Method for detecting quality of thorny elaeagnus leaves, method for establishing HPLC (High Performance Liquid Chromatography) characteristic peak spectrum of thorny elaeagnus leaves and method for establishing characteristic spectrum of thorny elaeagnus leaf medicinal materials, decoction pieces, standard decoction and formula granules

Abstract

The invention provides a method for detecting the quality of thorny elaeagnus leaves. According to the method, high performance liquid chromatography is adopted for detection. The invention also provides a construction method of characteristic chromatograms of elaeagnus pungens leaf medicinal materials, decoction pieces, standard decoction and formula granules, and a method for judging the stability of the elaeagnus pungens leaf formula granules. Single variety research is performed on the elaeagnus pungens leaf formula granules, the elaeagnus pungens leaves are scientifically identified, the method has the characteristics of comprehensive detection and simple operation, the elaeagnus pungens leaf formula granules can be effectively identified, and the method is suitable for quality control of each link in the preparation process of the elaeagnus pungens leaf formula granules. The uniformity and stability of the quality of the thorny elaeagnus leaf formula granules can be ensured. The method is suitable for elaeagnus pungens leaf medicinal materials, decoction pieces, standard decoction and formula granules.

Description

TECHNICAL FIELD

[0001] The application relates to a construction method of a characteristic chromatogram of Elaeagnus pungens Thunb. BACKGROUND

[0002] Elaeagnus pungens Thunb. is a dry leaf of Elaeagnus pungens Thunb. of Elaeagnaceae. The leaf is collected in autumn. The main chemical components of the leaf are flavonoids, alkaloids, fatty acids, volatile oils, steroids, terpenes, oligopeptides, amino acids and tannins. The leaf has the effects of astringing lung, relieving asthma and relieving cough. The leaf is mainly used for cough and asthma, and hemoptysis. However, the leaf is usually used in the form of a preparation in clinical application. At present, there is no quality standard for the preparation granules of the leaf.

[0003] Application No. 202410414810.5 and the invention name: detection method of Elaeagnus pungens Thunb. characteristic chromatogram and determination method of effective component content, wherein the high performance liquid chromatography method is adopted for determination; wherein the chromatographic conditions include: taking acetonitrile as mobile phase A, and taking formic acid solution or phosphoric acid solution as mobile phase B; gradient elution, and the gradient elution program includes: 0-90 min, the volume percentage of mobile phase A is 10% to 21%; the volume percentage of mobile phase B is 90% to 79%. The chromatographic conditions further include: the mobile phase B is 0.05-0.15% formic acid solution or 0.05-0.15% phosphoric acid solution; and / or, the column temperature is 30-40 DEG C; and / or, the flow rate is 0.8-1.2 mL / min; and / or, the detection wavelength is 220-380 nm; and / or, the injection amount is 5-15 mu L; and / or, the chromatographic column is waters XSelect HSS T3, the specification is 250 mm x 4.6 mm, 5 mu m. The method has poor peak separation, long time and less peak number.

[0004] Zhang Weixin, et al., HPLC fingerprint of Elaeagnus pungens Thunb. medicinal materials, Chinese Experimental Prescriptions Journal, February 2017, the disclosed conditions are as follows: high performance liquid chromatography is adopted, the chromatographic conditions are Agilent ZOTBAX XDB-PhenylC18 chromatographic column (4.6 mm x 250 mm, 5 mu m), mobile phase 0.1% phosphoric acid water-acetonitrile gradient elution, column temperature 30 DEG C, flow rate 1.0 mL·min-1, detection wavelength 360 nm. 16 common peaks are determined to constitute the fingerprint characteristic fingerprint spectrum of 12 batches of Elaeagnus pungens Thunb. medicinal materials. The literature adopts similarity, has no relative retention time of characteristic peaks, and is not useful for quality control of medicinal materials. SUMMARY

[0005] The application provides a quality detection method of Elaeagnus pungens leaves, an HPLC characteristic peak spectrum establishment method of Elaeagnus pungens leaves and a characteristic spectrum construction method of Elaeagnus pungens leaf medicinal materials, decoction pieces, standard decoctions and formula granules.

[0006] The application provides a quality detection method of Elaeagnus pungens leaves, which comprises the following steps:

[0007] a. Preparation of test sample solution: the test sample raw material is dissolved and extracted by a solvent to obtain a to-be-tested solution;

[0008] b. The to-be-tested solution is determined by high performance liquid chromatography to obtain a high performance liquid chromatogram of Elaeagnus pungens leaves;

[0009] The high performance liquid chromatography has the following chromatographic conditions: a C18 column is used as the chromatographic column, acetonitrile is used as the mobile phase A, and a 0.1% phosphoric acid solution is used as the mobile phase B, and gradient elution is performed according to the following table:

[0010]

[0011] The preparation method of the test sample solution is as follows: Elaeagnus pungens leaves are taken, methanol water solution is added, ultrasonic treatment is performed, it is cooled, shaken, filtered, and the continuous filtrate is obtained.

[0012] The step further comprises preparation of a reference solution, including preparation of a control medicinal material reference solution and a control reference solution; the preparation method of the control medicinal material reference solution is as follows:

[0013] Elaeagnus pungens leaves are taken, water decoction is performed, filtration is performed, the filtrate is evaporated to dryness, the residue is added with methanol water solution, ultrasonic treatment is performed, it is cooled, shaken, filtered, and the continuous filtrate is obtained;

[0014] The preparation method of the control reference solution is as follows: a control reference solution of tannic acid is prepared by accurately weighing and adding methanol to prepare a solution containing 50 μg per 1 ml.

[0015] The chromatographic column is as follows: octadecylsilane-bonded silica gel is used as the filler (the column length is 250 mm, the inner diameter is 4.6 mm, and the particle size is 5 μm); the flow rate is 1.0 ml per minute; the column temperature is 25 DEG C; the detection wavelength is 270 nm; and the theoretical plate number calculated according to the tannic acid peak should be not less than 5000;

[0016] The application further provides an HPLC characteristic peak spectrum establishment method of Elaeagnus pungens leaves, which is to establish a characteristic spectrum by using the quality detection method, and the characteristic spectrum comprises eight common chromatographic peaks, and the relative retention times of the eight common chromatographic peaks are as follows:

[0017] Peak 1: 0.23 min, peak 2: 0.60 min, peak 3: 0.74 min, peak 4: 0.75 min, peak 5: 1 min, peak 6: 1.05 min, peak 7: 1.28 min, peak 8: 1.46 min, the relative retention time fluctuates within the range of ±10% of the specified value.

[0018] Peak 1 is gallic acid, peak 5 is ellagic acid, and peak 8 is silver forging glycoside.

[0019] The application provides a construction method of a characteristic spectrum of Elaeagnus pungens leaf medicinal material, decoction piece, standard decoction and formula granule.

[0020] The application also provides a method for judging the stability of the formula granule of the Elaeagnus pungens leaf, which is a characteristic spectrum constructed by the construction method of the characteristic spectrum of the Elaeagnus pungens leaf medicinal material, decoction piece, standard decoction and formula granule.

[0021] It comprises the following steps:

[0022] a. Pre-treating the sample to be detected;

[0023] b. Detecting the characteristic peaks, if none of the peaks 1-8 has a peak, it is judged that the formula granule is unqualified.

[0024] The application performs single-species research on the formula granule of the Elaeagnus pungens leaf, scientifically identifies the Elaeagnus pungens leaf, has the characteristics of comprehensive detection and simple operation, can effectively identify the formula granule of the Elaeagnus pungens leaf, is suitable for quality control of each link in the preparation process of the formula granule of the Elaeagnus pungens leaf, and can ensure the uniformity and stability of the quality of the formula granule of the Elaeagnus pungens leaf. The method is suitable for the Elaeagnus pungens leaf medicinal material, decoction piece, standard decoction and formula granule. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Reference characteristic spectrum (peak 1: gallic acid; peak 5 (S): ellagic acid; peak 8: silver forging glycoside);

[0026] Figure 2 Elaeagnus pungens leaf chromatogram-210 nm;

[0027] Figure 3 Elaeagnus pungens leaf granule chromatogram-230 nm;

[0028] Figure 4 Elaeagnus pungens leaf granule chromatogram-250 nm;

[0029] Figure 5 Elaeagnus pungens leaf granule chromatogram-270 nm;

[0030] Figure 6Elution profile of Elsholtziae Semen granules at 310 nm;

[0031] Figure 7 Elution profile of Elsholtziae Semen granules at 330 nm;

[0032] Figure 8 Investigation of Elsholtziae Semen granules at different column temperatures;

[0033] Figure 9 Investigation of flow rate;

[0034] Figure 10 Investigation of Elsholtziae Semen granules for delay;

[0035] Figure 11 Investigation of extraction solvent;

[0036] Figure 12 Investigation of extraction solvent;

[0037] Figure 13 Investigation of extraction time;

[0038] Figure 14 Investigation of solvent addition amount;

[0039] Figure 15 Peak identification;

[0040] Figure 16 Different instruments;

[0041] Figure 17 Different chromatographic columns;

[0042] Figure 18 Verification figures of characteristic chromatograms of 3 batches of Elsholtziae Semen granules (from bottom to top: HTZY-KL-01, HTZY-KL-02, HTZY-KL-03);

[0043] Figure 19 Characteristic chromatogram of Elsholtziae Semen medicinal material;

[0044] Figure 20 Characteristic chromatogram of Elsholtziae Semen decoction pieces;

[0045] Figure 21 Characteristic chromatogram of Elsholtziae Semen standard decoction;

[0046] Figure 22 Controlled characteristic chromatogram of Elsholtziae Semen granules (in which, peak 1: gallic acid; peak 5 (S): ellagic acid; peak 8: elsholtzynin);

[0047] Figure 23 Controlled characteristic chromatogram of Elsholtziae Semen medicinal material (in which, peak 1: gallic acid; peak 5 (S): ellagic acid; peak 8: elsholtzynin);

[0048] Figure 24Elsholtziae Folium quality control characteristic spectrum (in which, peak 1: gallic acid; peak 5 (S): ellagic acid; peak 8: silibinin).

[0049] Peak 8: silibinin.

[0050] Figure 25 Elsholtziae Folium quality control characteristic spectrum (in which, peak 1: gallic acid; peak 5 (S): ellagic acid; peak 8: silibinin). DETAILED DESCRIPTION

[0051] Elsholtziae Folium is the dry leaf of Elaeagnus pungens Thunb. of Elaeagnaceae. It has the effects of astringing lung, relieving asthma and relieving cough. The standard decoction and formula granules are the freeze-dried powder prepared from the processed medicinal materials. In order to ensure the uniformity and stability of the quality of Elsholtziae Folium medicinal materials, pieces, standard decoction and formula granules, a new characteristic spectrum method is established to control the quality.

[0052] Example 1 Quality detection method of Elsholtziae Folium in the application

[0053] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel is used as the filler (the column length is 250 mm, the inner diameter is 4.6 mm, and the particle size is 5 μm); acetonitrile is used as the mobile phase A, and 0.1% phosphoric acid solution is used as the mobile phase B, which is gradient eluted according to the following table; the flow rate is 1.0 ml per minute; the column temperature is 25 °C; and the detection wavelength is 270 nm. The theoretical plate number should not be less than 5000 calculated according to the ellagic acid peak.

[0054]

[0055] Preparation of reference solution: 1 g of Elsholtziae Folium control medicinal materials is added into 25 ml of water, decocted for 30 minutes, filtered, and the filtrate is evaporated to dryness; the residue is added into 25 ml of 50% methanol, ultrasonically treated (power 600 W, frequency 40 kHz) for 30 minutes, cooled, shaken uniformly, filtered, and the continuous filtrate is taken as the reference solution of the control medicinal materials. In addition, an appropriate amount of ellagic acid reference substance is accurately weighed and determined, methanol is added to prepare a solution containing 50 μg per 1 ml, which is taken as the reference solution of the reference substance.

[0056] Preparation of test solution: an appropriate amount of Elsholtziae Folium formula granules is finely ground, 0.1 g of which is taken into a conical flask with a plug, 25 ml of 50% methanol is added, ultrasonically treated (power 600 W, frequency 40 kHz) for 30 minutes, cooled, shaken uniformly, filtered, and the continuous filtrate is obtained.

[0057] Determination: 10 μl of the reference solution and the test solution are precisely taken respectively, injected into the liquid chromatograph, and determined, and the quality of Elsholtziae Folium is obtained.

[0058] The test sample chromatogram should present 8 characteristic peaks, which should correspond to the retention time of the 8 characteristic peaks in the chromatogram of the reference substance of the control medicinal material. Peak 5 should correspond to the retention time of the reference peak of the control substance. The peak corresponding to the reference peak of the control substance is the S peak. The relative retention time of each characteristic peak to the S peak is calculated, which should be within ±10% of the specified value. The specified value is: 0.23 (peak 1), 0.60 (peak 2), 0.74 (peak 3), 0.75 (peak 4), 1.05 (peak 6), 1.28 (peak 7), 1.46 (peak 8). (See Figure 1 )

[0059] Example 2: Selection of test method condition parameters of the present application

[0060] 1. Experimental instruments and materials

[0061] High performance liquid chromatograph: Shimadzu LC-20AD type high performance liquid chromatograph, Agilent 1260 type high performance liquid chromatograph, Waters e2695 type high performance liquid chromatograph;

[0062] Electronic balance: ME204E / 02, XPE26 (Mettler-Toledo Instruments Co., Ltd.);

[0063] Ultrapure water machine: CelluSafe 1810A (Shanghai Moore Scientific Instruments Co., Ltd.);

[0064] Ultrasonic cleaner: KQ600DB type (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0065] Chromatographic column: Kromasil 100-5-C18, 250x4.6mm; GL Sciences InertSustain AQ-C18 5μm, 250x4.6mm; Shim-pack GIST C18 4.6*250mm, 5μm;

[0066] Acetonitrile and phosphoric acid are chromatographically pure, water is ultrapure water, and the rest of the reagents are analytically pure.

[0067] Gallic acid reference substance (China Institute for Drug Control, batch number: 110831-201906, purity: 91.5%);

[0068] Tannic acid reference substance (China Institute for Drug Control, batch number: 111959-201903, purity: 88.8%);

[0069] Silver linden glycoside: (Chengdu Desit Biotechnology Co., Ltd., batch number: DSTDD004402, purity: 98.75%);

[0070] Elsholtziae Semen control drug (Chengdu Deest Biological Technology Co., Ltd., batch number: DSTYH012301);

[0071] Elsholtziae Semen dispensing granules: HTZY-KL-01, HTZY-KL-02, HTZY-KL-03;

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

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

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

[0075] 2. Detection wavelength point determination

[0076] On the basis of the above proposed experimental conditions, the diode array detector was used to scan the full wave band of the test solution, and the chromatograms of the test solution at 210 nm, 230 nm, 250 nm, 270 nm, 310 nm, 330 nm wavelength were extracted respectively. See Figures 2-7 .

[0077] The results showed that when the detection wavelength was 270 nm, the chromatographic peak information was larger, and the chromatogram baseline was more stable, so the detection wavelength was determined as 270 nm.

[0078] 3. Column temperature investigation

[0079] Based on the above experimental conditions, the column temperature was investigated at 25℃, 30℃ and 35℃, respectively. As shown in Table 1. Figure 8

[0080] Table 1. Column temperature investigation - relative retention time ratio of characteristic peaks

[0081]

[0082] The column temperature investigation results show that when the column temperature is 25℃, the chromatogram peak shape is relatively symmetrical, and the separation degree is good, so 25℃ is finally determined as the column temperature of the characteristic spectrum method of Elaeagnus pungens leaf dispensing granules.

[0083] 4. Flow rate investigation

[0084] Based on the above experimental conditions, the flow rate was investigated at 0.7ml / min, 1.0ml / min and 1.2ml / min, respectively. As shown in Table 2. Figure 9

[0085] Table 2. Flow rate - relative retention time

[0086]

[0087] The results show that when the flow rate is 1.0ml / min, the chromatogram peak shape is good, and the separation degree is moderate. Therefore, the flow rate is determined as 1.0ml / min.

[0088] 5. Delay test

[0089] Based on the above experimental conditions, the chromatogram acquisition time was extended to 150min. As shown in Table 3. Figure 10

[0090] The results show that the sample has basically no chromatographic peak after 75min, so the sample detection time is determined as 75min.

[0091] 6. Extraction solvent investigation

[0092] Take Elaeagnus pungens leaf dispensing granules (batch number: HTZY-KL-01) 0.1g, grind finely, and put into a conical flask with a plug, respectively add 25ml of methanol, 80% methanol, 50% methanol, 30% methanol, water, 50% ethanol, ethanol, ultrasonic treatment (power 600W, frequency 40kHz) for 30 minutes, cool, shake evenly, filter, take the filtrate, as shown in Table 4. Figure 11

[0093] The results show that when the extraction solvent is 50% methanol, the chromatographic peak information is large, so the test sample extraction solvent is determined as 50% methanol.​​​​

[0094] 7. Extraction mode investigation

[0095] Take Elaeagnus paliachatus leaf dispensing granules (batch number: HTZY-KL-01) 0.1 g, grind finely, add 50% methanol 25 ml, respectively, reflux, ultrasonic investigation, extraction time 30 minutes, cool, shake, filter, take the filtrate, that is, as shown in Figure 12 .

[0096] The results show that the effect is consistent when the test sample is extracted by ultrasonic extraction and reflux extraction. Since ultrasonic extraction is more convenient to operate, the test sample extraction method is determined to be ultrasonic extraction.

[0097] 8. Extraction time investigation

[0098] Take Elaeagnus paliachatus leaf dispensing granules (batch number: HTZY-KL-01) 0.1 g, grind finely, place in a conical flask with a stopper, add 50% methanol 25 ml, ultrasonic extraction of the test sample for 30 minutes, 45 minutes, 60 minutes, respectively, cool, shake, filter, take the filtrate, that is, as shown in Figure 13 .

[0099] The results show that when the extraction time is 30 minutes, it can be fully extracted. Therefore, the extraction time of the test sample is determined to be 30 minutes.

[0100] 9. Solvent addition amount investigation

[0101] Take Elaeagnus paliachatus leaf dispensing granules (batch number: HTZY-KL-01) 0.1 g, grind finely, place in a conical flask with a stopper, add 50% methanol 15 ml, 25 ml, 50 ml, respectively, tightly stop, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, shake, filter, take the filtrate, that is, as shown in Figure 14 .

[0102] The results show that when the solvent addition amount is 25 ml, the chromatographic peak area of the characteristic spectrum is moderate. Therefore, the solvent addition amount of the test sample is determined to be 25 ml.

[0103] 10. Determination of test sample preparation method

[0104] Take Elaeagnus paliachatus leaf dispensing granules 0.1 g, grind finely, place in a conical flask with a stopper, add 50% methanol 25 ml, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake, filter, take the filtrate, that is, as shown in

[0105] 11. Methodology investigation

[0106] 11.1 Chromatographic peak identification

[0107] Preparation of the test solution: Prepare the test solution of Elaeagnus pungens leaf granules according to the experimental conditions proposed above.

[0108] Preparation of the reference herb solution: Take 1g of Elaeagnus pungens leaf reference herb, add 25ml of water, decoct for 30 minutes, filter, evaporate the filtrate to dryness, add 25ml of 50% methanol to the residue, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, shake well, filter, and take the filtrate as the reference herb solution. Separately, take appropriate amounts of gallic acid reference standard, ellagic acid reference standard, and linalool reference standard, accurately weigh them, add methanol to prepare a solution containing 50μg per ml, as the reference standard solution.

[0109] Preparation of negative control solution: Prepare negative control solution of Elaeagnus pungens leaf-deficient formula granules according to the experimental conditions proposed above.

[0110] The characteristic peaks of the Elaeagnus pungens leaf formulation granules were located. For example... Figure 15 As shown.

[0111] Depend on Figure 15 It can be seen that peak 1 is gallic acid, peak 5 is ellagic acid, and peak 8 is linalool.

[0112] 11.2 Precision Test

[0113] Take the Elaeagnus pungens leaf granule test solution and inject it 6 times consecutively according to the proposed experimental method, 10 μL each time, and calculate the retention time of each characteristic peak. As shown in Tables 3 and 4.

[0114] Table 3 Precision Examination - Retention Time

[0115]

[0116] The results show that the retention time RSD of each characteristic peak is 0.01%-0.04%, indicating that the method has good precision.

[0117] 11.3 Repeatability Test

[0118] Six portions of the Elaeagnus pungens leaf granules were accurately weighed and prepared and measured according to the proposed experimental method. See Tables 5 and 6 for details.

[0119] Table 5 Repeatability Tests - Relative Retention Time

[0120]

[0121] The results show that the relative retention time (RSD) of each characteristic peak is 0.04%–0.23%. The method exhibits good repeatability.

[0122] 11.4 Intermediate Precision Examination

[0123] 11.4.1 Different instrument investigation

[0124] On the basis of the above experimental conditions, three portions of Elaeagnus pungens leaf dispensing granules were precisely weighed, and the test sample solutions were prepared, which were determined on Agilent 1260, Shimadzu LC-20AD, and Waters e2695 type high performance liquid chromatographs, respectively. Figure 16 , Table 7 and Table 8.

[0125] Table 7 Instrument durability investigation- relative retention time ratio

[0126]

[0127] The results showed that when the test sample was detected by the above three instruments, the RSD range of the relative retention time of each characteristic peak was 0.60%-3.56%, and the instrument durability was good.

[0128] 11.4.2 Different personnel and time investigation

[0129] On the basis of the above experimental conditions, two portions of Elaeagnus pungens leaf dispensing granules were weighed by different personnel (A1, A2) at different times (T1, T2), and the test samples were prepared and determined. As shown in Table 9 and Table 10.

[0130] Table 9 Personnel and time investigation- relative retention time

[0131]

[0132] The results showed that the determination of the same sample by different personnel at different times had good method stability.

[0133] 11.5 Durability investigation

[0134] 11.5.1 Column durability investigation

[0135] On the basis of the above experimental conditions, the columns Kromasil 100-5-C18, 250*4.6mm; GL Sciences InertSustain AQ-C18 5μm, 250*4.6mm; Shim-pack GIST C18 4.6*250mm, 5μm were investigated, respectively. As shown in Table 11 and Table 12. Figure 17

[0136] Table 11 Column durability investigation- relative retention time

[0137]

[0138] From the above results, it can be seen that the relative retention time of each characteristic peak was within the range of 0.60%-3.56%, and the column durability was good. Figure 17 ​It can be seen that the peak separation of each chromatogram is good and the peak is clear, and the durability of the chromatographic column is good.

[0139] 11.5.2 Stability Investigation

[0140] Based on the above experimental conditions, the same test solution was taken and measured at 0 h, 4 h, 8 h, 12 h, 16 h, and 24 h. As shown in Tables 13 and 14.

[0141] Table 13 Stability Investigation - Retention Time

[0142]

[0143] The results show that the RSD of the corresponding characteristic peak retention time is 0.03% to 0.10%, and the sample solution is stable within 24 hours.

[0144] 11.6 Verification Results of 3 Batches of Elaeagnus Pungens Leaf Formula Granules

[0145] The proposed method was used to determine the characteristic spectrum of 3 batches of samples, and the relative retention time was calculated. As shown in Figure 18 , Tables 15 and 16.

[0146] Table 2 Relative Retention Time of 3 Batches of Elaeagnus Pungens Leaf Formula Granules

[0147]

[0148]

[0149] According to the principles of relative retention time stability and detection of each batch of samples with relatively high peaks, a total of 8 peaks with good reproducibility were selected as characteristic peaks. The results show that when peak 5 is used as the S peak, the relative retention time RSD of the characteristic peaks of 3 batches of Elaeagnus Pungens Leaf Formula Granules is less than 1%.

[0150] Establishment of Relative Retention Time Limit

[0151] The results of the methodological investigation and verification are shown in Table 17:

[0152] Table 3 Summary of RSD% of Methodological Results - Relative Retention Time

[0153]

[0154] Therefore, the relative retention time of each peak is temporarily set as ±10%.

[0155] Finally, it is specified that the RSD of the relative retention time of each characteristic peak meets the requirements in the above investigations, and the method is good. The test sample characteristic spectrum should present 8 characteristic peaks, of which the peak corresponding to the reference of tannic acid is the S peak.

[0156] Results of 16 batches of Elaeagnus pungens leaf medicinal materials

[0157] The characteristic chromatograms of 16 batches of samples were determined by the proposed method, and the relative retention times were calculated. As shown in Table 18 and Table 19. Figure 19

[0158] Table 4 Relative retention times of 21 batches of Elaeagnus pungens leaf medicinal materials

[0159]

[0160]

[0161] Results of 16 batches of Elaeagnus pungens leaf medicinal materials

[0162] The characteristic chromatograms of 16 batches of samples were determined by the proposed method, and the relative retention times were calculated. As shown in Table 20 and Table 21. Figure 20

[0163] Table 20 Relative retention times of 21 batches of Elaeagnus pungens leaf medicinal materials

[0164]

[0165]

[0166] Results of 16 batches of Elaeagnus pungens leaf medicinal materials

[0167] The characteristic chromatograms of 16 batches of samples were determined by the proposed method, and the relative retention times were calculated. As shown in Table 22 and Table 23. Figure 21

[0168] Table 22 Relative retention times of 21 batches of Elaeagnus pungens leaf medicinal materials

[0169]

[0170]

[0171] Example 3 Verification test of the detection method of the present application

[0172] Granules:

[0173] Chromatographic conditions and system suitability test The octadecylsilane-bonded silica gel was used as the filler (the column length was 250 mm, the inner diameter was 4.6 mm, and the particle size was 5 μm); acetonitrile was used as the mobile phase A, and 0.1% phosphoric acid solution was used as the mobile phase B, which was gradient eluted according to the following table; the flow rate was 1.0 ml per minute; the column temperature was 25°C; and the detection wavelength was 270 nm. The theoretical plate number calculated according to the peak of gallic acid should not be less than 5000. ​​​

[0174]

[0175] Reference solution preparation: 1 g of the control medicinal material of Elaeagnus pungens leaves was added to 25 ml of water, decocted for 30 minutes, filtered, and the filtrate was evaporated to dryness. The residue was added to 25 ml of 50% methanol, ultrasonically treated (power 600 W, frequency 40 kHz) for 30 minutes, allowed to cool, shaken well, filtered, and the filtrate was taken as the reference solution of the control medicinal material. An appropriate amount of the reference substance of gallic acid was precisely weighed, dissolved in methanol to prepare a solution containing 50 μg per 1 ml, and the solution was taken as the reference solution of the control substance.

[0176] Test solution preparation: an appropriate amount of the formula granules of Elaeagnus pungens leaves was finely ground, 0.1 g of the powder was taken, added to a conical flask with a stopper, 25 ml of 50% methanol was added, ultrasonically treated (power 600 W, frequency 40 kHz) for 30 minutes, allowed to cool, shaken well, filtered, and the filtrate was taken as the test solution.

[0177] Determination: 10 μl of the reference solution and the test solution were precisely taken, injected into the liquid chromatograph, and determined, and the test solution was obtained.

[0178] In the chromatogram of the test sample, 8 characteristic peaks should be present, and should correspond to the retention times of the 8 characteristic peaks in the chromatogram of the reference solution of the control medicinal material. Peak 5 should correspond to the retention time of the peak of the reference substance of the control substance. The peaks corresponding to the peaks of the reference substance of gallic acid are S peaks. The relative retention times of the characteristic peaks and the S peaks were calculated, and the relative retention times should be within ±10% of the specified values. The specified values are: 0.23 (peak 1), 0.60 (peak 2), 0.74 (peak 3), 0.75 (peak 4), 1.05 (peak 6), 1.28 (peak 7), and 1.46 (peak 8) (see Table 1). Figure 22

[0179] Medicinal material:

[0180] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel was used as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as the mobile phase A, and 0.1% phosphoric acid solution was used as the mobile phase B, which was gradient eluted according to the following table; the flow rate was 1.0 ml per minute; the column temperature was 25°C; and the detection wavelength was 270 nm. The theoretical plate number calculated according to the peak of gallic acid should not be less than 5000.

[0181]

[0182]

[0183] ​Reference solution preparation: 1 g of the control drug of Elaeagnus pungens leaves was added to 25 ml of water, decocted for 30 minutes, filtered, and the filtrate was evaporated to dryness. The residue was added to 25 ml of 50% methanol, ultrasonically treated (power 600 W, frequency 40 kHz) for 30 minutes, allowed to cool, shaken well, filtered, and the filtrate was collected as the reference solution of the control drug. An appropriate amount of the control of gallic acid was precisely weighed, added to methanol to prepare a solution containing 50 μg per 1 ml, and used as the reference solution of the control.

[0184] Test solution preparation: 1 g of the powder of Elaeagnus pungens medicinal material (passed through a No. 2 sieve) was added to 25 ml of water, decocted for 30 minutes, filtered, and the filtrate was evaporated to dryness. The residue was added to 25 ml of 50% methanol, ultrasonically treated (power 600 W, frequency 40 kHz) for 30 minutes, allowed to cool, shaken well, filtered, and the filtrate was collected as the test solution.

[0185] Determination: 10 μl of each of the reference solution and the test solution was precisely taken and injected into the liquid chromatograph for determination.

[0186] In the chromatogram of the test solution, 8 characteristic peaks should be present and should correspond to the retention times of the 8 characteristic peaks in the chromatogram of the reference solution of the control drug. Peak 5 should correspond to the retention time of the peak of the reference of the control. The peak corresponding to the peak of the reference of the control of gallic acid is the S peak. The relative retention times of each characteristic peak and the S peak were calculated, which should be within ±10% of the specified values. The specified values are: 0.23 (peak 1), 0.60 (peak 2), 0.74 (peak 3), 0.75 (peak 4), 1.05 (peak 6), 1.28 (peak 7), and 1.46 (peak 8). (See Figure 23 )

[0187] Decoction pieces:

[0188] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel was used as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as the mobile phase A and 0.1% phosphoric acid solution was used as the mobile phase B, which was gradient eluted according to the following table; the flow rate was 1.0 ml per minute; the column temperature was 25°C; and the detection wavelength was 270 nm. The theoretical plate number calculated according to the peak of gallic acid should be not less than 5000.

[0189]

[0190] Reference solution preparation: 1 g of the control drug of Elaeagnus pungens leaves was added to 25 ml of water, decocted for 30 minutes, filtered, and the filtrate was evaporated to dryness. The residue was added to 25 ml of 50% methanol, ultrasonically treated (power 600 W, frequency 40 kHz) for 30 minutes, allowed to cool, shaken well, filtered, and the filtrate was collected as the reference solution of the control drug. An appropriate amount of the control of gallic acid was precisely weighed, added to methanol to prepare a solution containing 50 μg per 1 ml, and used as the reference solution of the control.

[0191] Preparation of test solution: 1 g of Elaeagnus pungens Thunb. powder (passed No. 2 sieve) was added to 25 ml of water, decocted for 30 minutes, filtered, and the filtrate was evaporated to dryness. The residue was added to 25 ml of 50% methanol, ultrasonically treated (power 600 W, frequency 40 kHz) for 30 minutes, allowed to cool, shaken well, filtered, and the filtrate was collected as the test solution.

[0192] Determination: 10 μl of the reference solution and the test solution were precisely pipetted into the liquid chromatograph, respectively, and determined to obtain the test solution.

[0193] In the chromatogram of the test solution, 8 characteristic peaks should be present, and should correspond to the retention times of the 8 characteristic peaks in the chromatogram of the reference solution of the control medicinal material. Peak 5 should correspond to the retention time of the reference peak of the control. The peak corresponding to the reference peak of the control is the S peak. The relative retention times of the characteristic peaks to the S peak were calculated, and the relative retention times should be within ±10% of the specified values. The specified values are: 0.23 (peak 1), 0.60 (peak 2), 0.74 (peak 3), 0.75 (peak 4), 1.05 (peak 6), 1.28 (peak 7), and 1.46 (peak 8). (See Figure 24 )

[0194] Standard decoction:

[0195] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel was used as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile was used as the mobile phase A, and 0.1% phosphoric acid solution was used as the mobile phase B, which was gradient eluted according to the following table; the flow rate was 1.0 ml per minute; the column temperature was 25°C; and the detection wavelength was 270 nm. The theoretical plate number calculated according to the peak of ellagic acid should not be less than 5000.

[0196]

[0197] Preparation of reference solution: 1 g of Elaeagnus pungens Thunb. leaf control medicinal material was added to 25 ml of water, decocted for 30 minutes, filtered, and the filtrate was evaporated to dryness. The residue was added to 25 ml of 50% methanol, ultrasonically treated (power 600 W, frequency 40 kHz) for 30 minutes, allowed to cool, shaken well, filtered, and the filtrate was collected as the reference solution of the control medicinal material. An appropriate amount of ellagic acid control was precisely weighed, dissolved in methanol to prepare a solution containing 50 μg per 1 ml, and the solution was used as the reference solution of the control.

[0198] Preparation of test solution: 0.1 g of Elaeagnus pungens Thunb. standard decoction was placed in a conical flask with a stopper, 25 ml of 50% methanol was added, the flask was tightly stoppered, ultrasonically treated (power 600 W, frequency 40 kHz) for 30 minutes, allowed to cool, shaken well, filtered, and the filtrate was collected as the test solution.

[0199] The assay method is as follows: precisely pipette 10 μl of the reference solution and the sample solution, respectively, inject into the liquid chromatograph, and determine, and then obtain.

[0200] The sample chromatogram should show 8 characteristic peaks, which should correspond to the retention time of the 8 characteristic peaks in the reference chromatogram of the control drug material. Peak 5 should correspond to the retention time of the reference peak of the control. The peak corresponding to the reference peak of the control is the S peak. Calculate the relative retention time of each characteristic peak to the S peak, which should be within ±10% of the specified value. The specified value is: 0.23 (peak 1), 0.60 (peak 2), 0.74 (peak 3), 0.75 (peak 4), 1.05 (peak 6), 1.28 (peak 7), 1.46 (peak 8). (See Figure 25 ).

Claims

1. A method for quality testing of Elaeagnus angustifolia leaves, characterized in that: It includes the following steps: a. Preparation of the test solution: Dissolve and extract the raw material of the test sample using a solvent to obtain the test solution; b. The test solution was analyzed by high performance liquid chromatography to obtain the high performance liquid chromatogram of Elaeagnus pungens leaves; The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column, acetonitrile is used as mobile phase A, 0.1% phosphoric acid solution is used as mobile phase B, and gradient elution is performed according to the specifications in the table below; 2. The method for quality detection of Elaeagnus angustifolia leaves according to claim 1, characterized in that: The method for preparing the test solution is as follows: take Elaeagnus pungens leaves, add methanol aqueous solution, sonicate, cool, shake well, filter, and take the filtrate to obtain the test solution.

3. The method for quality detection of Elaeagnus angustifolia leaves according to claim 2, characterized in that: The ultrasonic treatment conditions are: power 600W, frequency 40kHz.

4. The method for quality detection of Elaeagnus angustifolia leaves according to claim 1, characterized in that: The steps also include the preparation of a reference solution, including the preparation of a reference solution for a control medicinal material and a reference solution for a control standard; wherein, the method for preparing the reference solution for the control medicinal material is as follows: Take the leaves of Elaeagnus angustifolia, decoct them in water, filter them, evaporate the filtrate to dryness, add methanol-water solution to the residue, sonicate, cool, shake well, filter, and collect the filtrate to obtain the product. The preparation method of the reference solution is as follows: accurately weigh ellagic acid reference standard and add methanol to make a solution.

5. The method for quality testing of Elaeagnus angustifolia leaves according to any one of claims 1-4, characterized in that: The chromatographic column is constructed with octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); the flow rate is 1.0 ml per minute; the column temperature is 25 °C; the detection wavelength is 270 nm; and the theoretical plate number, calculated based on the ellagic acid peak, should be no less than 5000.

6. A method for establishing HPLC characteristic peak chromatograms of Elaeagnus pungens leaves, characterized in that: It establishes a characteristic chromatogram using the quality detection method described in any one of claims 1-5, wherein the characteristic chromatogram contains 8 common chromatographic peaks with relative retention times as follows: Peak 1: 0.23 min, Peak 2: 0.60 min, Peak 3: 0.74 min, Peak 4: 0.75 min, Peak 5: 1 min, Peak 6: 1.05 min, Peak 7: 1.28 min, Peak 8: 1.46 min, with relative retention times fluctuating within ±10% of the specified value.

7. The method for establishing the HPLC characteristic peak spectrum of Elaeagnus angustifolia leaves according to claim 6, characterized in that: Peak 1 is gallic acid, peak 5 is ellagic acid, and peak 8 is silicoside.

8. A method for constructing characteristic maps of Elaeagnus pungens leaf medicinal materials, processed slices, standard decoctions, and formulated granules, characterized in that: It constructs a feature map using the quality detection method for Elaeagnus angustifolia leaves as described in any one of claims 1-5.

9. A method for determining the stability of Elaeagnus pungens leaf granules, characterized in that: It is a feature spectrum constructed by the method for constructing feature spectrum of Elaeagnus pungens leaf medicinal material, decoction pieces, standard decoction, and formula granules as described in claim 8, and is used to evaluate the stability of Elaeagnus pungens leaf formula granules.

10. The method for determining the stability of Elaeagnus angustifolia leaf formulation granules according to claim 9, characterized in that: It includes the following steps: a. Pre-treatment of the sample to be tested; b. Detect characteristic peaks. If no peak is observed in any of peaks 1-8, the formula granules are judged to be unqualified.

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