A method for constructing a characteristic map of lotus seeds and a method for distinguishing between heart-removed lotus seeds and non-heart-removed lotus seeds

Abstract

The application discloses a method for constructing a characteristic spectrum of lotus seeds and a method for distinguishing between heart-removed lotus seeds and non-heart-removed lotus seeds, and comprises the following steps: adopting an ultra-high performance liquid chromatography method, and reasonably controlling the chromatography method, taking acetonitrile as a mobile phase A and taking a 0.1% formic acid solution as a mobile phase B to perform gradient elution, and establishing seven common peaks; the whole picture of the characteristic spectrum of the lotus seeds is constructed; and the heart-removed lotus seeds and the non-heart-removed lotus seeds are compared and studied, the internal quality of the whole lotus seed medicinal materials, decoction pieces, standard decoction, intermediates and formula granules is controlled, the characteristics of the lotus seeds can be comprehensively reflected, a new analysis means can be provided for internal quality control of the lotus seed standard decoction and the formula granules, and the purpose of identifying whether the lotus seeds are heart-removed or not is achieved. The method is simple, reproducible, accurate and reliable, convenient to operate, short in time, low in solvent consumption and small in environmental pollution.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for constructing a characteristic spectrum of lotus seeds and a method for distinguishing between lotus seeds with and without hearts, belonging to the technical field of traditional Chinese medicine identification. BACKGROUND

[0002] Lotus seeds are the dried mature seeds of Nelumbo nucifera Gaertn. of the Nelumbonaceae family, have the effects of tonifying the spleen to stop diarrhea, stopping leucorrhea, benefiting the kidney to astringe essence, nourishing the heart and tranquilizing the mind, and are used to treat spleen deficiency diarrhea, leucorrhea, spermatorrhea, palpitation and insomnia, etc. Modern pharmacological studies have shown that lotus seeds have the effects of antioxidant, anti-aging, and enhancing immunity, etc.

[0003] In existing literature, the detection of the quality of lotus seeds is mostly for lotus seed medicinal materials. For example, Wu Yanbin et al. established a fingerprint spectrum method of lotus seeds by high performance liquid chromatography to evaluate the quality of lotus seeds of different germplasm for standardized production (Wu Yanbin, Zheng Yuanbin, Wu Jinzhong. HPLC fingerprint spectrum evaluation of different germplasm of lotus seed quality [J]. Journal of Fujian University of Traditional Chinese Medicine, 2009, 19(2): 28-30); Yi Jun et al. established a fingerprint spectrum method of lotus seeds by high performance liquid chromatography to evaluate the quality of lotus seeds of different harvest times (Yi Jun, Zheng Yuanbin, Wu Jinzhong. HPLC fingerprint spectrum evaluation of different harvest times of lotus seed quality [J]. Fujian Traditional Chinese Medicine, 2010, 41(4): 37-39). The problems of the existing methods for detecting the quality of lotus seeds are long detection time, high detection cost, and uneven baseline of chromatogram, and there is no identification of chromatographic peaks, lack of characteristics, and cannot effectively control the quality of lotus seeds. There is no related research on the quality detection of lotus seed formula granules, decoction pieces, processed products, preparation intermediates or preparation finished products.

[0004] In addition, the current edition of Chinese Pharmacopoeia does not make clear requirements for lotus seed heart removal under lotus seed medicinal materials, and there are three sources of lotus seeds with heart, lotus seeds without heart or incomplete lotus seeds with heart in market circulation. However, it is clearly stipulated that when lotus seed decoction pieces are used, the heart is removed, slightly soaked, moistened, cut open, dried, or crushed and removed. The heart is directly used or crushed without heart. For traditional Chinese medicine preparation enterprises, it is undoubtedly the best choice to purchase lotus seeds with heart, but there is no quality control method for controlling the internal quality of lotus seed medicinal materials, decoction pieces and related preparation products. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a simple, fast and comprehensive method for constructing a characteristic spectrum of lotus seeds and a method for distinguishing between lotus seeds with and without hearts.

[0006] Technical scheme: To solve the above technical problems, the present application provides a method for constructing a characteristic spectrum of lotus seeds, comprising the following steps:

[0007] (1) respectively take lotus seed medicinal materials, decoction pieces, standard decoction or its formula granules powder, add organic solvent, extract, cool, filter, continue the filtrate as the test solution;

[0008] (2) take protocatechuic acid, add solvent to prepare the control solution;

[0009] (3) take the test solution and the control solution, respectively injected into the ultra-high performance liquid chromatograph, the characteristic spectrum is obtained;The characteristic spectrum obtained has 7 characteristic peaks, peak 4 is protocatechuic acid, the relative retention time of the rest of each characteristic peak and peak 4 is calculated, the relative retention time should be within ± 10% of the specified value, the specified value of peak 1-3 is calculated as follows: 0.30, 0.51, 0.80, and the specified value of peak 5-7 is calculated as follows: 1.44, 1.83, 2.03;The chromatographic conditions of the ultra-high performance liquid chromatograph are as follows: chromatographic column: CORTECS UPLC T3, Eclipse Plus C18 or Acquity UPLC HSS T3;Column temperature: 20-30℃;Flow rate: 0.15-0.20ml / min;Detection wavelength: 240-272nm;Gradient elution is carried out with acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B;Gradient elution is as follows: 0-7min, the volume fraction of mobile phase A is 1→2%, and the volume fraction of mobile phase B is 99→98%;7-30min, the volume fraction of mobile phase A is 2→10%, and the volume fraction of mobile phase B is 98→90%.

[0010] Among them, the organic solvent in step (1) includes water or 10% methanol.

[0011] Among them, the extraction method in step (1) includes ultrasonic, shaking or reflux;The extraction time is 15-60 minutes.

[0012] The application also provides a method for distinguishing between lotus seeds with and without core, comprising the following steps:

[0013] (1) respectively take lotus seed medicinal materials or decoction pieces powder, add organic solvent, extract, cool, filter, continue the filtrate as the test solution;The organic solvent includes water or 10% methanol;

[0014] (2) take protocatechuic acid and p-coumaric acid-4-O-β-D-glucopyranoside, respectively add solvent to prepare the control solution;

[0015] (3) take the test solution and the control solution, respectively injected into the ultra-high performance liquid chromatograph, and the chromatogram is recorded by ultra-high performance liquid chromatography detection;

[0016] (4) In the chromatogram, select the peak with stable relative retention time as the characteristic peak, and then compare it with the peak of the control solution to determine 7 characteristic peaks; peak 4 is protocatechuic acid, and the relative retention time of the remaining characteristic peaks to peak 4 should be within ±10% of the specified value; the specified values of peaks 1-3 are 0.30, 0.51 and 0.80 respectively, and the specified values of peaks 5-7 are 1.44, 1.83 and 2.03 respectively; peak A is p-coumaric acid-4-O-β-D-glucopyranoside;

[0017] (5) Calculate the K value by using the formula ; wherein, S A is the peak area of characteristic peak A, and S1 is the peak area of peak 1;

[0018] (6) When K≤0.15, the lotus seed medicinal material or decoction piece to be tested is a heart-removed lotus seed medicinal material or decoction piece; when K>0.15, the lotus seed medicinal material or decoction piece is a non-heart-removed lotus seed medicinal material or decoction piece.

[0019] In step (1), the chromatographic conditions of the ultra-high performance liquid chromatograph are as follows: a chromatographic column: CORTECS UPLCT3, Eclipse Plus C18 or Acquity UPLC HSS T3; column temperature: 20-30℃; flow rate: 0.15-0.20ml / min; detection wavelength: 240-272nm; gradient elution is performed with acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B; gradient elution is as follows: 0-7min, the volume fraction of mobile phase A is 1→2%, and the volume fraction of mobile phase B is 99→98%; 7-30min, the volume fraction of mobile phase A is 2→10%, and the volume fraction of mobile phase B is 98→90%.

[0020] In step (1), the extraction method includes ultrasonic extraction, shaking extraction or reflux extraction; the extraction time is 15-60 minutes.

[0021] The application also provides a method for distinguishing heart-removed lotus seeds and non-heart-removed lotus seeds standard decoction or formula granules, which comprises the following steps:

[0022] (1) Respectively take lotus seed standard decoction or formula granule powder to be tested, add an organic solvent, extract, cool, filter, and the filtered solution is the test solution; the organic solvent includes water or 10% methanol;

[0023] (2) Take protocatechuic acid and p-coumaric acid-4-O-β-D-glucopyranoside, and respectively add a solvent to prepare a control solution;

[0024] (3) Take the test solution and the control solution, respectively inject them into an ultra-high performance liquid chromatograph, perform ultra-high performance liquid chromatography detection, and record the chromatogram;

[0025] (4) In the chromatogram, select the peak with stable relative retention time as the characteristic peak, and then compare it with the peaks of the reference solution chromatogram to determine 7 characteristic peaks; peak 4 is protocatechuic acid, and the relative retention time of each characteristic peak to peak 4 should be within ±10% of the specified value. The specified values of peaks 1-3 are 0.30, 0.51, and 0.80, respectively, and the specified values of peaks 5-7 are 1.44, 1.83, and 2.03, respectively; peak A is p-coumaric acid-4-O-β-D-glucopyranoside;

[0026] (5) Calculate the K value using the formula ; wherein S A is the peak area of characteristic peak A, and S1 is the peak area of peak 1;

[0027] (6) When K≤0.17, the lotus seed standard decoction or formula granules to be tested are heartless lotus seed standard decoction or formula granules; if K>0.17, the lotus seed standard decoction or formula granules to be tested are not heartless lotus seed standard decoction or formula granules.

[0028] In step (1), the extraction method includes ultrasonic extraction, shaking extraction, or reflux extraction; the extraction time is 15-60 minutes.

[0029] In step (1), the chromatographic conditions of the ultra-high performance liquid chromatograph are as follows: chromatographic column: CORTECS UPLCT3, Eclipse Plus C18, or Acquity UPLC HSS T3; column temperature: 20-30°C; flow rate: 0.15-0.20 ml / min; detection wavelength: 240-272 nm; gradient elution is performed with acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B; gradient elution is as follows: 0-7 min, volume fraction of mobile phase A: 1→2%, volume fraction of mobile phase B: 99→98%; 7-30 min, volume fraction of mobile phase A: 2→10%, volume fraction of mobile phase B: 98→90%.

[0030] In step (1), the mass-volume ratio of lotus seed medicinal material, decoction piece, standard decoction, or formula granule powder to organic solvent can be within a reasonable range, and is preferably 1:(10-100) g / ml.

[0031] Further, in step (3), the extraction solvent for preparing the reference solution of the reference medicinal material is water or a 10-30% methanol aqueous solution, the extraction time is 30-90 minutes, and the extraction method is heating reflux.

[0032] The characteristic spectrum method for detecting lotus seeds can be applied to quality detection of lotus seed medicinal materials, decoction pieces, standard decoctions and formula granules, and can distinguish between heart-removed lotus seeds and non-heart-removed lotus seeds.

[0033] The application establishes a quality control method for lotus seeds, controls the internal quality of the whole lotus seed medicinal materials, decoction pieces and related preparation products, provides a new analysis means for the internal quality control of lotus seed preparation products, and achieves the purpose of identifying whether the heart-removal degree of lotus seeds meets the requirements.

[0034] Advantages: compared with the prior art, the application has the following remarkable advantages: 1. The method is simple, reproducible, accurate and reliable, easy to operate, short in time, low in solvent consumption, small in environmental pollution; 2. The application establishes seven common peaks by adopting the chromatographic method for comparing and researching the ingredient differences of heart-removed lotus seeds and non-heart-removed lotus seeds, adopting the ultra-high performance liquid chromatography method, and reasonably controlling the chromatographic method to perform gradient elution with acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B; the whole picture of the characteristic spectrum of lotus seeds is constituted, and the heart-removed lotus seeds and non-heart-removed lotus seeds are compared and researched, the internal quality of the whole lotus seed medicinal materials, decoction pieces, standard decoctions, intermediates and formula granules is controlled, the characteristics of lotus seeds can be comprehensively reflected, a new analysis means can be provided for the internal quality control of lotus seed standard decoctions and formula granules, and the purpose of identifying whether the lotus seeds are heart-removed or not is achieved. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 The Isoabsorbance Plot graph of the lotus seed formula granule;

[0036] Figure 2 The UPLC chromatogram of the lotus seed formula granule under different detection wavelengths;

[0037] Figure 3 The UPLC graph investigated by different extraction solvents;

[0038] Figure 4 Comparison of extraction efficiency of different extraction solvents (peak area / sample weight);

[0039] Figure 5 The UPLC graph investigated by different extraction methods;

[0040] Figure 6 Comparison of extraction efficiency of different extraction methods (peak area / sample weight);

[0041] Figure 7 The UPLC graph investigated by different extraction times;

[0042] Figure 8 Comparison of extraction efficiency of different extraction times (peak area / sample weight);

[0043] Figure 9 Specificity test of lotus seed formula granules;

[0044] Figure 10 Separation effect of different chromatographic columns;

[0045] Figure 11 Influence of column temperature on separation effect;

[0046] Figure 12 Influence of flow rate on separation effect;

[0047] Figure 13 Lotus seed medicinal material multi-batch characteristic spectrum;

[0048] Figure 14 Lotus seed standard decoction multi-batch characteristic spectrum;

[0049] Figure 15 Lotus seed formula granules multi-batch characteristic spectrum;

[0050] Figure 16 Lotus seed medicinal material, decoction piece, standard decoction, intermediate, formula granule characteristic spectrum;

[0051] Figure 17 Lotus seed formula granules and reference solution of reference substance UPLC diagram of control product;

[0052] Figure 18 Comparison of characteristic spectrum of heart-removed lotus seed medicinal material and non-heart-removed lotus seed medicinal material;

[0053] Figure 19 Comparison of characteristic spectrum of heart-removed lotus seed standard decoction and non-heart-removed lotus seed standard decoction. DETAILED DESCRIPTION

[0054] The technical solutions of the present application will be further described below in combination with the drawings.

[0055] Example 1

[0056] This example is used to confirm the optimal scheme of lotus seed quality evaluation method, specifically including comparison of chromatographic conditions, wavelength selection, preparation of reference solution, preparation scheme of test solution, and confirmation of feasibility of the method through methodological validation.

[0057] 1. Instruments and samples

[0058] Agilent 1290 UPLC ultra-high performance liquid chromatograph (Agilent Company); OpenLAB CDS workstation (Agilent Company); Waters-ACQUIYT-UPLC-H-Class, ultra-high performance liquid chromatography system; Waters-Quaternary-Solvent-Manager-four element pump; Sample-manager-FTN automatic sampler; Waters-UPLC-PDA detector; Empower-3 chromatography workstation; Agilent G6530 Accurate-Mass Q-TOF mass spectrometry (Agilent Company); Agilent Mass Hunter Workstation data acquisition and qualitative analysis software (Agilent Company); electronic analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); PL-J100 mechanical ultrasonic cleaner (Dongguan Kangshi Ultrasonic Technology Co., Ltd.); HY-4 speed-adjustable multi-purpose oscillator (Kangxin Instrument Factory, Jintan); HH-4 constant-temperature digital water bath (Guohua Electric Co., Ltd., Changzhou); Milli-Q water system (Millipore Corporation); TGL-16C centrifuge (Shanghai Anting Scientific Instrument Factory).

[0059] Lotus seed formula granules (batch numbers: S16(KL), S17(KL), S18(KL)) were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd.

[0060] Heart-removed lotus seed medicinal materials and lotus seeds without heart removal were provided by Jiangyin Tianjiang Pharmaceutical Co., Ltd., and both met the various requirements of Chinese Pharmacopoeia 2020 edition under lotus seeds, and the medicinal material information is shown in Table 1.

[0061] Table 1

[0062]

[0063] 2. Source of reference substance

[0064] Lotus seed reference medicinal material (batch number: 121121-201205) and protocatechuic acid (batch number: 110879-201906) were purchased from China Institute for Drug Control.

[0065] p-Coumaric acid-4-O-β-D-glucopyranoside (batch number: 10339) was purchased from Shanghai Shidanded Standard Technology Service Co., Ltd.

[0066] 3. Determined chromatographic conditions

[0067] The packing agent was octadecylsilane-bonded silica gel (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile was used as mobile phase A, and 0.1% formic acid solution was used as mobile phase B; gradient elution was performed according to the provisions in Table 2; the flow rate was 0.2 ml per minute; the column temperature was 25°C; and the detection wavelength was 254 nm. The theoretical plate number should not be less than 5000 calculated according to the protocatechuic acid peak.

[0068] Table 2 Gradient elution procedure in Example 1

[0069]

[0070] 4. Selection of wavelength

[0071] A suitable amount of Lianzi Dispensing Granules (batch number: S16(KL)) was finely ground, about 1 g was taken, and 2 portions were prepared in parallel. 10% methanol 25 ml was added, the bottle was tightly capped, and ultrasonic treatment (power 600 W, frequency 40 kHz) was performed for 30 minutes. After cooling, the mixture was shaken well and filtered. The filtrate was taken as the test sample solution. The test sample solution was precisely taken and injected into the liquid chromatograph (flow rate 0.2 ml per minute; column temperature 25°C). The packing agent was octadecylsilane-bonded silica gel (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile was used as mobile phase A, and 0.1% formic acid solution was used as mobile phase B; gradient elution was performed according to the provisions in Table 2; and the absorption spectrum in the range of 190-400 nm was recorded (see Figure 1 ).

[0072] As can be seen from Figure 2 , under the detection wavelength of 240-272 nm, the information of the characteristic chromatographic peaks of Lianzi is relatively rich, the response value is high, and the baseline is relatively smooth. Under the detection wavelength of 254 nm, the impurity peaks are small, and therefore the detection wavelength of 254 nm is preferred as the final detection wavelength.

[0073] 5. Preparation of reference solution

[0074] Lianzi control medicinal material 2 g was placed in a conical flask with a plug, 10% methanol 25 ml was added, and heating reflux was performed for 1 hour. After cooling, the mixture was shaken well and filtered. The filtrate was taken as the reference solution of the control medicinal material. Appropriate amounts of protocatechuic acid control and p-coumaric acid-4-O-β-D-glucopyranoside control were precisely weighed and measured, and 30% methanol was added to prepare a solution containing 40 μg per 1 ml, which was taken as the reference solution of the control.

[0075] 6. Preparation of test sample solution

[0076] (1) Investigation of different extraction solvents

[0077] Take lotus seed formula granules (batch number: S16 (KL) ) appropriate amount, grind fine, take about 1g, a total of 6 groups, 2 groups each, accurately weighed, placed in a conical flask with a plug, accurately add water, 10% methanol, 30% methanol, 50% methanol, 70% methanol, methanol 25ml respectively, tightly sealed, weighed, ultrasonic treatment (power 600W, frequency 40kHz) 30 minutes, cool, reweigh, respectively with the corresponding solvent to make up for the loss of weight, shake up, filter, take the filtrate, i.e. test sample solution. Respectively, accurately pipette each test sample solution 2μl, inject into liquid chromatograph, according to the above determined chromatographic conditions (octadecylsilane bonded silica as the filler (column length is 100mm, inner diameter is 2.1mm, particle size is 1.8μm) ; according to the gradient in table 2, flow rate is 0.2ml per minute; column temperature is 25℃, detection wavelength 254nm) for determination, results see Figures 3-4 And table 3 below.

[0078] Table 3 comparison of extraction efficiency of different extraction solvents (peak area / sample weight)

[0079]

[0080] When 30% methanol, 50% methanol, 70% methanol and methanol are used as extraction solvents, peaks 1-3 show obvious solvent effect; when water and 10% methanol are used as extraction solvents, the chromatographic peak shape is better, and the total extraction efficiency is relatively close. From the perspective of convenient sample preservation, 10% methanol solution is selected as the extraction solvent for the determination of lotus seed formula granules characteristic chromatogram.

[0081] (2) Investigation of different extraction methods

[0082] Take lotus seed formula granules (batch number: S16 (KL) ) appropriate amount, grind fine, take about 1g, a total of 3 groups, 2 groups each, accurately weighed, placed in a conical flask with a plug, accurately add 10% methanol 25ml, tightly sealed, weighed, ultrasonic treatment (power 600W, frequency 40kHz), shaking extraction, heating reflux 30 minutes, cool, reweigh, with 10% methanol to make up for the loss of weight, shake up, filter, take the filtrate, i.e. test sample solution. Respectively, accurately pipette each test sample solution 2μl, inject into liquid chromatograph, according to the above determined chromatographic conditions (flow rate is 0.2ml per minute; column temperature is 25℃, detection wavelength 254nm, octadecylsilane bonded silica as the filler (column length is 100mm, inner diameter is 2.1mm, particle size is 1.8μm) ; according to the gradient in table 2) for determination, results see Figures 5-6 And table 4.

[0083] Table 4 comparison of extraction efficiency of different extraction methods (peak area / sample weight)

[0084]

[0085] When using ultrasonic, shaking and reflux as the extraction method, the number of chromatographic peaks is consistent, the peak shape is good, and the total extraction efficiency is relatively close. From the perspective of simple operation, ultrasonic extraction is selected as the extraction method for the determination of the characteristic spectrum of Lianzi Dispensing Granules.

[0086] (3) Investigation of different extraction times

[0087] Take Lianzi Dispensing Granules (batch number: S16(KL)) in an appropriate amount, grind finely, take about 1 g, a total of 4 groups, 2 portions in each group, accurately weigh, place in a conical flask with a stopper, accurately add 10% methanol 25 ml, tightly seal, weigh the weight, ultrasonic treatment (power 600 W, frequency 40 kHz) for 15 minutes, 30 minutes, 45 minutes and 60 minutes respectively, shake well, filter, take the filtrate, and the test sample solution is obtained. Accurately pipette 2 μl of each test sample solution, inject into the liquid chromatograph, and determine according to the above determined chromatographic conditions (flow rate is 0.2 ml per minute; column temperature is 25°C, detection wavelength is 254 nm, octadecylsilane bonded silica gel is used as the filler (column length is 100 mm, inner diameter is 2.1 mm, particle size is 1.8 μm); according to the gradient in Table 2), the results are shown in Table 4 and Table 5. Figures 7-8

[0088] Table 5 Comparison of extraction efficiency of different extraction times (peak area / sample weight)

[0089]

[0090] When the ultrasonic time is 15 minutes, 30 minutes, 45 minutes and 60 minutes, the number of chromatographic peaks is consistent, the peak shape is good, and the total extraction efficiency is relatively close. From the perspective of simple operation and energy saving, the extraction time of Lianzi Dispensing Granules is determined to be 30 minutes.

[0091] (4) Determination of test sample solution preparation method

[0092] After investigation, the test sample solution preparation method for the determination of the characteristic spectrum of Lianzi Dispensing Granules is finally determined as follows: take the product in an appropriate amount, grind finely, take about 1 g, place in a conical flask with a stopper, add 10% methanol 25 ml, tightly seal, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool down, shake well, filter, and take the filtrate.

[0093] 7. Methodology verification

[0094] (1) Investigation of specificity

[0095] ​The above prepared lotus seed formula granule test sample solution, negative solution (prepared by the test sample preparation method), was injected into the liquid chromatograph, and detected under the chromatographic conditions determined in Example 1 and the detection wavelength of 254 nm. The chromatogram is shown in Figure 9 . Among them, the chromatographic column: Eclipse Plus C18, 2.1 mm x 100 mm, 1.8 μm, column temperature: 25 ℃, flow rate: 0.2 ml / min.

[0096] The experimental results show that the solvent and excipients do not interfere with the characteristic peaks of the lotus seed formula granule chromatogram.

[0097] (2) Precision test

[0098] The test sample solution was precisely taken, and detected under the above determined chromatographic conditions and the detection wavelength of 254 nm, and 6 times of continuous injection, 2 μl each time, with peak 4 as the reference peak S, the relative retention time and relative peak area of the rest of the characteristic peaks to S peak were calculated, and the results are shown in Table 6 and Table 7. Among them, the chromatographic column: Eclipse Plus C18, 2.1 mm x 100 mm, 1.8 μm, column temperature: 25 ℃, flow rate: 0.2 ml / min.

[0099] Table 6 Precision test results (relative retention time)

[0100]

[0101] Table 7 Precision test results (relative peak area)

[0102]

[0103] The results show that the relative retention time and relative peak area of each characteristic peak are less than 2%, and the instrument precision is good.

[0104] (3) Stability test

[0105] The test sample solution was precisely taken, and detected under the above determined chromatographic conditions and the detection wavelength of 254 nm, and 6 times of continuous injection, 2 μl each time, with peak 4 as the reference peak S, the relative retention time and relative peak area of the rest of the characteristic peaks to S peak were calculated, and the results are shown in Table 6 and Table 7. Among them, the chromatographic column: Eclipse Plus C18, 2.1 mm x 100 mm, 1.8 μm, column temperature: 25 ℃, flow rate: 0.2 ml / min.

[0106] Table 8 Stability test results (relative retention time)

[0107]

[0108] The results show that the relative retention time and relative peak area of each characteristic peak are less than 2%, and the instrument precision is good.

[0109] Table 9 Stability experiment results (relative peak area)

[0110]

[0111] The results showed that the relative retention time and relative peak area of each characteristic peak were less than 3%, and the test solution was stable within 24 hours.

[0112] (4) Repeatability investigation

[0113] Take 1 g of lotus seed formula granules (batch number: S16(KL)), accurately weigh, 6 parallel, respectively, according to the above test solution preparation method to prepare the test solution, respectively, 2 μl, with peak 4 as the reference peak S, calculate the relative retention time and relative peak area of the rest of the characteristic peaks and S peak, the results are shown in Table 10 and Table 11. Among them, the detection was carried out under the chromatographic conditions determined in Example 1 and the detection wavelength of 254 nm. Chromatographic column: Eclipse Plus C18, 2.1 mm x 100 mm, 1.8 μm, column temperature: 25 ℃, flow rate: 0.2 ml / min.

[0114] Table 10 Repeatability experiment results (relative retention time)

[0115]

[0116] Table 11 Repeatability experiment results (relative peak area)

[0117]

[0118]

[0119] The results showed that the relative retention time and relative peak area of each characteristic peak were less than 3%, and the repeatability test was good.

[0120] (5) Durability test

[0121] 1) Chromatographic column investigation

[0122] In this study, the separation effect of test solution of three different brands of chromatographic columns CORTECS UPLC T3 (Waters, 2.1 x 100 mm, 1.6 μm) (marked as Cortecs T3 in the figure), Eclipse Plus C18 (Agilent, 2.1 x 100 mm, 1.8 μm) (marked as EPC18 in the figure), Acquity UPLC HSS T3 (Waters, 2.1 x 100 mm, 1.8 μm) (marked as HSS T3 in the figure) was investigated, and the results are shown in Figure 10The detection was carried out under the chromatographic conditions determined in Example 1 and the detection wavelength of 254 nm. The flow rate was 0.2 ml / min; the column temperature was 25℃.

[0123] The results showed that the lotus seed sample could be well separated in CORTECS UPLC T3, Eclipse Plus C18 and Acquity UPLC HSST3 chromatographic columns, and the method had good durability for different chromatographic columns.

[0124] 2) Investigation of column temperature

[0125] The separation effect of lotus seed formula granules was investigated when the column temperature was 20℃, 25℃ and 30℃ respectively using Eclipse Plus C18 (Waters, 2.1x100mm, 1.8μm) chromatographic column. The results are shown in Figure 11 and Table 12. The detection was carried out under the chromatographic conditions determined in Example 1 and the detection wavelength of 254 nm, and the flow rate was 0.2 ml / min.

[0126] Table 12 Influence of column temperature on separation effect (relative retention time)

[0127]

[0128] The results showed that the analysis method had good durability when the column temperature was 20℃-30℃. The relative retention time of each characteristic peak was within the specified range, and small column temperature variation could meet the system suitability requirements.

[0129] 3) Investigation of flow rate

[0130] The separation effect of lotus seed formula granules was investigated when the flow rate was 0.15 ml / min, 0.20 ml / min and 0.25 ml / min respectively using Eclipse Plus C18 (Waters, 2.1x100mm, 1.8μm) chromatographic column. The results are shown in Figure 12 and Table 13. The detection was carried out under the chromatographic conditions determined in Example 1 and the detection wavelength of 254 nm, and the column temperature was 25℃.

[0131] Table 13 Influence of flow rate on separation effect (relative retention time)

[0132]

[0133] The results showed that when the flow rate was 0.15 ml / min-0.20 ml / min, the relative retention time of each characteristic peak was within the specified range, and small flow rate variation could meet the system suitability requirements. The analysis method had good durability.

[0134] Example 2

[0135] The optimal scheme identified in Example 1 was used to construct the ultra-high performance liquid chromatography characteristic map of the lotus seed sample, and the quality of lotus seed formula granules and its upstream raw materials was qualitatively analyzed. The characteristic map reflects the quality consistency and traceability of lotus seed medicinal materials, decoction pieces, standard decoction, intermediates and formula granules.

[0136] 1. Selection of common peaks

[0137] Using the determined chromatographic conditions and 254 nm detection wavelength, the preparation method of the test solution, and the chromatographic column: Eclipse Plus C18, 2.1 mm x 100 mm, 1.8 μm, column temperature: 25℃, flow rate: 0.2 ml / min, the characteristic maps of multiple batches of lotus seed medicinal materials, multiple batches of lotus seed standard decoction and multiple batches of lotus seed granules were determined, and the results are shown in Figures 13-15 The characteristic maps of lotus seed medicinal materials, decoction pieces, standard decoction, intermediates and formula granules obtained above were compared to determine the common peaks, and the results are shown in Figure 16 The preparation conditions of the test solution (lotus seed medicinal material solution, decoction piece solution, standard decoction solution, intermediate solution, formula granule solution) are the conditions determined in Example 1. The processing method of lotus seed decoction pieces under lotus seed in the 2020 edition of Chinese Pharmacopoeia is: for those with heart, slightly soak, moisten, cut open, remove the heart, dry; or crush, remove the heart. For those without heart, directly into medicine or crush. Therefore, 18 batches of lotus seed without heart can be directly used as decoction pieces in the present application, and the decoction pieces are the same as the medicinal materials. The products in the actual production process of the decoction pieces to the formula granules, the precursors of the formula granule granulation, and the intermediates are required to be consistent with the standard decoction.

[0138] 2. Identification of reference substance

[0139] An appropriate amount of protocatechuic acid reference substance and p-coumaric acid-4-O-β-D-glucopyranoside reference substance was precisely weighed and dissolved in 30% methanol to prepare a solution containing 40 μg per 1 ml as a reference solution.

[0140] 1 μl of the reference solution and 2 μl of the test solution were precisely taken and injected into the liquid chromatograph, and the determination was carried out according to the determination conditions of the test solution in this example, and the results are shown in Figure 17 ).

[0141] Combining the actual implementability and the identification of the reference substance, the final characteristic map specifies 7 common peaks. As the characteristic peaks of lotus seed, they are used to evaluate the quality, in which peak 4: protocatechuic acid; peak A: p-coumaric acid-4-O-β-D-glucopyranoside.

[0142] 3. Peak positioning-determination of relative retention time

[0143] The characteristic chromatogram of the lotus seed sample should present 7 characteristic peaks, which should correspond to the retention time of the 7 characteristic peaks in the chromatogram of the reference substance of the control. Peak 4 should correspond to the retention time of the protocatechuic acid control reference substance peak. The peak corresponding to the protocatechuic acid control reference substance peak is the S peak, and the relative retention time of the other characteristic peaks to the S peak is calculated, which should be within ±10% of the specified value: 0.30 (peak 1), 0.51 (peak 2), 0.80 (peak 3), 1.44 (peak 5), 1.83 (peak 6), 2.03 (peak 7).

[0144] 4. Application value of the characteristic map

[0145] The determination results of the characteristic map of the above multiple batches of medicinal materials, decoction pieces, standard decoction, intermediates, and formula granules have important application value and guiding significance. That is, from lotus seed medicinal materials / decoction pieces-standard decoction-intermediates-formula granules, all present 7 common characteristic peaks, indicating that the key quality of lotus seed formula granules is consistent with that of intermediates, standard decoction, and medicinal materials / decoction pieces.

[0146] Example 3

[0147] In this example, the ultra-high performance liquid chromatography characteristic map of the lotus seed sample constructed using the optimal scheme confirmed in Example 1 is used for qualitative analysis of the quality of lotus seed formula granules and its upstream raw materials, and to distinguish between heartless lotus seeds and lotus seeds with heart through the characteristic map.

[0148] 1. Identification of difference peaks between heartless lotus seeds and lotus seeds with heart

[0149] Heartless lotus seed medicinal materials and lotus seed medicinal materials with heart meet the requirements of each item under lotus in the Chinese Pharmacopoeia 2020 edition.

[0150] The characteristic maps of heartless lotus seed medicinal materials and lotus seed medicinal materials with heart were compared, and the results are shown in Figure 18 .

[0151] 2. Comparison between heartless lotus seed medicinal materials and lotus seed medicinal materials with heart

[0152] K value calculation formula:

[0153] Among them, S A is the peak area of characteristic peak A, and S1 is the peak area of peak 1.

[0154] Table 14 Relative peak areas (K values) of peak A and peak 1 in the characteristic map of 18 batches of heartless lotus seed medicinal materials

[0155]

[0156]

[0157] Table 15 Relative peak area (K value) of peak A and peak 1 in 3 batches of uncored Plukenetia volubilis L. medicinal materials

[0158]

[0159] The relative peak area (K value) of peak A and peak 1 in Plukenetia volubilis L. medicinal materials / drinks is calculated. If K≤0.15 in the detected Plukenetia volubilis L. medicinal materials / drinks, the detected sample is cored Plukenetia volubilis L. medicinal materials / drinks; if K>0.15 in the detected Plukenetia volubilis L. medicinal materials / drinks, the detected sample is uncored Plukenetia volubilis L. medicinal materials / drinks. The difference is obvious, and Plukenetia volubilis L. medicinal materials / drinks can be distinguished according to the K value.

[0160] (see Figure 18 and Tables 14-15)

[0161] 3. Comparison of cored Plukenetia volubilis L. standard decoction and uncored Plukenetia volubilis L. standard decoction

[0162] Table 16 Relative peak area (K value) of peak A and peak 1 in 18 batches of cored Plukenetia volubilis L. standard decoction

[0163]

[0164]

[0165] Table 17 Relative peak area (K value) of peak A and peak 1 in 3 batches of uncored Plukenetia volubilis L. standard decoction

[0166]

[0167] Table 18 Relative peak area (K value) of peak A and peak 1 in 3 batches of cored Plukenetia volubilis L. formula granules

[0168]

[0169] The relative peak area (K value) of peak A and peak 1 in Plukenetia volubilis L. standard decoction / formula granules is calculated. If K≤0.17 in the detected Plukenetia volubilis L. standard decoction / formula granules, the detected sample is cored Plukenetia volubilis L. standard decoction / formula granules; if K>0.17 in the detected Plukenetia volubilis L. standard decoction / formula granules, the detected sample is uncored Plukenetia volubilis L. standard decoction / formula granules. The difference is obvious, and Plukenetia volubilis L. standard decoction / formula granules can be distinguished according to the K value (see Figure 19 and Tables 16-18).

Claims

1. A method for constructing a lotus seed feature map, characterized in that, Includes the following steps: (1) Take lotus seed medicinal material, decoction pieces, standard decoction or formula granule powder respectively, add extraction solvent, extract, cool, filter, and the filtrate is the test solution; the extraction solvent is water or 10% methanol; (2) Take protocatechuic acid and p-coumaric acid-4- O-β-D -Pyranoside, added to a solvent to prepare a reference solution; (3) Take the test solution and the reference solution and inject them into the ultra-high performance liquid chromatograph to obtain characteristic chromatograms; The obtained characteristic chromatogram has 7 characteristic peaks, with peak 4 being protocatechuic acid. The relative retention times of the remaining characteristic peaks and peak 4 were calculated, and their relative retention times should all be within ±10% of the specified values. The specified values ​​for peaks 1-3 were calculated to be 0.30, 0.51, and 0.80, respectively, and the specified values ​​for peaks 5-7 were calculated to be 1.44, 1.83, and 2.03, respectively. The chromatographic conditions of the ultra-high performance liquid chromatograph were as follows: column: CORTECS UPLC T3, Eclipse Plus C18, or Acquity UPLC HSS T3; column temperature: 20-30℃; flow rate: 0.15-0.20 ml / min; detection wavelength: 240-272 nm; gradient elution was performed using acetonitrile as mobile phase A and 0.1% formic acid solution as mobile phase B; the gradient elution time was: 0-7 min, with the volume fraction of mobile phase A being 1-2% and the volume fraction of mobile phase B being 99-98%; 7-30 min... At min, the volume fraction of mobile phase A is 2→10%, and the volume fraction of mobile phase B is 98→90%.

2. The construction method according to claim 1, characterized in that, The extraction methods described in step (1) include ultrasound, shaking, or reflux; the extraction time is 15 to 60 minutes.

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