Characteristic chromatogram construction method of kadsura pepper stem medicinal material or preparation thereof and content determination method of kadsura pepper stem formula granules

Abstract

The invention provides a method for constructing a characteristic chromatogram of a kadsura pepper stem medicinal material or a preparation of the kadsura pepper stem medicinal material. The method is suitable for detecting the high performance liquid characteristic chromatogram of kadsura pepper stem medicinal materials, decoction pieces, standard decoction and formula granules, can integrally control the characteristic components in the kadsura pepper stem medicinal materials, decoction pieces, standard decoction and formula granules, ensures the overall stability of the quality of the kadsura pepper stem medicinal materials, decoction pieces, standard decoction and formula granules, and is simple to operate, high in precision, good in stability and high in accuracy. The repeatability is good and the accuracy is high.

Description

Technical Field

[0001] This invention relates to a method for constructing a characteristic spectrum of Piper kadsura (sea kadsura) medicinal material or its preparations, and a method for determining the content of Piper kadsura formula granules, belonging to the field of drug detection. Background Technology

[0002] Piper kadsura (Choisy) Ohwi, a plant in the Piperaceae family, is a dried vine with properties that dispel wind and dampness, unblock meridians, and relieve pain. Clinically, it is mainly used for wind-cold-dampness arthralgia, joint pain, muscle spasms, and difficulty in flexion and extension. Modern research shows that Piper kadsura contains lignans, alkaloids, volatile oils, flavonoids, epoxides, and steroids. Among these, neolignans and amide alkaloids are its main chemical components. Literature review indicates that the amide alkaloid component, kadsuraine, has anti-inflammatory, antipyretic, antibacterial, and vascular protective effects. Simultaneously, as a TRPV1 receptor antagonist, it has good analgesic effects, corresponding to the pain-relieving properties of Piper kadsura.

[0003] Currently, there are numerous publications on the quality testing of Piper kadsura, such as CN202210400951.2, entitled "Detection Method and Quality Control Method for Piper kadsura and its Preparations," which relates to detection methods and quality control methods for Piper kadsura and its preparations. The detection method uses ultra-high performance liquid chromatography (UHPLC), and the test sample solution used in the detection process contains notopterygium alcohol. The detection method for Piper kadsura and its preparations provided by this invention uses notopterygium alcohol as an internal standard, and the obtained fingerprint spectrum shows a notopterygium alcohol peak. However, this detection method is an internal standard method, and its characteristic spectrum does not identify the characteristic peaks in the Piper kadsura sample; furthermore, the preparation of the test sample for this detection method is complex.

[0004] CN202210105802.3, Invention Title: Application of Herba davidii alkaloids in the Quality Detection of Piper kadsura samples and corresponding quality detection methods, relates to the application of herba davidii alkaloids in the quality detection of Piper kadsura samples and corresponding quality detection methods. This invention provides a quality detection method for Piper kadsura samples, comprising the following steps: preparation of a reference solution, preparation of a test solution, liquid chromatography detection, and data analysis, wherein the data analysis includes calculating the content of herba davidii alkaloids in the Piper kadsura sample. This detection method is only a content detection method. Due to the complex composition of Piper kadsura, this method cannot achieve quality control of Piper kadsura medicinal materials.

[0005] There are many studies on the fingerprint chromatograms of Piper kadsura, but no chromatographic peak identification has been performed, and the similarity of chromatographic peaks has been used as the quality evaluation standard for Piper kadsura. For example, the literature includes: Fan Weifeng, et al., Study on HPLC fingerprint chromatogram of Piper kadsura from Guangxi, Journal of Hubei University of Traditional Chinese Medicine, August 2019, Vol. 21, No. 4; Liu Yanju, et al., Study on HPLC fingerprint chromatogram of Piper kadsura, Chinese Pharmacist, Vol. 12, No. 11, 2009; Li Bin, et al., Source and quality analysis of Piper kadsura and Piper zedoaria based on UHPLC fingerprint chromatogram and multi-component quantification, Chinese Journal of Modern Applied Pharmacy, October 2024, Vol. 41, No. 19 (using Piper kadsura ketone, white magnolia tin B, and kadsura quinol as marker components). Summary of the Invention

[0006] This invention provides a method for constructing a characteristic spectrum of Piper kadsura (sea kadsura) medicinal material or its preparations, and a method for determining the content of Piper kadsura formula granules.

[0007] This invention discloses a method for constructing a characteristic spectrum of Piper kadsura (a type of medicinal herb) or its preparations, comprising the following steps:

[0008] a. Preparation of the test solution:

[0009] Take the *Piper kadsura* sample, dissolve and extract it using a solvent to obtain the test solution;

[0010] b. The test solution was determined by high performance liquid chromatography to obtain the characteristic chromatogram of Piper kadsura or its preparations;

[0011] The characteristic chromatographic conditions for the *Hypericum perforatum* medicinal material or its preparations are as follows: a C18 column; acetonitrile as mobile phase A, water as mobile phase B, gradient elution, and the elution conditions are as follows:

[0012]

[0013] The method for preparing the test solution in step a is as follows:

[0014] Take the herb *Piper kadsura* or its preparation, add methanol, sonicate, filter, and take the filtrate as the test solution.

[0015] The ultrasonic treatment conditions are: power 600W, frequency 40kHz, and treatment time 20 minutes.

[0016] The C18 column has a length of 150 mm, an inner diameter of 2.1 mm, and a particle size of 1.6 μm; the flow rate is 0.3 mL per minute; the column temperature is 30 °C; the detection wavelength is 260 nm; and the theoretical plate number, calculated based on the alkaloid peak, should be no less than 10,000.

[0017] This also includes the preparation of reference solutions, with reference materials including *Piper kadsura* reference medicinal material and wallwort alkaloids;

[0018] Among them, the preparation method of the reference medicinal material solution of Piper kadsura is as follows:

[0019] Take the reference medicinal material of Piper kadsura, add methanol and perform ultrasonic treatment, let it cool, shake well, filter, and take the continuous filtrate as the reference medicinal material reference solution;

[0020] The preparation method of the pellitorine reference solution is: Take the reference substance of pellitorine and prepare it into a reference solution with methanol aqueous solution.

[0021] Among them, the ultrasonic treatment conditions are: power 600W, frequency 40kHz.

[0022] Among them, the characteristic chromatogram contains 6 characteristic peaks, and the relative retention times are respectively: Peak 1: 0.52, Peak 2: 0.70, Peak 3: 0.73, Peak 4: 0.87, Peak 5: 0.98, and the relative retention time fluctuates within the range of ±10%; Peak 6 is pellitorine.

[0023] Among them, the Piper kadsura medicinal material or its preparation is Piper kadsura medicinal material, cut crude drug, standard decoction or formula granule.

[0024] The present invention provides a method for determining the content of Piper kadsura formula granule, which detects the content of pellitorine by using the characteristic chromatogram construction method of the Piper kadsura medicinal material or its preparation.

[0025] Among them, the Piper kadsura formula granule contains 0.13 - 0.36 mg / g of pellitorine.

[0026] The present invention provides a method for constructing the characteristic chromatogram of Piper kadsura medicinal material, its cut crude drug, standard decoction and formula granule, which can solve the problem that it is difficult to comprehensively evaluate and control the technological process from Piper kadsura medicinal material to the finished product of Piper kadsura formula granule in the prior art, so as to fill the blank in the prior art.

[0027] Compared with the literature of the detection methods reported currently, the present invention identifies a new index component (pellitorine), and can qualitatively and quantitatively detect this component, and the chromatographic conditions are simpler, the preparation operation of the test sample is more convenient, it can better save the experimental cost, and is convenient for rapid experiment and analysis of experimental results. Description of the Drawings

[0028] Figure 1 Reference characteristic chromatogram of formula granule (Peak 6(S): Pellitorine; Chromatographic column: Omega PS C18 150mm×2.1mm, 1.6μm);

[0029] Figure 2 Chromatograms of Piper kadsura formula granule at different wavelengths;

[0030] Figure 33D graph of kadsurae caulis formula granules;

[0031] Figure 4 Column temperature investigation;

[0032] Figure 5 Flow rate investigation;

[0033] Figure 6 Delayed chromatogram;

[0034] Figure 7 Extraction solvent investigation;

[0035] Figure 8 Extraction method investigation;

[0036] Figure 9 Extraction time investigation;

[0037] Figure 10 Solvent amount investigation;

[0038] Figure 11 Kadsurae caulis formula granules characteristic chromatogram peak identification;

[0039] Figure 12 Wall grass alkali spectrum- reference substance;

[0040] Figure 13 Wall grass alkali spectrum- formula granules;

[0041] Figure 14 Different instrument investigation;

[0042] Figure 15 Different chromatographic column;

[0043] Figure 16 Kadsurae caulis formula granules characteristic chromatogram- KL-HFT-01;

[0044] Figure 17 Kadsurae caulis formula granules characteristic chromatogram- KL-HFT-02;

[0045] Figure 18 Kadsurae caulis formula granules characteristic chromatogram- KL-HFT-03;

[0046] Figure 19 Kadsurae caulis formula granules characteristic chromatogram (Note: Each chromatogram from bottom to top represents batch Kadsurae caulis formula granules KL-HFT-01, KL-HFT-02, KL-HFT-03);

[0047] Figure 20 Kadsurae caulis formula granules control chromatogram (chromatographic column: Omega PS C18 150mmx2.1mm, 1.6μm);

[0048] Figure 21Reference characteristic chromatogram (Peak 6(S): pellitorine; chromatographic column: Omega PS C18 150 mm × 2.1 mm, 1.6 μm);

[0049] Figure 22 Reference characteristic chromatogram (Peak 6(S): pellitorine; chromatographic column: Omega PS C18 150 mm × 2.1 mm, 1.6 μm);

[0050] Figure 23 Reference characteristic chromatogram of Piper kadsura cut pieces (Peak 6(S): pellitorine; chromatographic column: Omega PSC18 150 mm × 2.1 mm, 1.6 μm);

[0051] Figure 24 Comparison chart of reference characteristic chromatograms of Piper kadsura herbs, Piper kadsura cut pieces, standard decoction, and finished products (Peak 6(S): pellitorine);

[0052] Figure 25 UPLC chromatogram of Piper kadsura formula granules. Specific implementation manners

[0053] Example 1 Method for constructing characteristic chromatogram of Piper kadsura herbs or its preparations

[0054]

Characteristic chromatogram

[0055] Chromatographic conditions and system suitability test Use octadecylsilyl silica gel as the filler (column length is 150 mm, inner diameter is 2.1 mm, particle size is 1.6 μm); use acetonitrile as mobile phase A and water as mobile phase B, and perform gradient elution according to the regulations in the following table; the flow rate is 0.3 mL per minute; the column temperature is 30 °C; the detection wavelength is 260 nm. The number of theoretical plates calculated based on the pellitorine peak should be not less than 10000.

[0056]

[0057]

[0058] Preparation of reference substance solution Take about 1 g of Piper kadsura reference herb, place it in a stoppered conical flask, add 20 ml of methanol, ultrasonically treat (power 600 W, frequency 40 kHz) for 20 minutes, cool, shake well, filter, and take the continuous filtrate as the reference herb reference substance solution; take an appropriate amount of pellitorine reference substance, accurately weigh it, and make a solution containing 10 μg of pellitorine per 1 ml with methanol as the reference substance solution.

[0059] Preparation of test solution: about 1 g of powder (pass through No. 3 sieve) of crude drug (cut drug) was taken into 20 mL of methanol, and ultrasonic treatment (power 600 W, frequency 40 kHz) was conducted for 20 minutes. After cooling, shaking and filtration, the filtrate was obtained.

[0060] Preparation of test solution: about 1 g of powder (pass through No. 3 sieve) of crude drug (cut drug) was taken into 20 mL of methanol, and ultrasonic treatment (power 600 W, frequency 40 kHz) was conducted for 20 minutes. After cooling, shaking and filtration, the filtrate was obtained.

[0061] Preparation of test solution: about 1 g of powder (pass through No. 3 sieve) of crude drug (cut drug) was taken into 20 mL of methanol, and ultrasonic treatment (power 600 W, frequency 40 kHz) was conducted for 20 minutes. After cooling, shaking and filtration, the filtrate was obtained.

[0062] Preparation of test solution: about 1 g of powder (pass through No. 3 sieve) of crude drug (cut drug) was taken into 20 mL of methanol, and ultrasonic treatment (power 600 W, frequency 40 kHz) was conducted for 20 minutes. After cooling, shaking and filtration, the filtrate was obtained.

[0063] Preparation of test solution: about 1 g of powder (pass through No. 3 sieve) of crude drug (cut drug) was taken into 20 mL of methanol, and ultrasonic treatment (power 600 W, frequency 40 kHz) was conducted for 20 minutes. After cooling, shaking and filtration, the filtrate was obtained. Figure 1 )

[0064] Example 2 Method for constructing characteristic chromatogram of Litseae Folium or its preparation

[0065] 1. Establishment of characteristic chromatogram of Litseae Folium preparation granules

[0066] 1.1 Experimental instruments and materials

[0067] High performance liquid chromatograph: Agilent 1290 type high performance liquid chromatograph, Waters H-class type high performance liquid chromatograph;

[0068] Electronic balance: ME204E / 02, MS205DM, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0069] Ultrapure water machine: cell type 1810A (Shanghai Moore Scientific Instruments Co., Ltd.);

[0070] Ultrasonic cleaner: KQ5200DB type (600 W, 40 kHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0071] Chromatographic column: Omega PSC18 150×2.1mm 1.6μm.

[0072] Acetonitrile was of chromatographic grade, water was ultrapure water, and all other reagents were of analytical grade.

[0073] Wallwort alkaloid (Chengdu Desite Biotechnology Co., Ltd., batch number: DST230721-160, purity: 98%)

[0074] Reference material of Piper kadsura (China National Institutes for Food and Drug Control, batch number: 121635-202104);

[0075] Piper kaempferi medicinal materials (batch numbers: YC-HFT-01, YC-HFT-02, YC-HFT-03, YC-HFT-04, YC-HFT-05, YC-HFT-06, YC-HFT-07, YC-HFT-08, YC-HFT-09, YC-HFT-10, YC-HFT-11, YC-HFT-12, YC-HFT-13, YC-HFT-14, YC-HFT-15, YC-HFT-16)

[0076] Haifengteng pieces (batch number: YP-HFT-01, YP-HFT-02, YP-HFT-03, YP-HFT-04, YP-HFT-05, YP-HFT-06, YP-HFT-07, YP-HF T-08, YP-HFT-09, YP-HFT-10, YP-HFT-11, YP-HFT-12, YP-HFT-13, YP-HFT-14, YP-HFT-15, YP-HFT-16)

[0077] Piper kadsura standard decoction (batch numbers: BT-HFT-01, BT-HFT-02, BT-HFT-03, BT-HFT-04, BT-HFT-05, BT-HFT-06, BT-HFT-07, BT-HFT-08, BT-HFT-09, BT-HFT-10, BT-HFT-11, BT-HFT-12, BT-HFT-13, BT-HFT-14, BT-HFT-15, BT-HFT-16)

[0078] Sea Breeze Vine Formula Granules (Batch No.: KL-HFT-01, KL-HFT-02, KL-HFT-03).

[0079] 1.2 Chromatographic conditions

[0080] The octadecylsilane-bonded silica gel was used as the filler (column length 150 mm, inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile was used as the mobile phase A, water was used as the mobile phase B, gradient elution was carried out according to the table below; the flow rate was 0.3 mL per minute; the column temperature was 30 ℃; the detection wavelength was 260 nm.

[0081]

[0082] 1.3 Preparation of reference solution

[0083] Take about 1 g of Litsea rotundifolia reference material, put it in a conical flask with a plug, add 20 ml of methanol, ultrasonic treat (power 600 W, frequency 40 kHz) for 20 minutes, cool, shake well, filter, take the filtrate, as the reference solution of the reference material; take the appropriate amount of wall grass alkaloid control, accurately weigh, add methanol to prepare a solution containing 10 μg of wall grass alkaloid per 1 ml, as the reference solution of the control.

[0084] 1.4 Preparation of test solution

[0085] Take the formula granules, grind finely, about 0.5 g, add 20 mL of methanol, ultrasonic treat (power 600 W, frequency 40 kHz) for 20 minutes, cool, shake well, filter, take the filtrate, and get it.

[0086] 1.5 Determination method

[0087] Accurately take 2 μl of the reference solution and the test solution, inject into the liquid chromatograph, determine, and get it.

[0088] 1.6 Chromatographic conditions and system suitability test

[0089] 1.6.1 Wavelength selection

[0090] On the basis of the above proposed experimental conditions, the test solution was scanned by diode array detector, and the chromatograms of the test solution at 220 nm, 260 nm, 300 nm, and 330 nm were extracted, respectively. See Figure 2 、 3 .

[0091] The results show that when the detection wavelength is 260 nm, the amount of chromatographic peak information is larger, and the baseline of the chromatogram is more stable, so the detection wavelength is determined as 260 nm.

[0092] 1.6.2 Column temperature investigation

[0093] On the basis of the above proposed experimental conditions, the column temperature was investigated at 25 ℃, 30 ℃, and 35 ℃, respectively. See Figure 4 .

[0094] The results of column temperature investigation showed that when the column temperature was 25-30°C, the peak shape of the chromatogram was relatively symmetrical and the resolution was good, so 30°C was temporarily determined as the column temperature of the characteristic chromatogram method of the Formulation Granules of Seaflame.

[0095] 1.6.3 Flow rate investigation

[0096] On the basis of the above proposed experimental conditions, the flow rates of 0.25 mL / min, 0.3 mL / min and 0.35 mL / min were investigated, respectively. See Figure 5 .

[0097] The results showed that when the flow rate was 0.3 mL / min, the peak shape of the chromatogram was good and the resolution was moderate. Therefore, the flow rate was determined to be 0.3 mL / min.

[0098] 7.1.2.12.3.4 Delayed experiment

[0099] On the basis of the above proposed experimental conditions, the chromatogram acquisition time was extended to 70 min. The results are as follows Figure 6 .

[0100] The results showed that after 38 minutes of chromatogram acquisition, the chromatographic peaks were completely acquired, so the chromatogram acquisition time was determined to be 38 minutes.

[0101] In summary, the chromatographic conditions of the characteristic chromatogram of the Formulation Granules of Seaflame were determined as follows: octadecylsilane-bonded silica gel as the filler (column length 150 mm, inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile as the mobile phase A, water as the mobile phase B, gradient elution according to the following table; flow rate 0.3 mL / min; column temperature 30°C; detection wavelength 260 nm.

[0102]

[0103] 1.7 Investigation of the preparation of the test solution

[0104] 1.7.1 Investigation of the extraction solvent

[0105] Take about 0.5 g of the Formulation Granules (batch number: KL-HFT-01) powder, a total of four, and place them in conical bottles with stoppers, respectively add 20 mL of methanol, water, 30% methanol and 70% methanol solution, ultrasonic treatment (power 600 W, frequency 40 kHz) for 20 minutes, cool down, shake well, filter, and take the filtrate, i.e. the result is obtained. See Figure 7 .

[0106] The results showed that when the extraction solvent was methanol and 70% methanol, the chromatographic peaks of the chromatogram had little difference, and it was easy to operate, so methanol was selected as the extraction solvent for the characteristic chromatogram of the Formulation Granules of Seaflame.

[0107] 1.7.2 Examination of Extraction Methods

[0108] Take approximately 0.5g of the formula granules (batch number: KL-HFT-01) powder, in two portions, and place them in stoppered conical flasks. Add 20mL of methanol. Examine the extraction methods for the sample using reflux extraction and ultrasonic extraction, respectively, for 20 minutes. After cooling, shake well, filter, and collect the filtrate. See [link to sample preparation]. Figure 8 .

[0109] The results showed that there was no significant difference in peak shape and peak area between ultrasonic extraction and reflux extraction of the test sample. Therefore, the simple ultrasonic method was selected as the extraction method for preparing the test sample solution of Piper kadsura formula granules.

[0110] 7.1.2.12.4.3 Examination of Extraction Time

[0111] Take approximately 0.5g of the formula granules (batch number: KL-HFT-01) powder, divide into three portions, and place each portion in a stoppered conical flask. Add 20mL of methanol to each flask, and sonicate (power 600W, frequency 40kHz). Examine the extraction time of the sample at 20 minutes, 30 minutes, and 50 minutes, respectively. Cool, shake well, filter, and collect the filtrate as the final product. See [link to product details]. Figure 9 .

[0112] The results showed that a extraction time of 20 minutes was sufficient for complete extraction, resulting in a chromatogram with good peak shape. Therefore, the extraction time for the test sample was determined to be 20 minutes.

[0113] 7.1.2.12.4.4 Investigation of solvent dosage

[0114] Take approximately 0.5g of the formula granules (batch number: KL-HFT-01) powder, in three portions, and place them in stoppered conical flasks. Add methanol, seal tightly, and sonicate (600W power, 40kHz frequency) for 20 minutes. Test the solvent volume of the sample at 10mL, 20mL, and 50mL respectively. Cool, shake well, filter, and collect the filtrate as the final product. See [link to product details]. Figure 10 .

[0115] The results showed that when the solvent volume was 20 mL, the chromatographic peak area was moderate and the peak shape was good. Therefore, the solvent volume for the test sample was determined to be 20 mL.

[0116] In summary, the preparation method of the test solution of the granule characteristic spectrum of Piper kadsura is determined as follows: Take an appropriate amount of granule, grind it finely, take about 0.5g, place it in a stoppered conical flask, add 20mL of methanol, sonicate (power 600W, frequency 40kHz) for 20 minutes, cool, shake well, filter, and take the filtrate to obtain the product.

[0117] 1.8 Methodological Examination

[0118] 1.8.1 Chromatographic peak assignment

[0119] Preparation of test solution: The test solution of the sample was prepared according to the experimental conditions as above.

[0120] Preparation of reference solution: About 1 g of the reference material of the sample was taken in a conical flask with a stopper, 20 ml of methanol was added, and ultrasonic treatment (power 600 W, frequency 40 kHz) was carried out for 20 minutes. After cooling, it was shaken well and filtered. The filtrate was taken as the reference solution of the reference material.

[0121] Preparation of reference solution: About 1 g of the reference material of the sample was taken in a conical flask with a stopper, 20 ml of methanol was added, and ultrasonic treatment (power 600 W, frequency 40 kHz) was carried out for 20 minutes. After cooling, it was shaken well and filtered. The filtrate was taken as the reference solution of the reference material.

[0122] Preparation of test solution: The test solution of the sample was prepared according to the experimental conditions as above.

[0123] The characteristic peaks of the sample were located. The results are shown in Table 1-2. Figure 11-13 .

[0124] The results showed that the chromatographic peaks of the reference material and the sample were consistent. Figure 1 In the following methodological investigation, 6 peaks in the sample were investigated

[0125] 1.8.2 Precision test

[0126] The test solution of the sample was taken, 6 times of 2 μl were continuously injected according to the experimental method, and the relative retention time and relative peak area of each characteristic peak were calculated. See Table 1-2.

[0127] Table 1 Precision investigation- relative retention time

[0128]

[0129] Table 2 Precision investigation- relative peak area

[0130]

[0131]

[0132] The results showed that the relative retention time RSD of each characteristic peak was 0.01% to 0.09%, indicating that the precision of the instrument was good.

[0133] 1.8.3 Reproducibility investigation

[0134] The sample was precisely weighed, and the preparation and determination were carried out according to the experimental method. See Table 3-4.

[0135] Table 3 Reproducibility investigation- relative retention time

[0136]

[0137] Table 4 Reproducibility Investigation - Relative Peak Area

[0138]

[0139] The results show that the relative retention time RSD of the 6 samples is 0.01% to 0.32%, indicating that the method has good reproducibility.

[0140] 1.8.4 Intermediate Precision Investigation

[0141] 1.8.4.1 Different Instrument Investigation

[0142] Based on the above experimental conditions, the sample solution was prepared by precisely weighing the Litsea cubeba granules, and was determined on Waters and Agilent 1290 type high performance liquid chromatograph, respectively. See Table 5-6. Figure 14

[0143] Table 5 Intermediate Precision Investigation - Relative Retention Time

[0144]

[0145]

[0146] Table 6 Intermediate Precision Investigation - Relative Peak Area

[0147]

[0148] The results show that the RSD of the relative retention time of each characteristic peak is 0.3% to 2.6% when the same sample is determined by different personnel at different times, indicating that the method has good stability.

[0149] 1.8.5 Robustness Investigation

[0150] Based on the above experimental conditions, the column 1 Luna Omega PS C18 150mm x 2.1mm, 1.6μm (S / N: H22-010689) and the column 2 Luna Omega PS C18 150mm x 2.1mm, 1.6μm (S / N: H20-153838) were investigated, respectively. See Table 7-8. Figure 15

[0151] Table 7 Column Robustness Investigation - Relative Retention Time

[0152]

[0153] ​​​​Table 8. Column robustness study - relative peak area

[0154]

[0155] The results showed that when using the three chromatographic columns described above to detect samples, the RSD of the relative retention time of the characteristic peaks ranged from 0.16% to 4.29%, and the RSD of the relative peak area ranged from 4.67% to 28.08%.

[0156] 1.8.6 Stability

[0157] Based on the experimental conditions proposed above, the same test solution was taken and measured at 0h, 4h, 8h, 12h, and 16h, respectively. See Table 9-10.

[0158] Table 9 Stability Study - Retention Time

[0159]

[0160] Table 10 Stability Study - Peak Area

[0161]

[0162] The results showed that the RSD of the relative retention time of the characteristic peak was between 0.03% and 0.83%, and the sample solution was stable within 16 hours.

[0163] In summary, 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 above six characteristic peaks will be included in subsequent investigations.

[0164] 1.8.7 Determination of characteristic peaks and establishment of reference spectra

[0165] 1.8.7.1 Validation results of three batches of Piper kadsura formula granules

[0166] The characteristic spectra of three batches of *Piper kadsura* granules were determined using the proposed method, and the relative retention time and relative peak area were calculated. (See attached image.) Figure 16-19 Table 11-12.

[0167] Table 11 Relative retention times of three batches of *Haifengteng* (a type of herb) formulation granules

[0168]

[0169] Table 12 Relative peak area of ​​granules in three batches of *Hypericum perforatum* formulation

[0170]

[0171] 1.8.7.2 Establishment of Limits for Relative Retention Time

[0172] Table 13 summarizes the methodological examination items and validation results:

[0173] Table 13 Summary of RSD% for Methodological Results – Relative Retention Time

[0174]

[0175] Based on the principles of relatively stable retention time, detectability in all batches of samples, and relatively high peak values, six peaks with good robustness were selected as characteristic peaks. According to the methodological evaluation results, the provisional theoretical plate number, calculated based on the alkaloid peak, should not be less than 10,000.

[0176] The final specification stipulates that the chromatogram of the test sample should show six characteristic peaks, and the retention times should correspond to the six characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the reference material is designated as the S peak. The relative retention times of each characteristic peak and the S peak should be calculated, and these relative retention times should be within ±10% of the specified values. The specified values ​​are 0.52 (peak 1), 0.70 (peak 2), 0.73 (peak 3), 0.87 (peak 4), and 0.98 (peak 5).

[0177] The chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012 version) was used to synthesize chromatograms of 16 batches of Piper kadsura standard decoction, and a reference chromatogram of the characteristic chromatograms of Piper kadsura standard decoction was established. See [link / reference]. Figure 20 .

[0178] Conclusion: The characteristic spectral method of Piper kadsura formula granules can effectively detect Piper kadsura formula granules, therefore this method is included in the main text of Piper kadsura formula granules.

[0179] 2. Establishment of the characteristic chromatogram of the standard decoction of Piper kadsura

[0180] 2.1 Method for Determining the Characteristic Spectra of Standard Decoctions

[0181] Chromatographic conditions and system suitability tests are the same as those for the formulation granules [characteristic chromatograms].

[0182] The preparation of the reference solution is the same as the [Characteristic Spectrum] item for the formulation particles.

[0183] Preparation of the test solution: Take about 0.5g of standard decoction powder, add 20mL of methanol, sonicate (power 600W, frequency 40kHz) for 20 minutes, cool, shake well, filter, and take the filtrate to obtain the test solution.

[0184] The determination method is the same as that for the [characteristic spectrum] of the formulation granules.

[0185] 2.2 Verification of the characteristic chromatograms of standard decoctions

[0186] The prepared method was used to determine the characteristic chromatogram of 16 batches of standard decoction of C. japonica, and the relative retention time and relative peak area were calculated, as shown in Tables 14-15.

[0187] Table 14 Relative retention time of C. japonica standard decoction

[0188]

[0189] Table 15 Relative peak area of C. japonica standard decoction

[0190]

[0191] According to the principles of stable relative retention time and detectable peak in each batch of samples, a total of 6 peaks with good durability were selected as characteristic peaks. According to the results of methodological investigation and verification of 16 batches of standard decoction, the theoretical plate number was tentatively determined to be not less than 10000 based on the calculation of wall grass alkaloid peak.

[0192] Finally, it was stipulated that 6 characteristic peaks should be present in the test sample chromatogram, and should correspond to the retention time of the 6 characteristic peaks in the reference material chromatogram of the control medicinal material, among which peak 6 should correspond to the retention time of the reference peak of the control. The peak corresponding to the wall grass alkaloid reference is S peak, and the relative retention time of each characteristic peak to S peak should be within ±10% of the specified value. The specified value is 0.52 (peak 1), 0.70 (peak 2), 0.73 (peak 3), 0.87 (peak 4), and 0.98 (peak 5).

[0193] The 16 batches of C. japonica standard decoction were synthesized using Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version), and the control chromatogram of C. japonica standard decoction characteristic chromatogram was established. See Figure 21 .

[0194] 3. Establishment of characteristic chromatogram of C. japonica medicinal material

[0195] 3.1 Preparation method of medicinal material characteristic chromatogram

[0196] The chromatographic conditions and system suitability test were the same as those in the preparation of granules [characteristic chromatogram].

[0197] The preparation of reference solution was the same as that in the preparation of granules [characteristic chromatogram].

[0198] The test sample solution was prepared by accurately weighing about 1.0 g of medicinal material powder (passed through No. 3 sieve) into a conical flask with a plug, accurately adding 20 mL of methanol, weighing, ultrasonic treatment (power 600 W, frequency 40 kHz) for 20 minutes, cooling, weighing again, supplementing the weight loss with methanol, shaking, filtering, and taking the filtrate.

[0199] The determination method was the same as that in the preparation of granules [characteristic chromatogram].

[0200] 3.2 Verification of medicinal material characteristic maps

[0201] The above method was used to measure 22 batches of samples, and the ratios of relative retention time and relative peak area were calculated. The results are shown in Table 16-17.

[0202] Table 16 Relative Retention Time of Characteristic Images of Piper kadsura (Haifengteng) Medicinal Material

[0203]

[0204] Table 17 Relative Peak Area of ​​Characteristic Spectra of Piper kadsura (Haifengteng)

[0205]

[0206]

[0207] Based on the principles of stable relative retention time, detectability in all batches of samples, and relatively high peak values, six peaks with good repeatability were selected as characteristic peaks. The results showed that the relative peak area (RSD) of the characteristic peaks in the medicinal materials varied too much to specify a relative peak area, therefore these peaks were not included in the main text of the quality standard.

[0208] The final specification stipulates that the chromatogram of the test sample should show six characteristic peaks, and the retention times should correspond to the six 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 reference standard is designated as peak S. The relative retention times of each characteristic peak and peak S should be calculated and should be within ±10% of the specified values. The specified values ​​are 0.52 (peak 1), 0.70 (peak 2), 0.73 (peak 3), 0.87 (peak 4), and 0.98 (peak 5).

[0209] The similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine (2012 version) was used to synthesize 16 batches of Piper kadsura (sea kadsura) medicinal materials, and a reference characteristic chromatogram for Piper kadsura medicinal materials was established. See [link / reference]. Figure 22 .

[0210] 4. Establishment of a characteristic atlas of *Haifengteng* (a type of herb) slices

[0211] 4.1 Method for Determining the Characteristic Atlas of Medicinal Herbs

[0212] Chromatographic conditions and system suitability tests are the same as those for the formulation granules [characteristic chromatograms].

[0213] The preparation of the reference solution is the same as the [Characteristic Spectrum] item for the formulation particles.

[0214] The preparation of the test solution is the same as that of the [Characteristic Chromatography] section for medicinal materials.

[0215] The determination method is the same as that for the [characteristic spectrum] of the formulation granules.

[0216] 4.2 Verification of the characteristic chromatogram of cut crude drugs

[0217] Twenty batches of samples were determined according to the above method, and the ratios of relative retention time and relative peak area were calculated. The results are shown in Tables 18 - 19.

[0218] Table 18 Relative retention time of the characteristic chromatogram of Piper kadsura cut crude drugs

[0219]

[0220]

[0221] Table 19 Relative peak area of the characteristic chromatogram of Piper kadsura cut crude drugs

[0222]

[0223] According to the principle of stable relative retention time, all batches of samples can be detected and the peaks are relatively high, a total of 6 peaks with good repeatability were selected as characteristic peaks. The results showed that the RSD of the relative peak area of the characteristic peaks of the medicinal materials was too different to stipulate the relative peak area, so it was not included in the main text of the quality standard. The RSD of the relative retention time of the 6 characteristic peaks of 16 batches of Piper kadsura medicinal materials was less than 1.0%.

[0224] Finally, it was stipulated that: six characteristic peaks should be presented in the chromatogram of the test sample, and the retention times of the six characteristic peaks should correspond to those in the chromatogram of the reference crude drug reference substance. Among them, the peak corresponding to the pellitorine reference substance was the S peak, and the relative retention time of each characteristic peak and the S peak was calculated, and its relative retention time should be within ±10% of the specified value. The specified values were 0.52 (peak 1), 0.70 (peak 2), 0.73 (peak 3), 0.87 (peak 4), 0.98 (peak 5).

[0225] Sixteen batches of Piper kadsura cut crude drug characteristic chromatograms were synthesized using the Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (2012 Edition) to establish a reference chromatogram of the characteristic chromatogram of Piper kadsura cut crude drugs. See Figure 23 .

[0226] 5 Results of the correlation between Piper kadsura medicinal materials, cut crude drugs, standard decoctions, and formula granules

[0227] Using the Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (2012 Edition), the characteristic chromatograms of 16 batches of Piper kadsura medicinal materials were synthesized into a reference chromatogram of medicinal materials, the characteristic chromatograms of 16 batches of Piper kadsura cut crude drugs were synthesized into a reference chromatogram of cut crude drugs, the characteristic chromatograms of 16 batches of Piper kadsura standard decoctions were synthesized into a reference chromatogram of standard decoctions, and the characteristic chromatograms of 3 batches of Piper kadsura formula granules were synthesized into a reference chromatogram of formula granules. The above reference chromatograms of Piper kadsura medicinal materials, cut crude drugs, standard decoctions, and formula granules were compared, and the results are shown in Figure 24 .

[0228] The results showed that six characteristic peaks could be detected in the raw materials, processed slices, standard decoctions, and formula granules of *Piper kadsura*, and their material basis was consistent. The method of this invention can accurately and efficiently detect the characteristic components in the raw materials, processed slices, standard decoctions, and formula granules of *Piper kadsura*, and achieve the purpose of overall quality control of the raw materials, processed slices, standard decoctions, and formula granules of *Piper kadsura*.

[0229] In summary, this high-performance liquid chromatography (HPLC) method for detecting the characteristic components of *Piper kadsura* medicinal materials, processed slices, standard decoctions, and formulated granules can comprehensively control the characteristic components in these materials, ensuring the overall stability of their quality. Furthermore, the method is simple to operate, highly precise, stable, repeatable, and accurate.

[0230] Example 3: Method for detecting the content of Piper kadsura (sea breeze vine) or its preparations

[0231] 1. Materials

[0232] 1.1 Instruments

[0233] Waters H-Class ultra-high performance liquid chromatograph (Waters Corporation), Agilent 1290 high performance liquid chromatograph. Omega PS C18 column (150mm × 2.1mm, 1.6μm), ME204E analytical balance (Mettler-Toledo), XP26 analytical balance (Mettler-Toledo), ME204E analytical balance (Mettler-Toledo), KQ5200DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), Cellular 1810A ultrapure water system (Shanghai Moller Scientific Instrument Co., Ltd.)

[0234] 1.2 Test Drugs

[0235] Herba salsa reference standard (batch number: DST230721-160); Piper kadsura reference medicinal material (China National Institutes for Food and Drug Control, batch number 121635-202104). Acetonitrile was chromatographically pure; all other reagents were analytically pure; water was ultrapure water. All 16 batches of Piper kadsura granules were from Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd., and the sample numbers were S1 to S16.

[0236] 2. Methods and Results

[0237] 2.1 Determination of the content of wall grass alkaloid

[0238] 2.1.1 Chromatographic conditions

[0239] Chromatographic column: Omega PSC18 (150 mm x 2.1 mm, 1.6 μm); mobile phase: acetonitrile (A) - water (B); gradient elution (0-5 min, 10%→32% A; 5-15 min, 32% A, 15-32 min, 32%→52% A, 32-33 min, 52%→10% A, 33-38 min, 10% A) detection wavelength: 260 nm; flow rate: 0.3 mL / min; column temperature: 30 °C; injection volume: 2 μL.

[0240] 2.1.2 Preparation of the control solution

[0241] An appropriate amount of the control sample of Tetrahydrocolumbianine was precisely weighed, and dissolved in methanol to prepare a solution containing 10 μg per 1 mL, which was used as the control solution.

[0242] 2.1.3 Preparation of the test solution

[0243] An appropriate amount of the Huifengteng Dispensing Granules was finely ground, and about 0.5 g was precisely weighed into a conical flask with a stopper, and 20 mL of methanol was precisely added. The flask was tightly stoppered, and the weight was determined. The sample was ultrasonically treated (power 600 W, frequency 40 kHz) for 20 minutes. The flask was allowed to cool, and the weight was determined again. The lost weight was made up with methanol, and the mixture was shaken and filtered. The filtrate was collected, and the test solution was obtained.

[0244] 2.1.4 Investigation of specificity

[0245] An appropriate amount of the control solution, the test solution, and the blank solvent were determined by HPLC under the chromatographic conditions described in item 2.1.1. The results showed that the method had good specificity. See Table 2.1.1. Figure 25 .

[0246] 2.1.5 Investigation of linearity

[0247] An appropriate amount of the control sample of Tetrahydrocolumbianine (purity 98%) was dissolved in a 10 mL volumetric flask with methanol to prepare a solution containing 349.076 μg per 1 mL. The solution was diluted to prepare control solutions containing 0.3490762 μg, 1.396304 μg, 3.49076 μg, 6.98152 μg, 27.92608 μg, and 139.6304 μg per 1 mL, respectively. The control solutions were determined by HPLC under the chromatographic conditions described in item 2.1.1. The standard curve was plotted with the control sample concentration (μg / mL) as the abscissa (X) and the peak area as the ordinate (Y). The linear regression equation of Tetrahydrocolumbianine was y = 43578.9098x + 6064.9944 (R = 1.0000), indicating that the concentration of Tetrahydrocolumbianine was linearly related in the range of 0.3490762-139.6304 μg / mL. 2

[0248] 2.1.6 Investigation of precision

[0249] ​Take the concentration of 10.023 μg / mL of the control solution, according to the "2.1.1" under the chromatographic conditions repeated injection 6 times, the RSD of the peak area of jatrophine was 0.8%, indicating that the precision of the instrument was good.

[0250] 2.1.7 Stability test

[0251] Take the sample of Litseae Folium formula granules (S1), prepare the test solution according to the method under "2.1.3", and measure at 0, 4, 8, 9, 12, and 24 h respectively according to the chromatographic conditions under "2.1.1". The RSD of the area of jatrophine was 0.5%, indicating that the test solution was stable within 24 h.

[0252] 2.1.8 Reproducibility test

[0253] Take the sample of Litseae Folium formula granules (S1), prepare the test solution according to the method under "2.1.3", and measure according to the chromatographic conditions under "2.1.1". The RSD of the content of jatrophine was 0.7%, indicating that the method was reproducible.

[0254] 2.1.9 Accuracy test

[0255] Take the sample of Litseae Folium formula granules (S1) with known content, grind finely, and accurately weigh 6 portions of about 0.25 g each into conical bottles with stoppers. Add jatrophine control solution corresponding to 100% content to each group, prepare the test solution according to the method under "2.1.3", and measure according to the chromatographic conditions under "2.1.1". The recovery rate of jatrophine was 100.9%-103.2%, the average recovery rate was 101.5%, and the RSD value was 1.2%. This indicates that the method is accurate.

[0256] 2.1.10 Sample detection

[0257] Take the samples of Litseae Folium formula granules (S1-S16), prepare the test solution according to the method under "2.1.3", and measure according to the chromatographic conditions under "2.1.1". The results are shown in Table 20.

[0258] Table 20 Content determination results of Litseae Folium formula granules

[0259]

Claims

1. A method for constructing a characteristic spectrum of Piper kadsura or its preparations, characterized in that: It includes the following steps: a. Preparation of the test solution: Take the *Piper kadsura* sample, dissolve and extract it using a solvent to obtain the test solution; b. The test solution was determined by high performance liquid chromatography to obtain the characteristic chromatogram of Piper kadsura or its preparations; The characteristic chromatographic conditions for the *Hypericum perforatum* medicinal material or its preparations are as follows: a C18 column; acetonitrile as mobile phase A, water as mobile phase B, gradient elution, and the elution conditions are as follows:

2. The method for constructing the characteristic spectrum of Piper kadsura or its preparations according to claim 1, characterized in that: The method for preparing the test solution in step a is as follows: Take the herb *Piper kadsura* or its preparation, add methanol, sonicate, filter, and take the filtrate as the test solution.

3. The method for constructing the characteristic spectrum of Piper kadsura or its preparations according to claim 2, characterized in that: The ultrasonic treatment conditions are: power 600W, frequency 40kHz, and treatment time 20 minutes.

4. The method for constructing the characteristic spectrum of Piper kadsura medicinal material or its preparation according to any one of claims 1-3, characterized in that: The C18 column has a length of 150 mm, an inner diameter of 2.1 mm, and a particle size of 1.6 μm; the flow rate is 0.3 mL per minute; the column temperature is 30 °C; the detection wavelength is 260 nm; and the theoretical plate number, calculated based on the alkaloid peak, should be no less than 10,000.

5. The method for constructing the characteristic spectrum of Piper kadsura medicinal material or its preparation according to any one of claims 1-3, characterized in that: It also includes the preparation of reference solutions, with references including *Piper kadsura* reference material and wallwort alkaloids; The preparation method of the reference herb solution of *Piper kadsura* is as follows: Take the reference herb Piper kadsura, add methanol and sonicate, cool, shake well, filter, and take the filtrate as the reference solution for the reference herb; The preparation method of the reference solution of cypermethrin is as follows: cypermethrin reference standard is prepared by adding methanol aqueous solution to prepare the reference solution.

6. The method for constructing the characteristic spectrum of Piper kadsura or its preparations according to claim 5, characterized in that: The ultrasonic treatment conditions are: power 600W, frequency 40kHz.

7. The method for constructing the characteristic spectrum of Piper kadsura medicinal material or its preparations according to any one of claims 1-6, characterized in that: The characteristic spectrum contains six characteristic peaks with relative retention times of: peak 1: 0.52, peak 2: 0.70, peak 3: 0.73, peak 4: 0.75, peak 5: 0.76, peak 6: 0.77, peak 7: 0.78, peak 8: 0.79, peak 9: 0.79, peak 10: 0.79, peak 11: 0.79, peak 12: 0.79, peak 13: 0.79, peak 14: 0.79, peak 15: 0.79, peak 16: 0.79, peak 17: 0.79, peak 18: 0.79, peak 19: 0.79, peak 12: 0.79, peak 13: 0.79, peak 14: 0.79, peak 15:

0. Peak 5: 0.98, with relative retention times fluctuating within ±10%; Peak 6 is oxaliplatin.

8. The method for constructing the characteristic spectrum of Piper kadsura medicinal material or its preparations according to any one of claims 1-7, characterized in that: The aforementioned *Piper kadsura* medicinal material or its preparations may be *Piper kadsura* medicinal material, processed slices, standard decoctions, or formulated granules.

9. A method for determining the content of *Hippophae rhamnoides* (sea bream) granules, characterized in that: It uses the characteristic spectrum construction method of Piper kadsura or its preparations as described in any one of claims 1-8 to detect the content of wallwort alkaloids.

10. The method for determining the content of *Piper kadsura* formulation granules according to claim 9, characterized in that: The aforementioned *Hypericum perforatum* formula granules contain 0.13-0.36 mg / g of wallwort alkaloid.

Images