Method for constructing characteristic chromatogram of stamen nelumbinis medicinal material or preparation thereof

Abstract

The invention provides a stamen nelumbinis medicinal material or a stamen nelumbinis preparation characteristic spectrum construction method which comprises the following steps: a, preparation of a test sample: taking a stamen nelumbinis test sample, and dissolving and extracting the stamen nelumbinis test sample by adopting a solvent to obtain a to-be-detected solution; b, determining by adopting a high performance liquid chromatography to obtain the characteristic chromatogram of the stamen nelumbinis medicinal material or the preparation thereof, acetonitrile is used as a mobile phase A, a 0.1% phosphoric acid solution is used as a mobile phase B, and gradient elution is carried out. The invention also provides a characteristic spectrum construction method of the stamen nelumbinis decoction pieces, the standard decoction and the formula granules. According to the method, single-variety research is carried out on the lotus stamen formula granules, scientific identification is carried out on the lotus stamen, the method has the characteristics of comprehensive detection and simple operation, the lotus stamen formula granules can be effectively identified, and uniformity and stability of the quality of the lotus stamen formula granules can be ensured. The method is suitable for stamen nelumbinis medicinal materials, decoction pieces, standard decoction and formula granules.

Description

Technical Field

[0001] This invention relates to a method for constructing a characteristic spectrum of lotus stamen medicinal material or its preparation. Background Technology

[0002] Lotus stamens are the dried stamens of *Nelumbo nucifera* Gaertn., a plant in the Nymphaeaceae family. They are harvested on sunny days during the summer when the flowers are in bloom. Traditional Chinese medicine texts record their kidney-tonifying, sperm-strengthening, and anti-prolapse effects. Modern pharmacological research indicates that lotus stamens also have whitening, anti-thrombotic, anti-ulcer, and anti-diarrheal effects. Studies show that the main chemical components of lotus stamens are flavonoids, including quercetin, luteolin, kaempferol, and isoquercitrin. Lotus stamens are often used clinically in the form of preparations. Currently, there is limited literature on the qualitative and quantitative analysis of related formulation granules. (Meng Xiansheng et al., HPLC determination of quercetin and kaempferol content in lotus stamens, *Traditional Chinese Medicine*, December 2003, Vol. 25, No. 12: Determination of quercetin and kaempferol content in lotus stamens using high-performance liquid chromatography.) Ma Yinhai, et al., Rapid determination of quercetin and kaempferol in lotus stamens by high performance liquid chromatography, Journal of Kunming Teachers College, 2005, 27(4):12-13. This study investigated the determination of quercetin and kaempferol in lotus stamens using solid-phase extraction pre-separation and high performance liquid chromatography. The Guangdong Provincial Standard for Quality of Traditional Chinese Medicine Formula Granules discloses a method for determining the characteristic chromatogram of lotus stamen formula granules. The detection conditions are as follows: octadecylsilane-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile as mobile phase A and 0.2% glacial acetic acid solution as mobile phase B, with gradient elution; flow rate 0.2 ml / min; column temperature 30℃; detection wavelength 300 nm. The characteristic chromatogram obtained by this method has poor peak shape, poor separation, and few peaks. Summary of the Invention

[0003] This invention provides a novel method for constructing characteristic maps of lotus stamen medicinal materials or their preparations.

[0004] This invention provides a method for constructing a characteristic spectrum of lotus stamen medicinal material or its preparations, which includes the following steps:

[0005] a. Preparation of the test sample:

[0006] Lotus stamens were used as a test sample, dissolved and extracted with a solvent to obtain the test solution;

[0007] b. The characteristic chromatograms of lotus stamens or its preparations were determined by high-performance liquid chromatography (HPLC). The chromatographic conditions were: C18 column; acetonitrile as mobile phase A; 0.1% phosphoric acid solution as mobile phase B; gradient elution. The gradient elution conditions were as follows:

[0008]

[0009] The preparation method of the test solution in step a is as follows: Take the lotus stamen test sample, grind it into a fine powder, add methanol aqueous solution, sonicate, cool, shake well, filter, and collect the filtrate to obtain the test solution. The sonication conditions are: power 600W, frequency 40kHz, sonication time 20-40min; methanol concentration 80%; the amount of lotus stamen and methanol used is 10-50ml of methanol per 0.5g of lotus stamen.

[0010] The ultrasound time is 30 minutes, and the amount of lotus stamen and methanol used is 10 ml of methanol per 0.5 g of lotus stamen.

[0011] In step b, the chromatographic column is a C18 column with a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm; the flow rate is 0.8-1.2 ml per minute; the column temperature is 25-35℃; the detection wavelength is 210-346 nm; and the theoretical plate number, calculated based on the uridine peak, should be no less than 8000. Preferably, in step b, the column temperature is 30℃, the flow rate is 1.0 ml per minute, and the detection wavelength is 346 nm.

[0012] It also includes the preparation of reference solutions, including reference medicinal materials, rutin reference standards, kaempferol reference standards, isorhamnetin reference standards, and quercetin reference standards;

[0013] The preparation method of the reference solution of the reference medicinal material is as follows: take lotus stamen reference medicinal material, add water and decoct, filter, evaporate the filtrate to dryness, add methanol to the residue, sonicate, cool, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material.

[0014] The preparation method of the rutin reference standard, kaempferol reference standard, isorhamnetin reference standard, and quercetin reference standard solution is as follows: take rutin reference standard, kaempferol reference standard, isorhamnetin reference standard, and quercetin reference standard respectively, and add methanol to prepare reference standard solutions.

[0015] The characteristic spectrum contains 7 characteristic peaks with relative retention times of: peak 1: 0.33, peak 2: 0.55, peak 3: 0.77, peak 4: 0.78, peak 5: 0.93, peak 6: 1.0, and peak 7: 1.01, with the relative retention times fluctuating within ±10%.

[0016] This invention provides a method for constructing characteristic spectra of lotus stamen slices, standard decoctions, and formulated granules, which uses the aforementioned method for constructing characteristic spectra of lotus stamen medicinal materials or their preparations.

[0017] This invention also provides a method for identifying lotus stamen slices, standard decoctions, and formulated granules, which includes the following steps:

[0018] a. Take the sample to be tested;

[0019] b. Construct a characteristic spectrum according to the method for constructing characteristic spectrum of lotus stamen medicinal material or its preparation;

[0020] c. Comparison: If the characteristic peaks are completely identical, then it is genuine lotus stamen.

[0021] This invention provides a single-variety study on lotus stamen granules, enabling scientific identification of lotus stamens. It features comprehensive testing, simple operation, and effective identification of lotus stamen granules, ensuring uniform and stable quality. This method is applicable to lotus stamen medicinal materials, processed slices, standard decoctions, and granules. Attached Figure Description

[0022] Figure 1 Compare with characteristic chromatograms (peak 2: rutin; peak 5: quercetin; peak 6 (S): kaempferol; peak 7: isorhamnetin);

[0023] Figure 2 Chromatogram of lotus stamen granules (from top to bottom: 210nm, 230nm, 250nm, 270nm, 300nm)

[0024] 346nm);

[0025] Figure 3 Investigation of different column temperatures for lotus stamen granule formulation;

[0026] Figure 4 Flow velocity study;

[0027] Figure 5 Delayed reaction study of lotus stamen granules;

[0028] Figure 6 Examining the extraction method;

[0029] Figure 7 Extraction solvent investigation;

[0030] Figure 8 Take time to investigate;

[0031] Figure 9 Investigation of solvent addition amount;

[0032] Figure 10 Chromatographic peak identification;

[0033] Figure 11 Different instruments;

[0034] Figure 12 Different chromatographic columns;

[0035] Figure 13 Characteristic spectrum verification images of three batches of lotus stamen formula granules (S1-S3 are LX-KL-01, LX-KL-02, etc. respectively).

[0036] LX-KL-03);

[0037] Figure 14 Characteristic atlas of lotus stamen medicinal materials (S1-S16 are LX-YC-01, LX-YC-02, LX-YC-03, respectively)

[0038] LX-YC-04, LX-YC-05, LX-YC-06, LX-YC-07, LX-YC-08, LX-YC-09, LX-YC-10, LX-YC-11, LX-YC-12, LX-YC-13, LX-YC-14, LX-YC-15, LX-YC-16);

[0039] Figure 15 Characteristic chromatograms of lotus stamen slices (S1-S16 are LX-YP-01, LX-YP-02, LX-YP-03, etc., respectively)

[0040] LX-YP-04, LX-YP-05, LX-YP-06, LX-YP-07, LX-YP-08, LX-YP-09, LX-YP-10,

[0041] LX-YP-11, LX-YP-12, LX-YP-13, LX-YP-14, LX-YP-15, LX-YP-16);

[0042] Figure 16 Characteristic chromatograms of lotus stamen standard decoction (S1-S16 are LX-BT-01, LX-BT-02, LX-BT-03, etc., respectively)

[0043] LX-BT-04, LX-BT-05, LX-BT-06, LX-BT-07, LX-BT-08, LX-BT-09, LX-BT-10,

[0044] LX-BT-11, LX-BT-12, LX-BT-13, LX-BT-14, LX-BT-15, LX-BT-16);

[0045] Figure 17 Compare with characteristic chromatograms (peak 2: rutin; peak 5: quercetin; peak 6 (S): kaempferol; peak 7: isorhamnetin);

[0046] Figure 18 Compare with characteristic chromatograms (peak 2: rutin; peak 5: quercetin; peak 6 (S): kaempferol; peak 7: isorhamnetin);

[0047] Figure 19 Compare with characteristic chromatograms (peak 2: rutin; peak 5: quercetin; peak 6 (S): kaempferol; peak 7: isorhamnetin);

[0048] Figure 20 Compare with characteristic chromatograms (peak 2: rutin; peak 5: quercetin; peak 6 (S): kaempferol; peak 7: isorhamnetin);

[0049] Figure 21 Method verification. Detailed Implementation

[0050] Example 1: Method for constructing characteristic maps of lotus stamen medicinal material or its preparations according to the present invention

[0051] Lotus stamens are the dried stamens of Nelumbo nucifera Gaertn., a plant in the Nymphaeaceae family. The standard decoction is a freeze-dried powder produced from the medicinal material through a fixed preparation process. To ensure the uniformity and stability of the quality of lotus stamens, processed medicinal pieces, and their standard decoction, this paper establishes a new characteristic chromatographic method for quality control.

[0052] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 346 nm. The theoretical plate number, calculated based on the kaempferol peak, should be no less than 5000.

[0053]

[0054] Preparation of the reference solution: Take 2g of lotus stamen reference material, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of methanol to the residue, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take an appropriate amount of kaempferol reference standard, accurately weigh it, add methanol to prepare a solution containing 50μg per ml, as the reference solution.

[0055] Preparation of the test solution: Take 0.5g of lotus stamen, grind it into a fine powder, place it in a stoppered conical flask, add 10ml of 80% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0056] The determination method involves precisely pipetting 10 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0057] The chromatogram of the test sample should show 7 characteristic peaks, and the retention times should correspond to the 7 characteristic peaks in the chromatogram of the reference medicinal material. Peak 6 should correspond to the retention time of the reference peak. The peak corresponding to the kaempferol reference peak is the S peak. Calculate the relative retention times of each characteristic peak and the S peak; the relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.33 (peak 1), 0.55 (peak 2), 0.77 (peak 3), 0.78 (peak 4), 0.93 (peak 5), and 1.01 (peak 7). (See...) Figure 1 )

[0058] Example 2: Screening test of the conditions for constructing the characteristic spectrum of lotus stamen medicinal material or its preparation according to the present invention.

[0059] 1. Experimental instruments and materials

[0060] High-performance liquid chromatographs: Shimadzu LC-20AD, Agilent 1260, and Waters e2695;

[0061] Electronic balances: ME204E / 02, XPE26 (Mettler-Toledo Instruments Ltd.);

[0062] Ultrapure water system: Cellular type 1810A (Shanghai Moler Scientific Instruments Co., Ltd.);

[0063] Ultrasonic cleaner: KQ600DB model (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0064] Chromatographic columns: Diamonsil Plus C18 5μm 250×4.6mm; Agilent ZORBAX Eclipse XDB-C18 250×4.6mm, 5μm; Kromasil 100-5-C18 5μm 250×4.6mm;

[0065] Acetonitrile and phosphoric acid were of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0066] Rutin reference standard (China National Institutes for Food and Drug Control, batch number: 100080-202012, purity: 92.2%);

[0067] Isorhamnetin reference standard (China National Institutes for Food and Drug Control, batch number: 110860-202012, purity: 99.1%);

[0068] Quercetin reference standard (China National Institutes for Food and Drug Control, batch number: 100081-201610, purity: 99.8%);

[0069] Kaempferol reference standard (China National Institutes for Food and Drug Control, batch number: 110861-202214, purity: 97.4%);

[0070] Lotus stamen reference material (China National Institutes for Food and Drug Control, batch number: 121392-202102);

[0071] Lotus stamen formula granules: LX-KL-01, LX-KL-02, LX-KL-03;

[0072] Lianxu medicinal materials: LX-YC-01, LX-YC-02, LX-YC-03, LX-YC-04, LX-YC-05, LX-YC-06, LX-YC-07, LX-YC- 08. LX-YC-09, LX-YC-10, LX-YC-11, LX-YC-12, LX-YC-13, LX-YC-14, LX-YC-15, LX-YC-16;

[0073] Lianxu decoction pieces: LX-YP-01, LX-YP-02, LX-YP-03, LX-YP-04, LX-YP-05, LX-YP-06, LX-YP-07, LX-YP- 08. LX-YP-09, LX-YP-10, LX-YP-11, LX-YP-12, LX-YP-13, LX-YP-14, LX-YP-15, LX-YP-16;

[0074] Lotus stamen standard decoction: LX-BT-01, LX-BT-02, LX-BT-03, LX-BT-04, LX-BT-05, LX-BT-06, LX-BT-07, LX-BT-08, LX-BT-09, LX-BT-10, LX-BT-11, LX-BT-12, LX-BT-13, LX-BT-14, LX-BT-15, LX-BT-16.

[0075] 2. Determination of detection wavelength point

[0076] Based on the above-specified experimental conditions, a diode array detector was used to perform a full-band scan of the test solution, and chromatograms of the test solution were extracted at wavelengths of 210 nm, 230 nm, 250 nm, 270 nm, 300 nm, and 346 nm. See [link to chromatogram]. Figure 2 .

[0077] The results showed that the chromatographic peak information was greater and the chromatographic baseline was more stable at a detection wavelength of 346 nm, so the detection wavelength was determined to be 346 nm.

[0078] 3. Column temperature investigation

[0079] Based on the above-planned experimental conditions, the effects were investigated at column temperatures of 25℃, 30℃, and 35℃. Figure 3 As shown in Figure 1 and Table 1.

[0080] Table 1. Column Temperature Investigation—Ratio of Relative Retention Time of Characteristic Peaks

[0081]

[0082] The column temperature investigation results showed that when the column temperature was 30℃, the peak shapes of the chromatograms were relatively symmetrical and the resolution was good. Therefore, 30℃ was finally determined as the column temperature for the characteristic chromatogram method of lotus stamen formula granules.

[0083] 4. Flow velocity investigation

[0084] Based on the above-established experimental conditions, the flow rates of 0.8 ml / min, 1.0 ml / min, and 1.2 ml / min were investigated. Figure 4 As shown in the figure and Table 2.

[0085] Table 2 Flow rate - relative retention time

[0086]

[0087]

[0088] The results showed that the chromatogram peak shape was good and the resolution was moderate at a flow rate of 1.0 ml / min. Therefore, the flow rate was determined to be 1.0 ml / min.

[0089] 5. Delayed testing

[0090] Based on the above-planned experimental conditions, the chromatogram acquisition time will be extended to 120 minutes. For example... Figure 5 As shown.

[0091] The results showed that the sample had virtually no chromatographic peaks after 60 minutes, so the sample detection time was set at 60 minutes.

[0092] 6. Examination of extraction methods

[0093] Take 0.5g of lotus stamen granules (LX-KL-01), grind them into a fine powder, place them in a stoppered conical flask, add 25ml of 50% methanol, reflux and sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the product. Figure 6 As shown.

[0094] The results showed that ultrasonic extraction and reflux extraction yielded consistent results for the test sample. Since ultrasonic extraction is simpler, it was chosen as the extraction method for the test sample.

[0095] 7. Investigation of extraction solvent

[0096] Take 0.5g of lotus stamen granules (LX-KL-01), grind them finely, place them in a stoppered conical flask, and add 30% methanol, 50% methanol, 80% methanol, methanol, 50% ethanol, and 25ml of water respectively. Sonicate the mixture (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and collect the filtrate. (The rest of the text appears to be a continuation of the previous sentence and is left untranslated.) Figure 7 As shown.

[0097] The results showed that 80% methanol was the best extraction solvent for the sample peak shape, so 80% methanol was chosen as the extraction solvent.

[0098] 8. Examination of extraction time

[0099] Take 0.5g of lotus stamen granules (LX-KL-01), grind them into a fine powder, place them in a stoppered conical flask, add 25ml of 80% methanol, and ultrasonically extract the sample for 20 minutes, 30 minutes, and 40 minutes respectively. Cool, shake well, filter, and collect the filtrate to obtain the product. Figure 8 As shown.

[0100] The results showed that extraction was complete at an extraction time of 30 minutes. Therefore, the extraction time for the test sample was determined to be 30 minutes.

[0101] 9. Investigation of Solvent Addition Amount

[0102] Take 0.5g of lotus stamen granules (LX-KL-01), grind them into a fine powder, and place them in stoppered conical flasks. Add 10ml, 25ml, and 50ml of 80% methanol respectively, seal tightly, and sonicate (600W power, 40kHz frequency) for 30 minutes. Shake well, filter, and collect the filtrate to obtain the final product. See [link to product description]. Figure 9 .

[0103] The results showed that the peak areas of the characteristic chromatograms were moderate when the solvent volume was 10 ml. Therefore, the solvent volume for the test sample was determined to be 10 ml.

[0104] 10. Determine the preparation method of the test sample.

[0105] Take 0.5g of lotus stamen granules, grind them into a fine powder, place them in a stoppered conical flask, add 10ml of 80% methanol, sonicate (600W power, 40kHz frequency) for 30 minutes, cool, shake well, filter, and collect the filtrate to obtain the product.

[0106] 11. Methodological Investigation

[0107] 11.1 Chromatographic Peak Identification

[0108] Preparation of the test solution: Prepare the lotus stamen formula granule test solution according to the experimental conditions proposed above.

[0109] Preparation of reference solution: Take appropriate amounts of rutin reference standard, kaempferol reference standard, isorhamnetin reference standard, and quercetin reference standard, accurately weigh them, and add methanol to prepare a solution containing 50 μg per ml, which is used as the reference solution.

[0110] Preparation of lotus stamen reference solution: Take 2g of lotus stamen reference material, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of methanol to the residue, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution of the reference material.

[0111] Preparation of negative control solution: Prepare negative control solution of lotus stamen-deficient formula granules according to the experimental conditions proposed above.

[0112] The characteristic peaks of the lotus stamen formula granules were located. For example... Figure 10 As shown.

[0113] As shown in the figure, peak 2 is rutin, peak 5 is quercetin, peak 6 is kaempferol, and peak 7 is isorhamnetin.

[0114] 11.2 Precision Test

[0115] Take the test solution of lotus stamen granules (batch number: LX-KL-01), inject it 6 times consecutively according to the proposed experimental method, and calculate the retention time of each characteristic peak. As shown in Table 3.

[0116] Table 3 Precision Examination - Retention Time

[0117]

[0118] The results show that the retention time RSD of each characteristic peak is 0.01%-0.11%. The method has good precision.

[0119] 11.3 Repeatability Test

[0120] Six portions of lotus stamen formula granules (batch number: LX-KL-01) were accurately weighed and prepared and measured according to the proposed experimental method. See Table 4 for details.

[0121] Table 4 Repeatability Tests - Relative Retention Time

[0122]

[0123]

[0124] The results show that the relative retention time (RSD) of each characteristic peak is 0.00%–0.35%. The method exhibits good repeatability.

[0125] 11.4 Intermediate Precision Examination

[0126] 11.4.1 Investigation with different instruments

[0127] Based on the above-planned experimental conditions, three portions of lotus stamen formula granules (batch number: LX-KL-01) were accurately weighed to prepare test solutions, which were then analyzed using Agilent 1260, Shimadzu LC-20AD, and Waters e2695 high-performance liquid chromatographs, respectively. Figure 11 As shown in Table 5.

[0128] Table 5. Instrument durability assessment - relative retention time ratio

[0129]

[0130] The results showed that when the above three instruments were used to detect the test samples, the RSD range of the relative retention time of each characteristic peak was 0.00%-8.34%, indicating that the methods and instruments had good durability.

[0131] 11.4.2 Investigations by different personnel and at different times

[0132] Based on the experimental conditions outlined above, two samples of lotus stamen formula granules (batch number: LX-KL-01) were precisely weighed by different personnel (A and B) at different times (T1 and T2) to prepare test samples for determination. The results are shown in Table 6.

[0133] Table 6. Personnel and Time Assessment - Relative Retention Time

[0134]

[0135] The results show that the method has good stability with different personnel and time.

[0136] 11.5 Durability Test

[0137] 11.5.1 Column robustness test

[0138] Based on the above-planned experimental conditions, the chromatographic columns Shimadzu Diamonsil Plus C18 5μm 250×4.6mm; Agilent ZORBAX Eclipse XDB-C18 250×4.6mm, 5μm; and Kromasil 100-5-C18 5μm 250×4.6mm were investigated. Figure 12 As shown in Table 7.

[0139] Table 7 Column robustness study - relative retention time

[0140]

[0141] Depend on Figure 12It can be seen that the chromatogram peaks of each column are well separated and the peaks are clear, indicating that the columns are durable.

[0142] 11.5.2 Stability Assessment

[0143] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 2h, 4h, 8h, 16h, and 24h, as shown in Table 8.

[0144] Table 8 Stability Study - Retention Time

[0145]

[0146] The results showed that the RSD of the corresponding characteristic peak retention time was between 0.02% and 0.14%, and the sample solution was stable within 24 hours.

[0147] Example 3: Verification Experiment of the Invention

[0148] Validation results of 1st and 3rd batches of lotus stamen formula granules

[0149] The proposed method was used to determine the characteristic spectra of three batches of this product, and the relative retention times were calculated. For example... Figure 13 As shown in Table 9.

[0150] Table 9. Relative retention times of three batches of lotus stamen granules.

[0151]

[0152]

[0153] Based on the principles of stable relative retention time, detectability in all batches of samples, and relatively high peak values, seven peaks with good repeatability were selected as characteristic peaks. The results showed that when peak 6 was an S peak, the relative retention time (RSD) of the characteristic peaks in all three batches of lotus stamen granule formulations was less than 2%.

[0154] 2. Establishment of limits for relative retention time

[0155] Table 10 summarizes the methodological examination items and validation results:

[0156] Table 2 Summary of RSD% for Methodological Results — Relative Retention Time

[0157]

[0158] The relative retention times of each characteristic peak are stable and within ±10% of the average value. Therefore, the specified range of relative retention times for each peak is tentatively set at ±10%.

[0159] The final requirement is that the RSD of the relative retention times of each characteristic peak meets the requirements in all the above tests, indicating that the method is effective. The chromatogram of the test sample should show seven characteristic peaks, with the peak corresponding to the kaempferol reference being the S peak.

[0160] 3. Results of 16 batches of lotus stamen medicinal material certificates

[0161] The proposed method was used to determine the characteristic spectra of 17 batches of this product, and the relative retention times were calculated. For example... Figure 14 As shown in Table 11.

[0162] Table 3. Relative retention times of 16 batches of lotus stamen medicinal materials

[0163]

[0164]

[0165] 4. Results of 17 batches of lotus stamen decoction pieces certificates

[0166] The proposed method was used to determine the characteristic spectra of 17 batches of this product, and the relative retention times were calculated. For example... Figure 15 As shown in Table 12.

[0167] Table 12 Relative retention times of 16 batches of lotus stamen slices

[0168]

[0169]

[0170] 5. Results of 17 batches of standard lotus stamen decoction prescriptions

[0171] The proposed method was used to determine the characteristic spectra of 16 batches of this product, and the relative retention times were calculated. For example... Figure 16 As shown in Table 13.

[0172] Table 13 Relative retention times of 16 batches of lotus stamen standard decoction

[0173]

[0174]

[0175] Example 3: Construction of characteristic spectra of lotus stamen formula granules, medicinal materials, processed slices, and standard decoctions of the present invention.

[0176] Formula granules:

[0177] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 346 nm. The theoretical plate number, calculated based on the kaempferol peak, should be no less than 5000.

[0178]

[0179] Preparation of the reference solution: Take 2g of lotus stamen reference material, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of methanol to the residue, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take an appropriate amount of kaempferol reference standard, accurately weigh it, add methanol to prepare a solution containing 50μg per ml, as the reference solution.

[0180] Preparation of the test solution: Take 0.5g of lotus stamen granules, grind them into a fine powder, place them in a stoppered conical flask, add 10ml of 80% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0181] The determination method involves precisely pipetting 10 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0182] The chromatogram of the test sample should show 7 characteristic peaks, and the retention times should correspond to the 7 characteristic peaks in the chromatogram of the reference medicinal material. Peak 6 should correspond to the retention time of the reference peak. The peak corresponding to the kaempferol reference peak is the S peak. Calculate the relative retention times of each characteristic peak and the S peak; the relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.33 (peak 1), 0.55 (peak 2), 0.77 (peak 3), 0.78 (peak 4), 0.93 (peak 5), and 1.01 (peak 7). (See...) Figure 17 )

[0183] Medicinal materials:

[0184] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 346 nm. The theoretical plate number, calculated based on the kaempferol peak, should be no less than 5000.

[0185]

[0186]

[0187] Preparation of the reference solution: Take 2g of lotus stamen reference material, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of methanol to the residue, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take an appropriate amount of kaempferol reference standard, accurately weigh it, add methanol to prepare a solution containing 50μg per ml, as the reference solution.

[0188] Preparation of the test solution: Take 1g of lotus stamen powder (passed through a No. 3 sieve), add 25ml of 80% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0189] The determination method involves precisely pipetting 10 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0190] The chromatogram of the test sample should show 7 characteristic peaks, and the retention times should correspond to the 7 characteristic peaks in the chromatogram of the reference medicinal material. Peak 6 should correspond to the retention time of the reference peak. The peak corresponding to the kaempferol reference peak is the S peak. Calculate the relative retention times of each characteristic peak and the S peak; the relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.33 (peak 1), 0.55 (peak 2), 0.77 (peak 3), 0.78 (peak 4), 0.93 (peak 5), and 1.01 (peak 7). (See...) Figure 18 )

[0191] Medicinal slices:

[0192] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 346 nm. The theoretical plate number, calculated based on the kaempferol peak, should be no less than 5000.

[0193]

[0194] Preparation of the reference solution: Take 2g of lotus stamen reference material, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of methanol to the residue, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take an appropriate amount of kaempferol reference standard, accurately weigh it, add methanol to prepare a solution containing 50μg per ml, as the reference solution.

[0195] Preparation of the test solution: Take 1g of lotus stamen powder (passed through a No. 3 sieve), add 25ml of 80% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0196] The determination method involves precisely pipetting 10 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0197] The chromatogram of the test sample should show 7 characteristic peaks, and the retention times should correspond to the 7 characteristic peaks in the chromatogram of the reference medicinal material. Peak 6 should correspond to the retention time of the reference peak. The peak corresponding to the kaempferol reference peak is the S peak. Calculate the relative retention times of each characteristic peak and the S peak; the relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.33 (peak 1), 0.55 (peak 2), 0.77 (peak 3), 0.78 (peak 4), 0.93 (peak 5), and 1.01 (peak 7). (See...) Figure 19 )

[0198] Standard decoction:

[0199] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength 346 nm. The theoretical plate number, calculated based on the kaempferol peak, should be no less than 5000.

[0200]

[0201] Preparation of the reference solution: Take 2g of lotus stamen reference material, add 50ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of methanol to the residue, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference solution. Separately, take an appropriate amount of kaempferol reference standard, accurately weigh it, add methanol to prepare a solution containing 50μg per ml, as the reference solution.

[0202] Preparation of the test solution: Take 0.5g of standard decoction, grind it into a fine powder, place it in a stoppered conical flask, add 25ml of methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0203] The determination method involves precisely pipetting 10 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0204] The chromatogram of the test sample should show 7 characteristic peaks, and the retention times should correspond to the 7 characteristic peaks in the chromatogram of the reference medicinal material. Peak 6 should correspond to the retention time of the reference peak. The peak corresponding to the kaempferol reference peak is the S peak. Calculate the relative retention times of each characteristic peak and the S peak; the relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.33 (peak 1), 0.55 (peak 2), 0.77 (peak 3), 0.78 (peak 4), 0.93 (peak 5), and 1.01 (peak 7). (See...) Figure 20 )

[0205] Comparative Example 1: Jiangsu Province Lotus Stamen Formula Granules Quality Standard

[0206] The method was followed for verification, and the results are shown below. Figure 21 .

[0207] Depend on Figure 21 It is known that this method cannot identify the four component peaks of the present invention.

Claims

1. A method for constructing a characteristic spectrum of lotus stamen medicinal material or its preparations, characterized in that: It includes the following steps: a. Preparation of the test sample: Lotus stamens were used as a test sample, dissolved and extracted with a solvent to obtain the test solution; b. The characteristic chromatograms of lotus stamens or its preparations were determined by high-performance liquid chromatography (HPLC). The chromatographic conditions were: C18 column; acetonitrile as mobile phase A; 0.1% phosphoric acid solution as mobile phase B; gradient elution. The gradient elution conditions were as follows:

2. The method for constructing the characteristic spectrum of lotus stamen medicinal material or its preparation according to claim 1, characterized in that: The preparation method of the test solution in step a is as follows: take lotus stamen test sample, grind it into a fine powder, add methanol aqueous solution, sonicate, cool, shake well, filter, and take the filtrate to obtain the solution.

3. The method for constructing the characteristic spectrum of lotus stamen medicinal material or its preparation according to claim 2, characterized in that: The ultrasonic treatment conditions described in step a are as follows: power 600W, frequency 40kHz, ultrasonic time 20-40min; methanol concentration 80%; the amount of lotus stamen and methanol is 10-50ml of methanol per 0.5g.

4. The method for constructing the characteristic spectrum of lotus stamen medicinal material or its preparation according to claim 3, characterized in that: The ultrasound time is 30 minutes, and the amount of lotus stamen and methanol used is 10 ml of methanol per 0.5 g of lotus stamen.

5. The method for constructing the characteristic spectrum of lotus stamen medicinal material or its preparation according to claim 1, characterized in that: The chromatographic column described in step b is a C18 column with a length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm; the flow rate is 0.8-1.2 ml per minute; the column temperature is 25-35℃; the detection wavelength is 210-346 nm; and the theoretical plate number calculated based on the uridine peak should be no less than 8000.

6. The method for constructing the characteristic spectrum of lotus stamen medicinal material or its preparation according to claim 4, characterized in that: Step b describes a column temperature of 30°C, a flow rate of 1.0 ml per minute, and a detection wavelength of 346 nm.

7. The method for constructing the characteristic spectrum of lotus stamen medicinal material or its preparation according to claim 1, characterized in that: This includes the preparation of reference solutions, which include reference medicinal materials, rutin reference standards, kaempferol reference standards, isorhamnetin reference standards, and quercetin reference standards; The preparation method of the reference solution of the reference medicinal material is as follows: take lotus stamen reference medicinal material, add water and decoct, filter, evaporate the filtrate to dryness, add methanol to the residue, sonicate, cool, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material. The preparation method of the rutin reference standard, kaempferol reference standard, isorhamnetin reference standard, and quercetin reference standard solution is as follows: take rutin reference standard, kaempferol reference standard, isorhamnetin reference standard, and quercetin reference standard respectively, and add methanol to prepare reference standard solutions.

8. The method for constructing the characteristic spectrum of lotus stamen medicinal material or its preparation according to any one of claims 1-7, characterized in that: The characteristic spectrum contains 7 characteristic peaks with relative retention times of: peak 1: 0.33, peak 2: 0.55, peak 3: 0.77, peak 4: 0.75, peak 5: 0.77, peak 6: 0.77, peak 7: 0.77, peak 8: 0.77, peak 9: 0.77, peak 10: 0.77, peak 11: 0.77, peak 12: 0.77, peak 13: 0.77, peak 14: 0.77, peak 15: 0.77, peak 16: 0.77, peak 17: 0.77, peak 17: 0.77, peak 18: 0.77, peak 19: 0.77, peak 12: 0.77, peak 19: 0.77, peak 12: 0 Peak 2: 0.78, Peak 5: 0.93, Peak 6: 1.0, Peak 7: 1.01, with relative retention times fluctuating within ±10%; Peak 2 is rutin; Peak 5 is quercetin; Peak 6 is kaempferol; Peak 7 is isorhamnetin.

9. A method for constructing characteristic chromatograms of lotus stamen slices, standard decoctions, and formulated granules, characterized in that: It is a method for constructing characteristic spectra of lotus stamen medicinal material or its preparations as described in any one of claims 1-8.

10. A method for identifying lotus stamen slices, standard decoctions, and formulated granules, characterized in that: It includes the following steps: a. Take the sample to be tested; b. Construct a characteristic spectrum according to the characteristic spectrum construction method of lotus stamen medicinal material or its preparation according to any one of claims 1-8; c. Comparison: If the characteristic peaks are completely identical, then it is genuine lotus stamen.

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