Method for constructing HPLC fingerprint spectrum of tetrastigma planicaule

Abstract

The invention relates to the technical field of drug detection, in particular to a construction method of a tetrastigma planicaule HPLC fingerprint spectrum. Comprising the following steps: (1) preparation of a test solution: taking a tetrastigma planicaule reference medicinal material, adding methanol, performing ultrasonic treatment, filtering, evaporating filtrate to dryness, adding water into residues for dissolving, extracting with ethyl acetate, combining ethyl acetate solutions, performing vacuum concentration until the solution is dry, fixing the volume with methanol, and filtering to obtain the test solution; (2) taking a resveratrol reference substance, and adding methanol to prepare a resveratrol reference substance solution; and (3) sucking the test solution and the resveratrol reference substance solution, injecting into a liquid chromatograph, and determining. The HPLC fingerprint spectrum established by the invention not only provides comprehensive and scientific technical support for quality evaluation of the tetrastigma planicaule, but also lays a foundation for pharmacological activity research and clinical application development of the tetrastigma planicaule. The method is easy and convenient to operate and good in reproducibility, has wide popularization value and can provide important reference for improvement of a traditional Chinese medicine quality control system.

Description

Technical Field

[0001] This invention relates to the field of drug detection technology, and in particular to a method for constructing an HPLC fingerprint of *Smilax china*. Background Technology

[0002] As a traditional Chinese medicine, *Cephalotaxus fortunei* (also known as *Cephalotaxus fortunei* var. *flavovirens*) possesses properties such as clearing heat and detoxifying, dispelling wind and dampness, and is widely used in folk medicine to treat rheumatic pain, traumatic injuries, and other ailments. Its pharmacological activity is mainly related to its phenolic acids, flavonoids, and stilbene compounds. However, current research on the quality control of *Cephalotaxus fortunei* var. *flavovirens* is relatively insufficient, lacking systematic chemical component analysis methods and standardized quality evaluation systems, which to some extent limits its clinical application and industrial development. Therefore, establishing a scientific and reliable HPLC fingerprinting method for *Cephalotaxus fortunei* var. *flavovirens* is of great significance for comprehensively evaluating its intrinsic quality and ensuring the stability of its efficacy.

[0003] High-performance liquid chromatography (HPLC) fingerprinting technology has become an important tool for the quality control of traditional Chinese medicine (TCM) due to its high separation efficiency and good reproducibility. This technology, through multi-component holistic characterization, can comprehensively reflect the characteristics of the chemical components of TCM, providing a basis for identifying authenticity and evaluating quality. However, existing HPLC analytical methods for *Cephalotaxus fortunei* are mostly limited to the content determination of single components, making it difficult to fully reflect its overall chemical characteristics. Furthermore, different extraction methods and chromatographic conditions can affect the stability and representativeness of fingerprint spectra; therefore, it is necessary to optimize sample pretreatment and chromatographic conditions to ensure the scientific validity and applicability of the methods.

[0004] Currently, there are few research reports on the HPLC fingerprinting of *Hedyotis diffusa*, especially regarding the lack of systematic exploration in areas such as gradient elution procedures, detection wavelength selection, and standard calibration. Existing methods may suffer from problems such as baseline drift, insufficient peak resolution, or excessively long analysis times. Therefore, developing an efficient and stable HPLC fingerprinting method for *Hedyotis diffusa*, combined with the simultaneous detection of multiple components (such as resveratrol), would help fill the technological gap in this field and provide technical support for improving the quality standards of *Hedyotis diffusa* and for subsequent pharmacological research. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing an HPLC fingerprint of *Symplocos buergeriana*.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for constructing an HPLC fingerprint of *Smilax china*, comprising the following steps:

[0008] (1) Preparation of test solution: Take the reference herb *Symplocos buergeriana*, add methanol and sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in water, extract with ethyl acetate 2-3 times, combine the ethyl acetate solutions, concentrate under reduced pressure to dryness, make up to volume with methanol, filter, and obtain the test solution.

[0009] (2) Take resveratrol reference standard and add methanol to prepare resveratrol reference standard solution;

[0010] (3) Take 8-12 μL of the test solution and the resveratrol reference solution and inject them into the liquid chromatograph for determination; wherein, octadecylsilane bonded silica gel is used as the stationary phase, acetonitrile is used as mobile phase A and 0.1% formic acid is used as mobile phase B.

[0011] The gradient elution procedure is as follows:

[0012] Table 1

[0013]

[0014] Preferably, the type of chromatographic column in step (3) is: CAPCELLPAK C18 MGIII, with a specification of 4.6×250mm and 5μm.

[0015] Preferably, the wavelength is set to 300-320 nm during chromatograph detection in step (3).

[0016] Preferably, the column temperature is set to 33-37°C during chromatograph detection in step (3).

[0017] Preferably, the ratio of the reference herb *Platycarpus stenoptera* in step (1) to methanol is 2g:90-110ml.

[0018] Preferably, the power of the ultrasound in step (1) is 450-550W and the time is 13-17min.

[0019] Preferably, the concentration of the resveratrol reference solution in step (2) is 45–55 μg / ml.

[0020] The present invention also provides an HPLC fingerprint of *Sedum aizoon* prepared by the above-described construction method. The HPLC fingerprint of *Sedum aizoon* includes four common chromatographic peaks: protocatechuic acid (relative retention time 15.9 min), protocatechuic aldehyde (relative retention time 20.6 min), leucine (relative retention time 39.3 min), and resveratrol (relative retention time 49.9 min).

[0021] This invention also provides the application of the HPLC fingerprint of *Cynanchum paniculatum* in the quality evaluation of *Cynanchum paniculatum* medicinal materials.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention achieves efficient separation and simultaneous detection of four major active components (protocatechuic acid, protocatechuic aldehyde, lecithin, and resveratrol) in *Trapa natans* by optimizing sample preparation, chromatographic conditions, and detection parameters. The method employs a gradient elution program, combined with a CAPCELLPAK C18 MGIII column and a 310 nm detection wavelength, significantly improving peak resolution and analytical efficiency. Experimental results show that the relative retention times (RSD) of all common peaks are less than 3.0%, and specificity tests confirm no interference from the negative control, indicating that this method possesses excellent stability, reproducibility, and specificity.

[0024] Methodological validation demonstrated the invention's excellent robustness and applicability. In repeatability, precision, and stability tests, the relative retention times of each chromatographic peak exhibited minimal fluctuations (RSD < 3.0%) and remained stable under varying column temperatures, flow rates, and column conditions. Furthermore, the similarity to 16 batches of *Smilax china* samples was all above 0.9, while the similarity to *Spatholobus suberectus* was below 0.5, indicating that this method effectively distinguishes *Smilax china* from easily confused medicinal materials. These characteristics make it a reliable tool for the quality control of *Smilax china* and suitable for the standardized evaluation of medicinal materials from different sources.

[0025] The innovation of this invention lies in the first-time identification of components such as protocatechuic aldehyde and leucine in the fingerprint spectrum of *Cephalotaxus fortunei*, filling a research gap in this field. The established HPLC fingerprint spectrum not only provides comprehensive and scientific technical support for the quality evaluation of *Cephalotaxus fortunei*, but also lays the foundation for its pharmacological activity research and clinical application expansion. This method is simple to operate, has good reproducibility, and has broad application value, providing an important reference for improving the quality control system of traditional Chinese medicinal materials. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0027] Figure 1 Chromatogram of specificity test; Peak 1: protocatechuic acid; Peak 2: protocatechuic aldehyde; Peak 3: leucine; Peak 4: resveratrol.

[0028] Figure 2 Repeatability test chromatography.

[0029] Figure 3 Chromatogram of precision test.

[0030] Figure 4Chromatogram of stability test.

[0031] Figure 5 Chromatograms were examined at different column temperatures.

[0032] Figure 6 Chromatograms were examined at different flow rates.

[0033] Figure 7 Chromatograms were examined using different chromatographic columns.

[0034] Figure 8 Chromatograms were examined using different instruments.

[0035] Figure 9 The characteristic chromatograms of 16 batches of *Biandan Teng* slices are shown in Table 8; the corresponding batch numbers of S1 to S16 are shown in Table 8, and R is the generated control characteristic chromatogram.

[0036] Figure 10 Comparison of images of *Symplocos buergeriana* and *Symplocos macrantha*; S4~S19 are *Symplocos buergeriana*, and S1~S3 are *Symplocos macrantha*.

[0037] Figure 11 Characteristic spectrum of *Ipomoea quamoclit*; Peak 1: protocatechuic acid; Peak 2: protocatechuic aldehyde; Peak 3: lecithin; Peak 4: resveratrol. Detailed Implementation

[0038] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1

[0040] 1. Instruments and reagents

[0041] Agilent 1260 high-performance liquid chromatograph (Agilent Technologies, USA); electronic analytical balance (model: GL224I-1SCN, Sartorius, Germany; XSR205DU, Mettler Toledo Group); rotary evaporator (model: RV10, Aika (Guangzhou) Instrument Equipment Co., Ltd.); CNC ultrasonic cleaner (KQ2200E, Kunshan Ultrasonic Instrument Co., Ltd.); digital display precision constant temperature water bath (DK-98-ⅡA, Tianjin Test Instrument Co., Ltd.).

[0042] Resveratrol reference substance (batch number 111535 - 201703, content calculated as 99.4%, National Institutes for Food and Drug Control), Piceatannol reference substance (batch number 2415698, content calculated as 98.0%, Shanghai Anpu Chuisheng Standard Technology Service Co., Ltd.); Protocatechuic acid reference substance (batch number 110809 - 202207, content calculated as 97.5%, National Institutes for Food and Drug Control); Protocatechualdehyde reference substance (batch number 110810 - 202210, content calculated as 99.9%, National Institutes for Food and Drug Control); 16 batches of Tetrastigma planicaule (Hook.) Merr. decoction pieces, 3 batches of Spatholobus suberectus Dunn decoction pieces (batch numbers 17 - 19), and their origins are shown in Table 2; Acetonitrile (chromatographic grade), water (ultrapure water), and other reagents are all of analytical grade.

[0043] Table 2 Batch numbers and origins of Tetrastigma planicaule (Hook.) Merr. and Spatholobus suberectus Dunn

[0044]

[0045] 2 Method

[0046] Chromatographic conditions and system suitability test Octadecylsilyl silica gel was used as the filler [recommended chromatographic column: CAPCELL PAK C18 MGIII (4.6×250 mm, 5 μm)]. Acetonitrile was used as mobile phase A, and 0.1% formic acid was used as mobile phase B. Gradient elution was carried out according to the regulations in Table 1. The detection wavelength was 310 nm, the flow rate was 0.8 ml / min, the column temperature was 35°C, and the number of theoretical plates calculated based on the resveratrol peak should be not less than 5000.

[0047] Preparation of test solution and resveratrol reference substance solution 2.0 g of the reference medicinal material of Tetrastigma planicaule (Hook.) Merr. was placed in a conical flask, added with 100 ml of methanol, ultrasonicated for 15 minutes, filtered, the filtrate was evaporated to dryness, the residue was dissolved in 15 ml of water, and extracted with ethyl acetate by shaking for 2 times, 20 ml each time. The ethyl acetate layers were combined, concentrated under reduced pressure to dryness, and made up to 5 ml with methanol, then filtered to obtain the test solution. Separately, an appropriate amount of resveratrol reference substance was accurately weighed and dissolved in methanol to make a solution containing 50 μg per 1 ml to obtain the resveratrol reference substance solution.

[0048] Determination method 10 μl of the reference substance solution and the test solution were accurately pipetted respectively and injected into the liquid chromatograph for determination to obtain the results.

[0049] Example 2

[0050] Methodology investigation (all based on the method of Example 1 below)

[0051] The powder of Tetrastigma planicaule (Hook.) Merr. decoction pieces with batch number 20 was used for methodology investigation. Resveratrol was selected as the reference peak, and the relative retention time and relative peak area of each common characteristic peak were calculated. 4 common peaks with good repeatability were selected as characteristic peaks.

[0052] 1. Specificity test

[0053] Take methanol as the negative sample, and inject 10 μl each of the test solution and the negative sample into the liquid chromatograph. Analyze the samples under the chromatographic conditions suitable for the system's suitability. The results are shown in the figure. Figure 1 .

[0054] The results showed that the negative sample had no corresponding chromatographic peak at the corresponding position of the analyte in the test sample, indicating that the solvent did not interfere with the detection and that the method had good specificity.

[0055] 2 Repeatability Test

[0056] Six parallel test solutions were prepared and measured to examine the repeatability of the experimental method. The relative retention times and relative peak areas of each characteristic peak were all less than 3.0%, indicating good repeatability. (See attached table). Figure 2 Table 3.

[0057] Table 3. Relative retention time and relative peak area for repeatability

[0058]

[0059]

[0060] 3. Precision test

[0061] The test solution was injected six times consecutively to assess the instrument's precision. The results showed that the relative retention times and relative peak areas of each characteristic peak measured in the six consecutive injections all had RSD values ​​less than 3.0%, indicating good instrument precision. (See...) Figure 3 Table 4.

[0062] Table 4. Precision: Relative Retention Time and Relative Peak Area

[0063]

[0064]

[0065] 4. Stability Test

[0066] The test solution was tested at 0, 8, 12, 20, 24, and 28 hours to assess its stability. The relative retention times (RSDs) of all characteristic peaks were less than 3.0%, indicating good stability of the test sample within 28 hours. (See attached image) Figure 4 Table 5.

[0067] Table 5. Relative retention time and relative peak area for stability

[0068]

[0069] 5. Durability Test

[0070] Based on the original chromatographic conditions, the robustness of the chromatographic conditions was investigated by changing the column temperature, flow rate, and column parameters to measure the test solution. The elution times of each peak were statistically analyzed, and the relative retention times and relative peak areas (RSDs) were calculated. The results are shown in [Figure number missing]. Figure 5 , Figure 6 , Figure 7 Table 6.

[0071] Table 6. Durability relative retention time and relative peak area

[0072]

[0073]

[0074] Column 1: CAPCELL PAK C18 MGIII (4.6 × 250 mm, 5 μm)

[0075] Column 2: YMC Hydrosphere C18 (4.6 × 250 mm, 5 μm)

[0076] Column 3: Welch Ultimate Plus C18 (4.6×250mm, 5μm)

[0077] The results show that the relative peak areas of the common peaks differ significantly when chromatographic conditions are varied within a small range. Therefore, it is recommended to specify only the relative retention time and not the relative peak area for the time being.

[0078] 6. Intermediate Precision Test

[0079] Based on the original chromatographic conditions, different instruments were used for injection to investigate the intermediate precision of the method. Elution times of each peak were statistically analyzed, and the relative retention times and relative peak areas (RSD values) were calculated. Results are shown below. Figure 8 Table 7.

[0080] Table 7. Intermediate Precision: Relative Retention Time and Relative Peak Area

[0081]

[0082]

[0083] Instrument 1: Agilent 1260 Infinity

[0084] Instrument 2: Agilent 1200

[0085] The results show that the RSD values ​​of the relative retention times of the common peaks are all less than 3.0%, and there are significant differences in the relative peak areas.

[0086] 7. Acquisition of Feature Maps

[0087] Sixteen batches of *Pinellia ternata* (a type of vine) and three batches of *Spatholobus suberectus* (another type of vine) were collected. Test solutions were prepared according to the method in Example 1, and the samples were injected and analyzed under the chromatographic conditions described in Example 1. Chromatograms were recorded. The results were imported into the *Research Version of the Chromatographic Fingerprint Similarity Evaluation System for Traditional Chinese Medicine* (2012 edition) developed by the National Pharmacopoeia Commission. Data processing was performed on the 16 batches of *Pinellia ternata* samples, and fingerprint chromatograms were established. (See attached image). Figure 9 The relative retention time and relative retention peak area of ​​the characteristic chromatograms of 16 batches of Biandan Teng slices are shown in Tables 8 and 9, and the similarity evaluation is shown in Table 10. Figure 10 A comparison of illustrations of *Symplocos buergeriana* and *Symplocos macrantha*.

[0088] Table 816 shows the relative retention time of characteristic images of *Biandan Teng* (a type of vine) slices.

[0089]

[0090]

[0091] Table 916 shows the relative peak areas of characteristic graphs of *Biandan Teng* (a type of vine) slices.

[0092]

[0093]

[0094] Table 10 Similarity Analysis of Carrying Pole Vine Samples

[0095]

[0096] By comparing with the reference standard, and through retention time and UV absorption spectrum comparison, four common chromatographic peaks were identified in the sample. (See below) Figure 11 Protocatechuic acid (15.9 min), protocatechuic aldehyde (20.6 min), leucine (39.3 min), resveratrol (49.9 min).

[0097] The results showed that the main chromatographic peaks of different batches of *Smilax china* samples were basically consistent, with the main difference being the peak area of ​​some peaks. The similarity results showed that the similarity of all 16 batches of *Smilax china* was greater than 0.9, indicating a high degree of consistency in the chemical composition of the 16 batches. The similarity calculation results with the three batches of *Spatholobus suberectus* were 0.441, 0.348, and 0.319, respectively, and the chromatograms were significantly different from the control fingerprint chromatogram of *Smilax china*, indicating that this method can effectively and quickly distinguish *Smilax china* from *Spatholobus suberectus*. Currently, there are few studies on the fingerprint chromatogram of *Smilax china*. The HPLC fingerprint chromatogram established in this study identified four common peaks and their components. Among them, protocatechuic aldehyde, lecithin, and resveratrol were identified in *Smilax china* for the first time.

[0098] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing an HPLC fingerprint of *Smilax china*, characterized in that, Includes the following steps: (1) Preparation of test solution: Take the reference herb *Symplocos buergeriana*, add methanol and sonicate, filter, evaporate the filtrate to dryness, dissolve the residue in water, extract with ethyl acetate 2-3 times, combine the ethyl acetate solutions, concentrate under reduced pressure to dryness, make up to volume with methanol, filter, and obtain the test solution. (2) Take resveratrol reference standard and add methanol to prepare resveratrol reference standard solution; (3) Take 8-12 μL of the test solution and the resveratrol reference solution and inject them into the liquid chromatograph for determination; wherein, octadecylsilane bonded silica gel is used as the stationary phase, acetonitrile is used as mobile phase A and 0.1% formic acid is used as mobile phase B. The gradient elution procedure is as follows: Table 1 2. The construction method according to claim 1, characterized in that, Step (3) The type of chromatographic column is: CAPCELLPAK C18 MGIII, with a specification of 4.6×250mm and 5μm.

3. The construction method according to claim 1, characterized in that, Step (3) The wavelength is set to 300-320 nm when the chromatograph is used for detection.

4. The construction method according to claim 1, characterized in that, Step (3) The column temperature is set to 33-37℃ during chromatograph detection.

5. The construction method according to claim 1, characterized in that, In step (1), the ratio of the reference herb *Smilax china* to methanol is 2g: 90-110ml.

6. The construction method according to claim 1, characterized in that, The ultrasound power in step (1) is 450-550W and the time is 13-17min.

7. The construction method according to claim 1, characterized in that, The concentration of the resveratrol reference solution in step (2) is 45–55 μg / ml.

8. The HPLC fingerprint of *Smilax china* obtained by the construction method according to any one of claims 1 to 7, characterized in that, The HPLC fingerprint of the *Ipomoea purpurea* vine included four common chromatographic peaks: protocatechuic acid (relative retention time 15.9 min), protocatechuic aldehyde (relative retention time 20.6 min), leucine (relative retention time 39.3 min), and resveratrol (relative retention time 49.9 min).

9. The application of the HPLC fingerprint of *Symplocos buergeriana* as described in claim 8 in the quality evaluation of *Symplocos buergeriana* medicinal materials.

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