sample characteristic patterns, determining the poria peel medicinal material characteristic pattern standard as follows: the characteristic spectrum should show 8 characteristic peaks, the peak corresponding to the uridine reference substance peak is S1 peak, the relative retention time of peak 1, peak 3-6 and S1 peak is calculated, the relative retention time should be within + -10% of the specified value; the peak corresponding to the adenosine reference peak is the S2 peak, the relative retention time of the peak 8 and the S2 peak is calculated, the relative retention time is within +/-10% of the specified value, and the specified value is: 0.95 (Peak 1), 1.15 (Peak 3), 1.43 (Peak 4), 1.66 (Peak 5), 1.79 (Peak 6), 1.05 (Peak 8).

According to the measurement results of 3 batches of Poria cocos sample characteristic patterns, determining Poria cocos medicinal material characteristic pattern standards as follows: the characteristic spectrum should show 8 characteristic peaks, the peak corresponding to the uridine reference substance peak is S1 peak, the relative retention time of peak 1-2, peak 4-5 and S1 peak is calculated, the relative retention time should be within + -10% of the specified value; the peak corresponding to the adenosine control reference peak was 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) were calculated.

6.4 differentiation and identification of Poria, poria skin and Poria cocos

As shown in fig. 23, the characteristic patterns of Poria cocos, poria cocos peel and Poria cocos have the same 4 common peaks, and compared with Poria cocos, poria cocos peel and Poria cocos peel each have a higher response chromatographic peak a at 39 minutes of chromatographic retention time, compared with Poria cocos peel, poria cocos peel has a higher response chromatographic peak B at 40 minutes of chromatographic retention time, so as to distinguish and identify Poria cocos, poria cocos peel and Poria cocos peel.

Table 4 comparison of the areas of the peaks of protocatechuic aldehyde and adenosine of Poria cocos, poria cocos peel and Poria cocos

As shown in the results of Table 4, the area ratio of protocatechuic aldehyde to adenosine peak in the poria cocos medicinal material ranges from 0.000 to 0.084, and the average value is 0.01; the peak area ratio of the poria peel medicinal material ranges from 0.167 to 1.359, and the average value is 0.54; the peak area ratio of the red poria medicinal material ranges from 5.903 to 7.996, and the average value is 6.90. The area ratio of protocatechuic aldehyde to adenosine peak in the red poria medicinal material is far higher than that of the poria medicinal material and the poria peel medicinal material, and a chromatographic peak with higher response is arranged at 40 minutes; the ratio of the poria peel medicinal material is obviously higher than that of the poria medicinal material, namely the protocatechuic aldehyde/adenosine peak area ratio: poria with red color > Poria skin > Poria. Therefore, the water-soluble characteristic spectrum method established by the invention can effectively distinguish the poria cocos medicinal material, the red poria cocos medicinal material and the poria cocos peel medicinal material.

While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that changes and modifications may be made without departing from the principles of the invention, and such changes and modifications are intended to be included within the scope of the invention.

Claims (10)

1. The construction method of the characteristic maps of the poria cocos, the red poria cocos and the poria cocos peel is characterized by comprising the following steps:

preparing a reference substance solution, wherein the reference substance solution comprises a uridine reference substance, an adenosine reference substance and a guanosine reference substance;

taking Poria, poria or Poria skin, and making into sample solution;

and respectively taking a preset amount of sample solution and a preset amount of reference substance solution, and injecting the sample solution and the reference substance solution into a liquid chromatograph for measurement, wherein the liquid chromatograph uses octadecylsilane chemically bonded silica 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 map.

2. The method for constructing characteristic maps of poria cocos wolf, poria cocos wolf and poria cocos wolf skin according to claim 1, wherein the gradient elution is carried out according to the following procedure:

0 to 10min, the mobile phase A is 0, and the mobile phase B is 100%;

10 min-15 min, mobile phase A from 0 to 4%, mobile phase B from 100 to 96%;

15-28 min, mobile phase A from 4-6% and mobile phase B from 96-94%;

28-40 min, mobile phase A from 6% -30%, mobile phase B from 94% -70%;

40-43 min, mobile phase A from 30% -50% and mobile phase B from 70% -50%.

3. The method for constructing the characteristic maps of poria cocos, poria cocos and poria cocos peel according to claim 1, wherein the concentration of the potassium dihydrogen phosphate solution is 2.5 mmol/L-3.5 mmol/L.

4. The method for constructing the characteristic maps of poria cocos, poria cocos and poria cocos peel according to claim 1, wherein the preset amount of the test solution or the reference solution is 9-11 mu L;

the liquid chromatograph takes octadecylsilane chemically bonded silica gel as a filler, the column length is 250mm, the inner diameter is 4.6mm, the particle size is 5 mu m, and the column temperature is 23-25 ℃;

when gradient elution is carried out, the flow speed of the mobile phase is 1.1 mL/min-1.3 mL/min;

the ultraviolet detection wavelength of the liquid chromatograph is 260 nm-270 nm.

5. The method for constructing characteristic maps of poria cocos, poria cocos and poria cocos peel according to claim 1, wherein the sample solution is prepared by the following method:

grinding Poria, poria or Poria skin, and reflux extracting with methanol under heating to obtain the test solution.

6. The method for constructing feature maps of poria cocos, red poria cocos and poria cocos peel according to claim 5, wherein 8% -12% of methanol is added into powder of poria cocos medicinal materials, red poria cocos medicinal materials or poria cocos peel medicinal materials in the heating reflux extraction treatment, and heating reflux is carried out for 2.5-3.5 hours.

7. The method for constructing characteristic maps of poria cocos, poria cocos and poria cocos peel according to claim 1, wherein the characteristic maps of poria cocos medicinal materials comprise 7 common peaks, wherein relative retention time of peak 3 uridine chromatographic peak as reference peak S, peak 1-peak 2, peak 4-peak 6, peak 7 and S peak is in accordance with: peak 1 was 0.31, peak 2 was 0.63, peak 4 was 1.20, peak 5 was 1.50, peak 6 was 1.81, peak 7 was 2.55, and the relative standard deviation was within.+ -. 10%.

8. The method for constructing the characteristic maps of poria cocos, red poria cocos and poria cocos peel according to claim 1, wherein the characteristic maps of poria cocos peel medicinal materials comprise 8 common peaks, wherein peak 2 uridine chromatographic peak is taken as a reference peak S1, and the relative retention time of peak 1, peak 3-peak 6 and S1 peak is consistent with that: 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, the relative standard deviation is within + -10%;

the relative retention times of peak 8 and S2 peaks were in agreement with the peak 7 adenosine chromatographic peak as reference peak S2: peak 8 was 1.05, within ±10% of the relative standard deviation.

9. The method for constructing characteristic maps of poria cocos, poria cocos and poria cocos peel according to claim 1, wherein the characteristic maps of poria cocos comprise 8 common peaks, wherein peak 3 uridine chromatographic peak is taken as a reference peak S1, and the relative retention time of peak 1-peak 2, peak 4-peak 5 and S1 peak is consistent with that: peak 1 was 0.32, peak 2 was 0.96, peak 4 was 1.20, peak 5 was 1.50, and the relative standard deviation was within ±10%;

the relative retention times of peak 7, peak 8 and S2 peaks, with the peak 6 adenosine chromatographic peak as reference peak S2, were in agreement: peak 7 was 1.04 and peak 8 was 1.06, with a relative standard deviation within + -10%.

10. The use of the method for constructing feature maps of Poria cocos, poria cocos and Poria cocos peel according to any one of claims 1 to 9 in (1) or (2):

(1) Identifying and controlling the quality of medicinal parts of Poria;

(2) Distinguishing and identifying poria cocos medicinal materials, red poria cocos medicinal materials and poria cocos skin medicinal materials.