Characteristic chromatogram construction method of piper wallichii medicinal material or preparation thereof

Abstract

The invention provides a characteristic spectrum construction method of a piper wallichii medicinal material or a preparation thereof, and further provides a true and false identification method of the piper wallichii medicinal material or the preparation thereof. According to the method, the single variety of the piper wallichii formula granules is researched, the piper wallichii is scientifically identified, the method has the characteristics of comprehensive detection and simplicity and convenience in operation, the piper wallichii formula granules can be effectively identified, and the uniformity and stability of the quality of the piper wallichii formula granules can be ensured. The method is suitable for piper wallichii medicinal materials, decoction pieces, standard decoction and formula granules.

Description

TECHNICAL FIELD

[0001] The application relates to a characteristic spectrum construction method of Piper wallichii (Miq.) Hand.-Mazz. medicinal material or a preparation thereof, and belongs to the field of drug quality detection. BACKGROUND

[0002] Piper wallichii (Miq.) Hand.-Mazz. is the dried whole plant of Piper wallichii (Miq.) Hand.-Mazz. in the Piperaceae family, which has the effects of expelling wind and dampness, strengthening the knees and abdomen, relieving pain, and relieving cough.

[0003] Piper wallichii (Miq.) Hand.-Mazz. mainly contains lignans, neolignans, amide alkaloids and the like, has the effects of expelling wind and cold, strengthening the knees and abdomen, and tonifying the kidney and strengthening yang, and is often used for treating rheumatic arthralgia, lumbago and the like. However, Piper wallichii (Miq.) Hand.-Mazz. is often used in the form of a preparation in clinical application. The existing literature research is mainly about the characteristic fingerprint spectrum and content determination of amide alkaloids in Piper wallichii (Miq.) Hand.-Mazz. (Cao Jie, et al., Piper wallichii (Miq.) Hand.-Mazz. HPLC characteristic spectrum research and amide component content measurement, Chinese Herbal Medicine, Vol. 45, No. 4, April 2022), and the method reported in the literature is to control the quality of Piper wallichii (Miq.) Hand.-Mazz. by taking pipersintenamide and wallenrootine as marker components. Pipersintenamide is a piperidine alkaloid and also belongs to an amide compound. The chemical structure of pipersintenamide is as follows: its systematic name is (2E,6E)-7-(1,3-benzodioxol-5-yl)-1-(piperidin-1-yl)hepta-2,6-dien-1-one, and the molecular formula is C 19 H 23 NO3. The structure contains a piperidine ring and an amide bond (-CON-), and therefore has the characteristics of piperidine alkaloids and amide compounds. Pipersintenamide has certain cytotoxicity and shows inhibitory effect on various cancer cell lines (such as HL-60, PC-3 and the like) (Cytotoxic amides from Piper sintenense. DOI:10.1055 / s-2002-35660). Taking alkaloids such as pipersintenamide and wallenrootine as marker components cannot reflect the quality of Piper wallichii (Miq.) Hand.-Mazz., and has limitations as a quality control index.

[0004] Because the chemical composition of Rhynchosia volubilis is complex (Ma Jiangxiong, et al. Research Progress on Chemical Constituents, Pharmacological Activities and Quality Control of Rhynchosia volubilis. Chinese Ethnic and Folk Medicine, February 2024, Vol. 33, No. 4), the wild resources of Rhynchosia volubilis cannot meet the production and clinical application, and similar varieties are often used instead. Quality evaluation and quality control research of Rhynchosia volubilis is imperative. The existing detection methods have limitations, such as using microscopic identification to distinguish the easily confused plants Hainan Rhynchosia and Rhynchosia volubilis; using thin layer scanning method to determine the content of Rhynchosia volubilis in the south, or using HPLC method with cephalanone B, aristolactam AII, and aristolactam AIIa as indicators. The existing methods cannot meet the requirements of quality control. SUMMARY

[0005] The application provides a characteristic spectrum construction method of Rhynchosia volubilis medicinal materials or preparations thereof.

[0006] The application provides a characteristic spectrum construction method of Rhynchosia volubilis medicinal materials or preparations thereof, which comprises the following steps:

[0007] a. Preparation of test sample solution:

[0008] The Rhynchosia volubilis medicinal materials or preparations are dissolved with a solvent, extracted, and a test solution is obtained;

[0009] b. The test solution is determined by high performance liquid chromatography to obtain the characteristic spectrum of Rhynchosia volubilis medicinal materials or preparations;

[0010] The high performance liquid chromatography conditions are as follows: the chromatographic column is a C18 column; acetonitrile is used as mobile phase A, and 0.2% formic acid solution is used as mobile phase B for gradient elution, and the gradient elution conditions are as follows:

[0011]

[0012] The preparation method of the test sample solution is as follows:

[0013] The Rhynchosia volubilis medicinal materials or preparations are finely ground, added with methanol, ultrasonically treated, cooled, shaken, filtered, and the filtered solution is obtained.

[0014] The ultrasonic treatment conditions are as follows: power 600 W, frequency 40 kHz, and treatment time 30 min.

[0015] It also comprises preparation of reference solution, and the reference is syringidin and bryonin;

[0016] The preparation method of the reference solution is as follows: the reference is added with methanol solution, and the reference solution is obtained.

[0017] The C18 column has a column length of 250mm, an inner diameter of 4.6mm and a particle size of 5um.

[0018] The chromatographic conditions are as follows: a flow rate of 1.0ml / min, a column temperature of 30 DEG C, a detection wavelength of 261nm, and a theoretical plate number of not less than 3000 calculated according to the peak of wallenrootine.

[0019] The characteristic spectrum contains 10 characteristic peaks, and the relative retention times are as follows:

[0020] Peak 1: 1.0, peak 2: 1.60, peak 3: 2.63, peak 4: 0.59, peak 5: 0.74, peak 6: 0.78, peak 7: 0.89, peak 8: 0.90, peak 9: 0.99, and the relative retention time is within the range of ± 10%.

[0021] The peak 1 is eugenol glucoside, and the peak 2 is wallenrootine.

[0022] The rhynchosia phaseoloides medicinal material or preparation thereof is a rhynchosia phaseoloides medicinal material, a decoction piece, a standard decoction, or a formula granule.

[0023] The application also provides a method for identifying the authenticity of the rhynchosia phaseoloides medicinal material or preparation thereof, which comprises the following steps:

[0024] a, taking a sample to be detected;

[0025] b, constructing a characteristic spectrum according to the characteristic spectrum construction method of the rhynchosia phaseoloides medicinal material or preparation thereof;

[0026] c, comparing the characteristic peaks, and if the characteristic peaks are completely consistent with those in the characteristic spectrum, the rhynchosia phaseoloides medicinal material is authentic.

[0027] The characteristic spectrum fingerprint method constructed by the application not only identifies the amide alkaloid component (wallenrootine) in the rhynchosia phaseoloides, but also newly identifies the antioxidant, anti-inflammatory and antibacterial component eugenol glucoside in the rhynchosia phaseoloides, which has potential application value and research significance in the field of pharmacy.

[0028] Compared with the literature: Cao Jie, et al., HPLC characteristic spectrum research and content measurement of amide components in rhynchosia phaseoloides, Chinese herbal medicine, vol. 45, No. 4, April 2022, the difference between the two is that the elution gradient of the chromatography is different, and the second difference is that the chromatographic peak of the method newly identifies eugenol glucoside; the newly identified antioxidant, anti-inflammatory and antibacterial component eugenol glucoside in the rhynchosia phaseoloides is a big advantage of the application, and the application establishes the characteristic spectrum of the rhynchosia phaseoloides by using the HPLC method, which aims to provide a basis for subsequent identification and quality evaluation of the rhynchosia phaseoloides.

[0029] The present application carries out single variety research on Rhynchosia minima formula granules, scientifically identifies Rhynchosia minima, has the characteristics of comprehensive detection and simple operation, can effectively identify Rhynchosia minima formula granules, and can ensure that the quality of Rhynchosia minima formula granules is uniform and stable. The method is suitable for Rhynchosia minima medicinal materials, decoction pieces, standard decoction and formula granules. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Control characteristic map (peak 1 (S1): syringic acid glucoside; peak 10 (S2): wall grass base);

[0031] Figure 2 Stone fruit granules chromatogram (from top to bottom, 320 nm, 300 nm, 261 nm, 240 nm, 230 nm);

[0032] Figure 3 Investigation of stone fruit granules at different column temperatures;

[0033] Figure 4 Flow rate investigation;

[0034] Figure 5 Investigation of stone fruit granules delay;

[0035] Figure 6 Investigation of extraction solvent;

[0036] Figure 7 Investigation of extraction method;

[0037] Figure 8 Investigation of extraction time;

[0038] Figure 9 Investigation of solvent addition amount;

[0039] Figure 10 Chromatographic peak identification;

[0040] Figure 11 Different instruments;

[0041] Figure 12 Different chromatographic columns;

[0042] Figure 13 3 batches of stone fruit granules characteristic map verification figure;

[0043] Figure 14 Stone fruit medicinal material characteristic map;

[0044] Figure 15 Stone fruit decoction piece characteristic map;

[0045] Figure 16 Stone fruit standard decoction characteristic map;

[0046] Figure 17Reference characteristic chromatogram (peak 1 (S1): syringic acid glucoside; peak 10 (S2): wallenrootine);

[0047] Figure 18 Reference characteristic chromatogram (peak 1 (S1): syringic acid glucoside; peak 10 (S2): wallenrootine);

[0048] Figure 19 Reference characteristic chromatogram (peak 1 (S1): syringic acid glucoside; peak 10 (S2): wallenrootine);

[0049] Figure 20 Reference characteristic chromatogram (peak 1 (S1): syringic acid glucoside; peak 10 (S2): wallenrootine);

[0050] Figure 21 Literature method detection result chart. DETAILED DESCRIPTION

[0051] Example 1: Method for constructing characteristic chromatogram of Piper wallichii (Miq.) Hand.-Mazz. medicinal material or its preparation

[0052] Piper wallichii (Miq.) Hand.-Mazz. is the dried whole plant of Piper wallichii (Miq.) Hand.-Mazz. in the Piperaceae family, which has the effects of expelling wind and dampness, strengthening the abdomen and knees, relieving pain, and relieving cough. The standard decoction is a lyophilized powder prepared from medicinal materials after processing according to a fixed preparation process. In order to ensure the uniformity and stability of the quality of Piper wallichii (Miq.) Hand.-Mazz. medicinal material, decoction pieces and standard decoction, a new characteristic chromatogram method is established in this paper to control the quality.

[0053] Chromatographic conditions and system suitability test: octadecylsilane-bonded silica gel as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A, 0.2% formic acid solution as mobile phase B, gradient elution according to the following table; flow rate 1.0 ml per minute; column temperature 30°C; detection wavelength 261 nm. The theoretical plate number should not be less than 3000 calculated according to the wallenrootine peak.

[0054]

[0055] Preparation of reference solution: take syringic acid glucoside and wallenrootine reference substances, accurately weigh, add methanol to prepare a solution containing 50 μg per 1 ml as the reference solution of the reference substance.

[0056] Preparation of test solution: take 0.1 g of Piper wallichii (Miq.) Hand.-Mazz. formula granules, grind finely, add 25 ml of methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake well, filter, and take the filtrate, which is obtained.

[0057] The assay method is to precisely pipette 10 μl of the reference solution and the test product solution, respectively, and inject into the liquid chromatograph for determination.

[0058] Ten characteristic peaks should appear in the test product chromatogram, of which peak 1 and peak 10 should correspond to the retention time of the corresponding reference peak of the control product. The peak corresponding to the reference peak of eugenol glucoside is S1 peak, and the relative retention time of peaks 2 and 3 to S1 peak is calculated; the peak corresponding to the reference peak of jatamansi is S2 peak, and the relative retention time of peaks 4-9 to S2 peak is calculated, which should be within ±10% of the specified value. The specified value is: 1.60 (peak 2), 2.63 (peak 3), 0.59 (peak 4), 0.74 (peak 5), 0.78 (peak 6), 0.89 (peak 7), 0.90 (peak 8), 0.99 (peak 9). (See Figure 1 )

[0059] 1. Experimental instruments and materials

[0060] High performance liquid chromatograph: Shimadzu LC-20AD, Agilent 1260 type, Waters se2695 type;

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

[0062] Ultrapure water machine: cell type 1810A (Shanghai Moore Scientific Instruments Co., Ltd.);

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

[0064] Chromatographic column: Kromasil-100-C18 5μm 250×4.6mm;

[0065] Acetonitrile, methanol, formic acid are chromatographically pure, water is ultrapure water, and the rest of the reagents are analytically pure.

[0066] Jatamansi control product (Chengdu Desite Biotechnology Co., Ltd., batch number: DSNT230721-160, purity: 98%);

[0067] Eugenol glucoside control product (CAS: 33228-65-8, OSNT, batch number: U40-7008658-02, purity: 100%);

[0068] Rhodotypos formula granules SNT-01, Rhodotypos formula granules SNT-02, SNT-03, SNT-04;

[0069] Rhizoma Rubi parvifolii: SNT-YC-01, SNT-YC-02, SNT-YC-03, SNT-YC-04, SNT-YC-05 SNT-YC-06, SNT-YC-07, SNT-YC-08, SNT-YC-09, SNT-YC-10, SNT-YC-11, SNT-YC-12, SNT-YC-13, SNT-YC-14, SNT-YC-15, SNT-YC-16;

[0070] Rhizoma Rubi parvifolii: SNT-YC-01, SNT-YC-02, SNT-YC-03, SNT-YC-04, SNT-YC-05 SNT-YC-06, SNT-YC-07, SNT-YC-08, SNT-YC-09, SNT-YC-10, SNT-YC-11, SNT-YC-12, SNT-YC-13, SNT-YC-14, SNT-YC-15, SNT-YC-16;

[0071] Rhizoma Rubi parvifolii: SNT-YC-01, SNT-YC-02, SNT-YC-03, SNT-YC-04, SNT-YC-05 SNT-YC-06, SNT-YC-07, SNT-YC-08, SNT-YC-09, SNT-YC-10, SNT-YC-11, SNT-YC-12, SNT-YC-13, SNT-YC-14, SNT-YC-15, SNT-YC-16;

[0072] 2. Detection wavelength point determination

[0073] On the basis of the above proposed experimental conditions, the diode array detector was used to scan the full waveband of the test solution, and the chromatograms of the test solution at 230 nm, 240 nm, 261 nm, 300 nm, 320 nm were extracted respectively. See Figure 2 .

[0074] The results showed that when the detection wavelength was 261 nm, the chromatographic peak information was larger, and the chromatogram baseline was more stable, so the detection wavelength was determined as 261 nm.

[0075] 3. Column temperature investigation

[0076] On the basis of the above proposed experimental conditions, the column temperature was investigated at 25℃, 30℃, 35℃ respectively. As Figure 3 shown and Table 1.

[0077] Table 1 Column temperature investigation—characteristic peak relative retention time ratio

[0078]

[0079] The results of column temperature investigation show that when the column temperature is 30°C, the peak shape of the chromatogram is relatively symmetrical, and the resolution is good, so 30°C is finally determined as the column temperature of the characteristic chromatogram method of Shishengteng Dispensing Granules.

[0080] 4. Flow rate investigation

[0081] On the basis of the above proposed experimental conditions, the flow rates of 0.8 ml / min, 1.0 ml / min and 1.2 ml / min were investigated. As shown in Table 2. Figure 4

[0082] Table 2 Flow rate-relative retention time

[0083]

[0084] The results show that when the flow rate is 1.0 ml / min, the peak shape of the chromatogram is good, and the resolution is moderate. Therefore, the flow rate is determined to be 1.0 ml / min.

[0085] 5. Delay test

[0086] On the basis of the above proposed experimental conditions, the chromatogram acquisition time was extended to 160 min. As shown in Table 3. Figure 5

[0087] The results show that the sample has basically no chromatographic peak after 80 min, so the sample detection time is determined to be 80 min.

[0088] 6. Investigation of extraction solvent

[0089] Take Shishengteng Dispensing Granules (SNT-01) in an appropriate amount, grind finely, take 0.1 g, and place it in a conical flask with a plug. Add 25 ml of methanol, 70% methanol, 50% methanol, 30% methanol, water, and ethanol, respectively. Ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool down, shake well, filter, and take the filtrate, i.e. the sample. As shown in Table 4. Figure 6

[0090] The results show that when methanol is used as the extraction solvent, the chromatographic peak information is large, so the extraction solvent of the test sample is determined to be methanol.

[0091] 7. Investigation of extraction method

[0092] Take Shishengteng Dispensing Granules (SNT-01) in an appropriate amount, grind finely, take 0.1 g, and place it in a conical flask with a plug. Add 25 ml of methanol, 70% methanol, 50% methanol, 30% methanol, water, and ethanol, respectively. Ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool down, shake well, filter, and take the filtrate, i.e. the sample. As shown in Table 4. Figure 7

[0093] ​​​​The results showed that the effect of ultrasonic extraction and reflux extraction was consistent. Since the ultrasonic extraction was more simple, the extraction method of the test sample was determined as ultrasonic extraction.

[0094] 8. Extraction time investigation

[0095] Take SNT-01 granules, grind finely, take 0.1 g, put into a conical flask with a stopper, add 50% methanol 25 ml, ultrasonic treatment (power 600 W, frequency 40 kHz) for 20 minutes, 30 minutes, 40 minutes respectively, shake well, filter, take the filtrate, as shown in Figure 8 .

[0096] The results showed that when the extraction time was 30 minutes, it was fully extracted. Therefore, the extraction time of the test sample was determined as 30 minutes.

[0097] 9. Solvent addition amount investigation

[0098] Take SNT-01 granules, grind finely, take 0.1 g, put into a conical flask with a stopper, add 50% methanol 15 ml, 25 ml, 50 ml respectively, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool down, shake well, filter, take the filtrate, as shown in Figure 9 .

[0099] The results showed that when the solvent addition amount was 25 ml, the chromatographic peak area of the characteristic spectrum was moderate. Therefore, the solvent addition amount of the test sample was determined as 25 ml.

[0100] 10. Determination of test sample preparation method

[0101] Take SNT-01 granules, grind finely, take 0.1 g, accurately weigh, put into a conical flask with a stopper, add methanol 25 ml, tightly stop, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool down, shake well, filter, take the filtrate, and get it.

[0102] 11. Methodology investigation

[0103] 11.1 Chromatographic peak identification

[0104] Preparation of test sample solution: Prepare the test sample solution of SNT-01 granules according to the above proposed experimental conditions.

[0105] Preparation of reference solution: Take appropriate amount of syringic acid glucoside and bryonoin reference substance, accurately weigh, add methanol to prepare a solution containing 50 μg per 1 ml as the reference substance solution of the control.

[0106] Preparation of negative control solution: Prepare the negative control solution of SNT-01 granules according to the above proposed experimental conditions.

[0107] The characteristic peaks of the Rhynchosia volubilis formula granules were located. As shown in Figure 10 .

[0108] 11.2 Precision test

[0109] The Rhynchosia volubilis formula granules (batch number: SNT-01) test sample solution was taken, 6 times of continuous sampling were carried out according to the proposed experimental method, 10 μL each time, and the retention time and peak area of each characteristic peak were calculated. As shown in Tables 3 and 4.

[0110] Table 3 Precision test-retention time

[0111]

[0112] Table 4 Precision test-peak area

[0113]

[0114] The results showed that the precision of each characteristic peak retention time RSD was 0.02%-0.10%, and the peak area RSD was 0.35%-12.31%. The method had good precision.

[0115] 11.3 Reproducibility test

[0116] The Rhynchosia volubilis formula granules (batch number: SNT-01) were accurately weighed 6 times, and prepared and determined according to the proposed experimental method. As shown in Tables 5 and 6.

[0117] Table 5 Reproducibility test-relative retention time

[0118]

[0119]

[0120] Table 6 Reproducibility test-relative peak area

[0121]

[0122] The results showed that the reproducibility of each characteristic peak relative retention time RSD was 0.01%-0.09%, and the relative peak area RSD was 1.25%-6.13%. The method had good reproducibility.

[0123] 11.4 Intermediate precision test

[0124] 11.4.1 Different instrument test

[0125] On the basis of the above experimental conditions, two portions of Shishan Teng Formula Granules (Batch No. SNT-01) were precisely weighed, and the test sample solution was prepared. The determination was performed on Shimadzu LC-20AD, Agilent 1260, and Waters se2695, respectively. As shown in Tables 7 and 8. Figure 11

[0126] Table 7 Instrument durability test - relative retention time ratio

[0127]

[0128] Table 8 Instrument durability test - relative peak area ratio

[0129]

[0130]

[0131] The results show that when the test sample is detected by the above three instruments, the RSD range of the relative retention time of each characteristic peak is 0.05%-3.15%, and the instrument durability of the method is good.

[0132] 11.4.2 Different personnel and time test

[0133] On the basis of the above experimental conditions, two portions of Shishan Teng Formula Granules (Batch No. SNT-01) were precisely weighed by different personnel (A, B) at different times (T1, T2), and the test sample was prepared for determination. As shown in Tables 9 and 10.

[0134] Table 9 Personnel and time test - relative retention time

[0135]

[0136] Table 10 Personnel and time test - relative peak area ratio

[0137]

[0138] The results show that the RSD range of the relative retention time of each characteristic peak is 0.01%-0.08%, and the RSD range of the relative peak area is 1.80%-4.01%, and the method has good stability of different personnel and time.

[0139] 11.5 Durability test

[0140] 11.5.1 Column durability test

[0141] On the basis of the above experimental conditions, Kromasil-100-C18 5 μm 250 x 4.6 mm of different batch numbers was tested, respectively. As shown in Tables 11 and 12. Figure 12 ​, Table 11 and Table 12.

[0142] Table 11 Column Durability Investigation-Relative Retention Time

[0143]

[0144] Table 12 Column Durability Investigation-Relative Peak Area

[0145]

[0146] From Figure 12 It can be seen that the chromatogram peak separation of each column is good and the peak is clear, and the column durability is good.

[0147] 11.5.2 Stability Investigation

[0148] On the basis of the above experimental conditions, the same test solution was taken and measured at 0 h, 3 h, 6 h, 12 h, 18 h, and 24 h. As shown in Table 13 and Table 14.

[0149] Table 13 Stability Investigation-Retention Time

[0150]

[0151] Table 14 Stability Investigation-Peak Area

[0152]

[0153]

[0154] The results show that the RSD of the corresponding characteristic peak retention time is 0.03%-0.09%, the peak area RSD is 0.50%-16.00%, and the sample solution is stable within 24 hours.

[0155] 12、Technical Effects

[0156] 21.13 batches of Rhizoma Rubi Formulation Granules Verification Results

[0157] The proposed method was used to determine the characteristic spectrum of 3 batches of Rhizoma Rubi Formulation Granules, and the relative retention time and relative peak area were calculated. As shown in Figure 13 , Table 15 and Table 16.

[0158] Table 2 Relative Retention Time of 3 Batches of Rhizoma Rubi Formulation Granules

[0159]

[0160] Table 3 Relative Peak Area of 3 Batches of Rhizoma Rubi Formulation Granules

[0161]

[0162] According to the principle of relative retention time stability and the detection of each batch sample with relatively high peaks, a total of 10 peaks with good reproducibility were selected as characteristic peaks. The results showed that when peak 1 was S1 peak and peak 10 was S2 peak, the relative retention time RSD of the characteristic peaks of the three batches of rhizoma smilacis glabrae granules was less than 2%.

[0163] 12.2 Establishment of the limit of the relative retention time

[0164] The results of each investigation item and validation of the methodology are shown in Table 17:

[0165] Table 4 Results of each item of the methodology RSD% summary standard - relative retention time

[0166]

[0167] The relative retention time of each characteristic peak was stable and within the average value ± 10%, so the relative retention time of each peak was temporarily set to ± 10%.

[0168] Final specification: 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 product characteristic spectrum should present 10 characteristic peaks, of which the peak corresponding to the eugenol glucoside reference is S1 peak and the peak corresponding to the bryonoin reference is S2 peak.

[0169] 12.3 Results of 16 batches of rhizoma smilacis glabrae medicinal materials

[0170] The proposed method was used to determine the characteristic spectrum of 16 batches of rhizoma smilacis glabrae medicinal materials, and the relative retention time and relative peak area were calculated. As shown in Table 5, Table 6, Table 18 and Table 19. Figure 14

[0171]

[0172]

[0173]

[0174] Table 6 Relative peak area of 16 batches of rhizoma smilacis glabrae medicinal materials

[0175]

[0176] 12.4 Results of 16 batches of rhizoma smilacis glabrae decoction pieces

[0177] The proposed method was used to determine the characteristic spectrum of 16 batches of rhizoma smilacis glabrae decoction pieces, and the relative retention time and relative peak area were calculated. As shown in Table 20 and Table 21. Figure 15

[0178] ​​​Table 20 Relative retention time of 16 batches of Rhizoma Rubi parvifolii

[0179]

[0180]

[0181] Table 21 Relative peak area of 16 batches of Rhizoma Rubi parvifolii

[0182]

[0183]

[0184] 12.5 Results of 16 batches of Rhizoma Rubi parvifolii standard decoction

[0185] The characteristic chromatogram of 16 batches of Rhizoma Rubi parvifolii standard decoction was determined by the developed method, and the relative retention time and relative peak area were calculated. As shown in Table 22 and Table 23. Figure 16

[0186] Table 22 Relative retention time of 16 batches of Rhizoma Rubi parvifolii standard decoction

[0187]

[0188] Table 23 Relative peak area of 16 batches of Rhizoma Rubi parvifolii standard decoction

[0189]

[0190]

[0191] Example 2 Characteristic chromatogram of Rhizoma Rubi parvifolii medicinal material or its preparation

[0192] 1. Formula granules:

[0193] The chromatographic conditions and system suitability test were as follows: octadecylsilane-bonded silica gel as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as the mobile phase A, 0.2% formic acid solution as the mobile phase B, gradient elution according to the following table; flow rate 1.0 ml per minute; column temperature 30°C; detection wavelength 261 nm. The theoretical plate number calculated according to the wall grass alkali peak should not be less than 3000.

[0194]

[0195] The reference solution was prepared by accurately weighing eugenol glucoside and wall grass alkali control substances, and adding methanol to prepare a solution containing 50 μg per 1 ml as the control reference solution.

[0196] ​Preparation of Test Solution: 0.1 g of the sample was finely ground and added to 25 ml of methanol. The mixture was ultrasonically treated (power 600 W, frequency 40 kHz) for 30 min, allowed to cool, shaken well, and filtered. The filtrate was collected to give the test solution.

[0197] Determination: 10 μl of the reference solution and 10 μl of the test solution were precisely taken and injected into the liquid chromatograph for determination.

[0198] In the chromatogram of the test solution, 10 characteristic peaks should be present, of which peak 1 and peak 10 should correspond to the retention time of the reference peaks of the corresponding control substances. The peak corresponding to the reference peak of syringic acid glucoside is S1 peak, and the relative retention times of peak 2 and peak 3 to S1 peak were calculated. The peak corresponding to the reference peak of wallenrootine is S2 peak, and the relative retention times of peak 4 to peak 9 to S2 peak were calculated, which should be within ±10% of the specified values. The specified values are: 1.60 (peak 2), 2.63 (peak 3), 0.59 (peak 4), 0.74 (peak 5), 0.78 (peak 6), 0.89 (peak 7), 0.90 (peak 8), and 0.99 (peak 9).

[0199] The three batches of the Formula Granules of Ledum were synthesized by using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version), and the control chromatogram of the characteristic chromatogram of the Formula Granules of Ledum was established, as shown in Figure 1. Figure 17

[0200] 2. Medicinal Material:

[0201] 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 mobile phase A, and 0.2% formic acid solution was used as 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 30 °C; and the detection wavelength was 261 nm. The theoretical plate number calculated according to the wallenrootine peak should not be less than 3000.

[0202]

[0203] Preparation of Reference Solution: appropriate amounts of syringic acid glucoside and wallenrootine control substances were precisely weighed and dissolved in methanol to prepare a solution containing 50 μg per 1 ml, which was used as the reference solution of the control substances.

[0204] Preparation of Test Solution: about 1.5 g of the Ledum palustre L. medicinal material powder (passed through a No. 2 sieve) was added to 25 ml of water, and boiled for 30 min. After centrifugation, the supernatant was collected, evaporated to dryness, and the residue was added to 25 ml of methanol. The mixture was ultrasonically treated (power 600 W, frequency 40 kHz) for 30 min, allowed to cool, shaken well, and filtered. The filtrate was collected to give the test solution.

[0205] ​Determination method, respectively, 10 μl of reference solution and test solution, injection liquid chromatograph, determination, namely.

[0206] The test sample characteristic spectrum should present 10 characteristic peaks, of which peak 1 and peak 10 correspond to the retention time of the corresponding reference peaks. The peak corresponding to the eugenol glucoside reference is S1 peak, and the relative retention time of peaks 2, 3 characteristic peaks and S1 peak is calculated. The peak corresponding to the wall grass alkali reference is S2 peak, and the relative retention time of peaks 4, 5, 6, 7, 8, 9 characteristic peaks and S2 peak is calculated, which should be within ±10% of the specified value. The specified value is: 1.60 (peak 2), 2.63 (peak 3), 0.59 (peak 4), 0.74 (peak 5), 0.78 (peak 6), 0.89 (peak 7), 0.90 (peak 8), 0.99 (peak 9).

[0207] The similarity evaluation system for traditional Chinese medicine chromatographic fingerprint (2012 version) was used to synthesize 16 batches of stone vine medicine, and the control spectrum of stone vine medicine characteristic spectrum was established. As shown in Figure 18 .

[0208] 3, decoction pieces:

[0209] Chromatographic conditions and system suitability test with octadecylsilane bonded silica gel as filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); with acetonitrile as mobile phase A, and 0.2% formic acid solution as mobile phase B, gradient elution according to the following table; flow rate is 1.0 ml per minute; column temperature is 30℃; detection wavelength is 261 nm. The theoretical plate number should not be less than 3000 according to wall grass alkali peak.

[0210]

[0211]

[0212] Preparation of reference solution: take eugenol glucoside and wall grass alkali reference substance, accurately weigh and determine, add methanol to prepare 50 μg per 1 ml solution as reference solution.

[0213] Preparation of test sample solution: take stone vine decoction piece powder (pass through No. 2 sieve) about 1.5 g, add water 25 ml, decoct for 30 minutes, centrifuge, take supernatant, evaporate to dryness, add methanol 25 ml to the residue, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cool, shake evenly, filter, take the filtrate, namely.

[0214] Determination method, respectively, 10 μl of reference solution and test solution, injection liquid chromatograph, determination, namely.

[0215] The test sample characteristic spectrum should present 10 characteristic peaks, wherein peak 1 and peak 10 correspond to the retention time of the corresponding reference peaks, respectively. The peak corresponding to the syringic acid glucoside reference is S1 peak, and the relative retention time of peaks 2 and 3 is calculated with respect to the S1 peak. The peak corresponding to the jateorrhizine reference is S2 peak, and the relative retention time of peaks 4-9 is calculated with respect to the S2 peak, which should be within ±10% of the specified value. The specified value is: 1.60 (peak 2), 2.63 (peak 3), 0.59 (peak 4), 0.74 (peak 5), 0.78 (peak 6), 0.89 (peak 7), 0.90 (peak 8), 0.99 (peak 9).

[0216] The 16 batches of Radix Rubi parvifolii samples were synthesized by using the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version), and the control spectrum of the Radix Rubi parvifolii sample characteristic spectrum was established, as shown in Figure 19

[0217] 4. Standard decoction:

[0218] The chromatographic conditions and system suitability test used octadecylsilane-bonded silica gel as the filler (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A, and 0.2% formic acid solution as mobile phase B, gradient elution according to the following table; flow rate was 1.0 ml per minute; column temperature was 30°C; detection wavelength was 261 nm. The theoretical plate number calculated according to the jateorrhizine peak should not be less than 3000.

[0219]

[0220] The reference solution was prepared by accurately weighing appropriate amounts of syringic acid glucoside and jateorrhizine reference substances, and adding methanol to prepare a solution containing 50 μg per 1 ml as the reference solution of the reference substance.

[0221] The test sample solution was prepared by taking 0.1 g of Radix Rubi parvifolii standard decoction, placing it in a conical flask with a plug, adding 25 ml of methanol, tightly sealing, ultrasonic treatment (power 600 W, frequency 40 kHz) for 30 minutes, cooling, shaking, filtering, and taking the filtrate as the test sample solution.

[0222] The determination method was to precisely take 10 μl of the reference solution and the test sample solution, respectively, and inject them into the liquid chromatograph for determination.

[0223] ​Ten characteristic peaks should be present in the chromatogram of the test sample, peak 1 and peak 10 should correspond to the retention time of the corresponding reference substance, respectively; the peak corresponding to the reference substance of eugenol glucoside is S1 peak, the relative retention time of peak 2 and peak 3 characteristic peaks to S1 peak is calculated, the peak corresponding to the retention time of the reference substance of wall grass alkali is S2 peak, the relative retention time of peaks 4-9 characteristic peaks to S2 peak is calculated, and the relative retention time should be within ±10% of the specified value, the specified value is: 1.60 (peak 2), 2.62 (peak 3), 0.59 (peak 4), 0.74 (peak 5), 0.78 (peak 6), 0.89 (peak 7), 0.90 (peak 8), 0.99 (peak 9).

[0224] The standard decoction of 16 batches of Shishan Teng was synthesized by using traditional Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version), and the control chromatogram of Shishan Teng standard decoction characteristic chromatogram was established. As shown in Figure 20

[0225] Comparative example 1

[0226] According to the literature (Cao Jie, et al., HPLC characteristic chromatogram of Shishan Teng and content measurement of amide components, Chinese herbal medicine, vol. 45, No. 4, April 2022) method, Shishan Teng medicinal materials, standard decoction, formula granules were detected, the results are shown in Figure 21 According to the experimental results, the literature method cannot identify eugenol glucoside.​

Claims

1. A method for constructing a characteristic chromatogram of a Rubus pungens Buch.-Ham or a preparation thereof, characterized by comprising the following steps: It comprises the following steps: ​ a. Preparation of test sample solution: Take the herb or preparation of Rhynchosia volubilis, dissolve it with solvent, extract it, and obtain the test solution; b. Determine the test solution by high performance liquid chromatography to obtain the characteristic chromatogram of the herb or preparation of Rhynchosia volubilis; The high performance liquid chromatography conditions are as follows: the chromatographic column is C18 column; acetonitrile is used as mobile phase A, and 0.2% formic acid solution is used as mobile phase B for gradient elution, and the gradient elution conditions are as follows:

2. The method for constructing the characteristic chromatogram of the herb of Asparagus cochinchinensis or its preparation according to claim 1, characterized in that: The preparation method of the test sample solution is as follows: Take the herb or preparation of Rhynchosia volubilis, grind it, add methanol, ultrasonically treat it, cool it, shake it, filter it, and take the filtrate to obtain it.

3. The method according to claim 2, wherein the method is characterized by: The ultrasonic treatment conditions are as follows: power 600W, frequency 40kHz, and treatment time 30min.

4. The method for establishing characteristic chromatogram of Shenshenteng medicinal material or its preparation according to claim 1, characterized in that: It also comprises preparation of reference solution, and the reference is syringidin and wallenrootine; The preparation method of the reference solution is as follows: take the reference, add methanol solution, and obtain it.

5. The method for establishing characteristic chromatogram of Shenshenteng medicinal material or its preparation according to claim 1, characterized in that: The chromatographic column C18 column has a column length of 250mm, an inner diameter of 4.6mm, and a particle size of 5μm.

6. The method for establishing characteristic chromatogram of Shenshenteng medicinal material or its preparation according to claim 1, characterized in that: The chromatographic conditions are as follows: flow rate 1.0ml / min, column temperature 30℃, detection wavelength 261nm, and theoretical plate number not less than 3000 calculated according to wallenrootine peak.

7. The method according to any one of claims 1-6, wherein the method is characterized by: The characteristic chromatogram comprises 10 characteristic peaks, and the relative retention times are as follows: Peak 1: 1.0, peak 2: 1.60, peak 3: 2.63, peak 4: 0.59, peak 5: 0.74, peak 6: 0.78, peak 7: 0.89, peak 8: 0.90, and peak 9: 0.99, and the relative retention times are within ±10% range. Peak 1 is syringidin, and peak 2 is wallenrootine.

8. The method according to claim 7, wherein the method is characterized by the following steps: 1) extracting the sample with 70% ethanol; 2) separating the extract by HPLC; 3) detecting the separated components by HPLC-ELSD; 4) comparing the obtained chromatogram with the reference chromatogram. The herb or preparation of Rhynchosia volubilis is the herb, decoction piece, standard decoction, and formula granule of Rhynchosia volubilis.

9. The method for establishing the characteristic chromatogram of the herb of Asparagus cochinchinensis or its preparation according to any one of claims 1-6, characterized in that: It comprises the following steps:

10. A method for identifying the authenticity of a Rubus suavissimus S. Lee medicinal material or preparation thereof, characterized in that: a. Take the sample to be tested; b. Construct the characteristic chromatogram according to the construction method of the characteristic chromatogram of the herb or preparation of Rhynchosia volubilis according to any one of claims 1-9; c. Compare the characteristic peaks, and if they are completely consistent with the characteristic peaks in the characteristic chromatogram, it is the genuine herb of Rhynchosia volubilis. ​

Images