Construction method of daphne odorata thumb. fingerprint spectrum

Abstract

The application discloses a kind of spider fragrant medicinal material fingerprint construction method, the application acetonitrile-0.1% formic acid solution is mobile phase, with water as test sample solvent, construct spider fragrant medicinal material fingerprint while determining the content of 8 components such as new chlorogenic acid, chlorogenic acid, cryptochlorogenic acid, coffee acid, isochlorogenic acid B, isochlorogenic acid A, hesperidin, isochlorogenic acid C.The construction method of spider fragrant fingerprint of the application method is optimized by extraction time, extraction method, so that the information amount of spider fragrant chromatographic peak is maximally exposed, and the detection sensitivity is high (among them, isochlorogenic acid B, isochlorogenic acid A, hesperidin, isochlorogenic acid C separation effect is obviously superior to prior art), with strong specificity and good reproducibility and stability, high accuracy, can quickly and efficiently carry out quality control to spider fragrant medicinal material.The content of cryptochlorogenic acid in the water extract of the application is determined for the first time, and the content control of coffee acyl derivatives in spider fragrant is further improved.

Description

Technical Field

[0001] This invention belongs to the field of fingerprinting technology of Chinese medicinal materials, specifically relating to a method for constructing a fingerprint spectrum of the medicinal material *Syzygium siceraria*. Background Technology

[0002] Spider Fragrance ( Valeriana jatamansi *Valeriana spp.* (also known as *Laojunxu*, *Matixiang*, *Douchicao*, and *Xinye Valeriana*) is a perennial herb belonging to the genus *Valeriana* of the family Valerianaceae. It is mainly distributed in Henan, Gansu, Shaanxi, Hunan, Sichuan, Guizhou, Yunnan, and Tibet. According to literature reports, the main chemical components of *Valeriana spp.* include iridoids, sesquiterpenes, flavonoids, lignans, phenolic acids, and alkaloids, exhibiting sedative, hypnotic, antidepressant, anti-anxiety, neuroprotective, antibacterial, anti-inflammatory, and antiviral effects. *Valeriana spp.* is included in the 1977, 2010, 2015, and 2020 editions of the *Chinese Pharmacopoeia*. Currently, the 2020 edition of the *Chinese Pharmacopoeia* only includes quality control items for *Valeriana spp.* such as moisture content, ash content, and thin-layer chromatography, lacking fingerprint spectroscopy and active ingredient content determination. Therefore, *Valeriana spp.* lacks a complete set of scientific quality control methods, making it impossible to truly control its quality.

[0003] Traditional Chinese medicine (TCM) fingerprinting is based on the chromatograms of characteristic components of TCM materials or TCM preparations. With the help of a TCM chromatographic fingerprinting similarity evaluation system, it reflects the data and variables of the intrinsic chemical components of TCM and its preparations, and realizes the overall effect of TCM material groups. Due to its strong specificity, good reproducibility and accurate analytical results, it has become an internationally recognized means of TCM quality control.

[0004] Literature review revealed that in the HPLC fingerprint study of *Symplocos rubra*, Fu Sihong et al. found that the established fingerprint baseline of *Symplocos rubra* was unstable; there were obvious large tailing peaks with poor peak shape; only 6 peaks were identified by the reference standard and there was no quantitative indication.

[0005] Lu Wenqin et al. used a Diamonsil® C18 column (4.6 mm × 250 mm, 5 μm) and a mobile phase of acetonitrile (A) - 0.1% formic acid aqueous solution (B) to establish a fingerprint spectrum in the HPLC fingerprint identification of spider lily medicinal material. The mobile phase was acetonitrile (A) - 0.1% formic acid aqueous solution (B), with gradient elution (0~18 min, 12%~30%A; 18~20 min, 30%~32%A; 20~23 min, 32%~40%A; 23~25 min, 40%A; 25~33 min, 40%~53%A; 33~90 min, 53%~85%). The chromatogram acquisition time of 90 min was relatively long and uneconomical. The chromatogram showed that although the peak exposure was rich, the peaks around caffeic acid in the small peaks had large interference, and the resolution did not reach the baseline separation. The requirements for equipment and chromatographic column technology are high, and the reproducibility may be poor, making it difficult to use as a quality control standard.

[0006] Cheng Shengyong et al. used an Agilent 1290 ultra-high performance liquid chromatograph and an Ultimate® UHPLC Polar-RP column (100mm×2.1mm, 1.8μm) to establish the UPLC fingerprint spectrum in their study of *Cymbidium goeringii*. The chromatograms showed that this method failed to achieve baseline separation of chlorogenic acid, caffeic acid, isochlorogenic acid A, isochlorogenic acid B, and isochlorogenic acid C in *Cymbidium goeringii*, indicating a lack of universality. Furthermore, the established method for simultaneously determining the content of nine index components in *Cymbidium goeringii* by HPLC took 90 minutes, failing to achieve rapid determination. Additionally, a peak appeared before chlorogenic acid in the chromatogram of the *Cymbidium goeringii* sample, and the resolution between isochlorogenic acid B, hesperidin, isochlorogenic acid A, and isochlorogenic acid C may not have reached 1.5, leading to inaccurate calculations of component content and preventing the determination of true values.

[0007] Traditional Chinese medicine decoctions have been used for thousands of years and are the carrier of traditional Chinese medicine to this day. Traditionally, all medicines are decocted in water. Most of the chemical components of *Symplocos rubra* reported in the literature are characterized as alcohol extracts, and no reports have been found of water extracts. Based on the traditional practice of decocting medicines in water, this invention aims to provide a method for constructing a fingerprint spectrum of the water extract of *Symplocos rubra*. Summary of the Invention

[0008] The purpose of this invention is to provide a method for constructing a fingerprint spectrum of the medicinal herb *Cymbidium goeringii*.

[0009] The objective of this invention is achieved by a method for constructing a fingerprint spectrum of the medicinal herb *Cymbidium goeringii*, which is implemented through the following steps: 1) Preparation of reference solutions: Weigh appropriate amounts of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, hesperidin, and isochlorogenic acid C reference standards, accurately weigh them, and add 50% methanol to prepare mixed reference solutions with concentrations of 40-50 µg / mL, 500-600 µg / mL, 100-150 µg / mL, 15-25 µg / mL, 20-30 µg / mL, 40-50 µg / mL, 30-40 µg / mL, and 35-45 µg / mL, respectively. 2) Preparation of test solution: Take 0.2~2g of spider lily powder that has passed through a No. 3 sieve, place it in a stoppered conical flask, accurately add 25~100mL of purified water, seal tightly, weigh, soak in an 80℃ water bath for 30~90min, then extract by ultrasonication for 30~90min, let it stand at room temperature, make up the weight loss with purified water, and filter through a 0.22µm microporous membrane to obtain the test solution; 3) The test solution and reference solution of spider lily were detected by high performance liquid chromatography to construct the fingerprint spectrum of spider lily. The chromatographic conditions were as follows: a C18 column, 250 mm × 4.6 mm, 5 μm; column temperature of 25–35 °C; gradient elution using acetonitrile-0.1% formic acid solution: 0–30 min, 6%–13% acetonitrile, 30–45 min, 13%–18% acetonitrile, 45–55 min, 18%–23% acetonitrile, 55–60 min, 23%–40% acetonitrile, 60–70 min, 40%–60% acetonitrile; flow rate of 1.0 μL / min; detection wavelength of 327 nm for 0–54 min and 284 nm for 54–70 min; injection volume of 5–20 µL.

[0010] The beneficial effects of this invention are as follows: This invention uses acetonitrile-0.1% formic acid solution as the mobile phase and water as the sample solvent to construct a fingerprint chromatogram of *Sedum aizoon* (spider vine) and simultaneously determine the content of eight components: neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, hesperidin, and isochlorogenic acid C. The method of constructing the *Sedum aizoon* fingerprint chromatogram of this invention optimizes the extraction time and method, maximizing the exposure of information from the chromatographic peaks, resulting in high detection sensitivity (among which, the separation effects of isochlorogenic acid B, isochlorogenic acid A, hesperidin, and isochlorogenic acid C are significantly better than existing technologies). It exhibits strong specificity, good reproducibility and stability, high accuracy, and can rapidly and efficiently control the quality of *Sedum aizoon*. This invention is the first to determine the content of cryptochlorogenic acid in *Sedum aizoon* water extract, further improving the control of caffeoyl derivative content in *Sedum aizoon*. Attached Figure Description

[0011] Figure 1 Characteristic chromatograms of 15 batches of Spider Incense medicinal materials; Figure 2 The HPLC chromatogram of the mixed reference solution shows that peak 1 is neochlorogenic acid, peak 4 is chlorogenic acid, peak 5 is cryptochlorogenic acid, peak 6 is caffeic acid, peak 13 is isochlorogenic acid B, peak 14 isochlorogenic acid A, peak 15 is hesperidin, and peak 16 isochlorogenic acid C. Figure 3 HPLC chromatogram of the test solution Figure 1 Peak 1 is neochlorogenic acid, peak 4 is chlorogenic acid, peak 5 is cryptochlorogenic acid, peak 6 is caffeic acid, peak 13 is isochlorogenic acid B, peak 14 is isochlorogenic acid A, peak 15 is hesperidin, and peak 16 is isochlorogenic acid C. Figure 4 This is the standard curve for chlorogenic acid reference standard; Figure 5 This is the standard curve for chlorogenic acid reference standard; Figure 6 This is the standard curve for cryptochlorogenic acid reference standard; Figure 7This is the standard curve for caffeic acid reference standard; Figure 8 This is the standard curve for isochlorogenic acid B reference standard; Figure 9 This is the standard curve for isochlorogenic acid A reference standard; Figure 10 This is the standard curve for hesperidin reference standard; Figure 11 This is the standard curve for isochlorogenic acid C reference standard. Detailed Implementation

[0012] The present invention will be further described below, but this is not intended to limit the invention in any way. Any modifications made based on the present invention are within the scope of protection of the present invention.

[0013] This invention discloses a method for constructing a fingerprint spectrum of the medicinal herb *Cymbidium goeringii*, which is implemented according to the following steps: 1) Preparation of reference solutions: Weigh appropriate amounts of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, hesperidin, and isochlorogenic acid C reference standards, accurately weigh them, and add 50% methanol to prepare mixed reference solutions with concentrations of 40-50 µg / mL, 500-600 µg / mL, 100-150 µg / mL, 15-25 µg / mL, 20-30 µg / mL, 40-50 µg / mL, 30-40 µg / mL, and 35-45 µg / mL, respectively. 2) Preparation of test solution: Take 0.2~2g of spider lily powder that has passed through a No. 3 sieve, place it in a stoppered conical flask, accurately add 25~100mL of purified water, seal tightly, weigh, soak in an 80℃ water bath for 30~90min, then extract by ultrasonication for 30~90min, let it stand at room temperature, make up the weight loss with purified water, and filter through a 0.22µm microporous membrane to obtain the test solution; 3) The test solution and reference solution of spider lily were detected by high performance liquid chromatography to construct the fingerprint spectrum of spider lily. The chromatographic conditions were as follows: a C18 column, 250 mm × 4.6 mm, 5 μm; column temperature of 25–35 °C; gradient elution using acetonitrile-0.1% formic acid solution: 0–30 min, 6%–13% acetonitrile, 30–45 min, 13%–18% acetonitrile, 45–55 min, 18%–23% acetonitrile, 55–60 min, 23%–40% acetonitrile, 60–70 min, 40%–60% acetonitrile; flow rate of 1.0 μL / min; detection wavelength of 327 nm for 0–54 min and 284 nm for 54–70 min; injection volume of 5–20 µL.

[0014] In step 1), the ultrasonic extraction power is 180W and the frequency is 40KHz.

[0015] This invention also provides a quality control method for spider lily medicinal materials, which is implemented according to the following steps: 1) Take the *Symplocos edulis* medicinal material to be tested and obtain its fingerprint spectrum according to the aforementioned method; 2) Compare the fingerprint spectrum obtained in step 1) with the standard fingerprint spectrum, calculate the similarity of each characteristic peak, and use a similarity of ≥0.995 as the quality control standard.

[0016] Example 1 1. Experimental Instruments: Agilent 1260 (Infinity II) high-performance liquid chromatograph (Agilent Technologies, USA); SQP electronic balance (Sartorius Scientific Instruments GmbH, Germany); Milli-Q IQ7000 ultrapure water system (Millipore, USA); XM-P101H ultrasonic cleaner (Kunshan Xiaomei Ultrasonic Instruments Co., Ltd.); HWS-24 electric thermostatic water bath (Shanghai Yiheng Scientific Instruments Co., Ltd.); Agilent ZORBAX SB-C18 (4.6mm×250mm 5µm) chromatographic column (Agilent Technologies, USA).

[0017] 2. Reagents and Materials: Neochlorogenic acid (batch number PS000974, mass fraction ≥98%), chlorogenic acid (batch number PS012822, mass fraction ≥98.3%), cryptochlorogenic acid (batch number PS001110, mass fraction ≥98%), caffeic acid (batch number PS012526, mass fraction ≥99.4%), isochlorogenic acid B (batch number PS012052, mass fraction ≥98%), isochlorogenic acid A (batch number PS001052, mass fraction ≥98%), hesperidin (batch number PS011595, mass fraction ≥97.9%), and isochlorogenic acid C (batch number PS012526, mass fraction ≥96%) were all purchased from Chengdu Pusi Biotechnology Co., Ltd.; acetonitrile and methanol were chromatographic grade and purchased from Merck Co., Ltd.; formic acid was analytical grade and purchased from Tianjin Fengchuan Chemical Reagent Technology Co., Ltd.; water was ultrapure water.

[0018] 3. Preparation of reference solutions: Weigh appropriate amounts of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, hesperidin, and isochlorogenic acid C reference standards, respectively, accurately weigh them, and add 50% methanol to prepare mixed reference solutions with concentrations of 40µg / mL, 580µg / mL, 100µg / mL, 20g / mL, 20µg / mL, 40µg / mL, 35µg / mL, and 40µg / mL, respectively.

[0019] 4. Preparation of the test solution: Take 0.5g of spider lily powder (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 25mL of purified water, seal tightly, weigh it, soak in an 80℃ water bath for 30min, ultrasonically extract (power 180W, frequency 40KHz) for 30min, let it stand at room temperature, make up the weight loss with purified water, filter through a 0.22µm microporous membrane to obtain the test solution.

[0020] 5. High Performance Liquid Chromatography (HPLC) Chromatographic conditions: Agilent ZORBAX SB-C18 (250 mm × 4.6 mm, 5 μm) column; mobile phase: acetonitrile (A) - 0.1% formic acid solution (B) gradient elution (0~30 min, 6%~13% A, 30~45 min, 13%~18% A, 45~55 min, 18%~23% A, 55~60 min, 23%~40% A, 60~70 min, 40%~60% A); flow rate: 1.0 mL / min; injection volume: 20 µL; column temperature: 30 °C; detection wavelength: 327 nm (0~54 min), 284 nm (54~70 min).

[0021] 6. Methodological review of the detection methods 1) Precision test: Take the spider fragrance test solution (S13), inject it continuously for 6 times according to the chromatographic conditions in step 5, and take the chromatographic peak of chlorogenic acid No. 4 as the reference peak (S). The relative retention time RSD of each common peak is calculated to be 0.06%~0.32%, and the relative peak area RSD is 0.66%~2.04%, indicating that the instrument precision is good.

[0022] 2) Repeatability test: Take an appropriate amount of spider lily test sample (S13), prepare 6 test sample solutions in parallel according to the method in step 4, and inject and determine according to the chromatographic conditions in step 5. With chlorogenic acid peak No. 4 as reference peak (S), the relative retention time RSD of each common peak was calculated to be 0.01%~0.08%, and the relative peak area RSD was 0.34%~1.11%, indicating that the method has good repeatability.

[0023] 3) Stability test: The spider fragrance test solution (S13) was injected and measured at 0, 4, 8, 12, 18 and 24 h according to the chromatographic conditions in step 5. The chlorogenic acid chromatographic peak No. 4 was used as the reference peak (S). The relative retention time RSD of each common peak was calculated to be 0.00%~0.01%, and the relative peak area RSD was 0.34%~2.67%, indicating that the test solution has good stability within 24 h.

[0024] 7. Establishment and similarity evaluation of fingerprint patterns Take 15 batches of spider lily samples, prepare spider lily test solutions according to the method in step (2), and inject and determine according to the chromatographic conditions in step (3). Record the chromatograms, import the data into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)", use the chromatogram of sample S1 as the reference chromatogram, use the median method, with a time window width of 0.1, perform multi-point correction, and generate superimposed chromatograms and reference chromatograms. Figure 1 A total of 16 common peaks were identified, and compared with the control standard (). Figure 2 The comparison identified eight common peaks: peak 1 (neochlorogenic acid), peak 4 (chlorogenic acid), peak 5 (cryptchlorogenic acid), peak 6 (caffeic acid), peak 13 (isochlorogenic acid B), peak 14 (isochlorogenic acid A), peak 15 (hesperidin), and peak 16 (isochlorogenic acid C). Peak 4, with its large peak area and good resolution, was used as the reference peak (S). The similarity results are shown in Table 1. The results indicate that the similarity between the chromatograms of all 15 batches of samples and the reference chromatogram was ≥0.995, suggesting relatively stable quality.

[0025] Table 1. Similarity Evaluation Results of Spider Fragrance

[0026] 8. Determination of the content of 8 components in spider fragrance 1) Examination of linear relationships Accurately pipette the reference stock solution prepared in step 3, and prepare a series of reference solutions using the serial dilution method. Inject each solution according to the chromatographic conditions in step (3), record the peak area values, and plot a standard curve with the concentration of the reference solution as the abscissa (X) and the peak area as the ordinate (Y). Figures 4-11 The detection limit was calculated with a signal-to-noise ratio of 3:1 and the quantitation limit was calculated with a signal-to-noise ratio of 10:1. The results are shown in Table 2.

[0027] Table 2. Results of linear relationship investigation of 8 components of Spider Fragrance

[0028] 2) Precision test Take the spider fragrance test solution (S13), and continuously inject and determine it 6 times under the chromatographic conditions in step 5. The RSDs of the peak areas of the above 8 components were calculated to be 1.91%, 0.78%, 1.04%, 1.92%, 1.83%, 1.93%, 0.85%, and 1.27%, respectively, indicating that the instrument has good precision.

[0029] 3) Repeatability test Take an appropriate amount of spider lily test sample (S13), prepare 6 test sample solutions in parallel according to the method in step 4, and inject them for determination according to the chromatographic conditions in step (3). The RSDs of the contents of the above 8 components were calculated to be 0.42%, 0.15%, 0.44%, 0.40%, 0.60%, 0.29%, 0.98%, and 0.46%, respectively, indicating that the method has good repeatability.

[0030] 4) Stability test The spider fragrance test solution (S13) was injected and measured at 0, 4, 8, 12, 18 and 24 h according to the chromatographic conditions in step 5. The calculated peak area RSDs of the above 8 components were 1.91%, 0.78%, 1.04%, 1.92%, 1.83%, 1.93%, 0.85% and 1.27%, respectively, indicating that the test solution had good stability within 24 h.

[0031] 5) Recovery test Six samples of spider licorice (S13) with known component content were accurately weighed, each 0.25 g. Appropriate amounts of eight reference standards were added, and the test solution was prepared according to the method described in step 4. The samples were then injected for determination. The results showed that the recoveries were all between 95% and 105%, and the RSDs of the eight components were 1.78%, 0.16%, 1.25%, 1.89%, 1.24%, 0.57%, 1.06%, and 0.82%, respectively, indicating good accuracy (see Table 3).

[0032] Table 3. Results of the recovery rate test of 8 components in Spider Fragrance (n=6)

[0033] 6) Determination of the content of 8 components Take 15 batches of spider lily medicinal material sample solutions, inject them according to the chromatographic conditions in step 5, and calculate the content of 8 components in the samples. The results are shown in Table 4.

[0034] Table 4. Results of determination of 8 components of Spider Fragrance (mg / g, n=3)

[0035] The above test results demonstrate that the method for constructing the fingerprint spectrum of spider licorice medicinal material in this invention, based on spider licorice water extract, not only reveals its fingerprint characteristics but also exhibits high precision, good stability, and good reproducibility. This invention also characterized the content of eight chemical components in spider licorice water extract, which is of great significance for accurately controlling the quality of spider licorice.

Claims

1. A method for constructing a fingerprint spectrum of the medicinal herb *Cymbidium goeringii*, characterized in that, Follow these steps to achieve the following: 1) Preparation of reference solutions: Weigh appropriate amounts of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isochlorogenic acid B, isochlorogenic acid A, hesperidin, and isochlorogenic acid C reference standards, accurately weigh them, and add 50% methanol to prepare mixed reference solutions with concentrations of 40-50 µg / mL, 500-600 µg / mL, 100-150 µg / mL, 15-25 µg / mL, 20-30 µg / mL, 40-50 µg / mL, 30-40 µg / mL, and 35-45 µg / mL, respectively. 2) Preparation of test solution: Take 0.2~2g of spider lily powder that has passed through a No. 3 sieve, place it in a stoppered conical flask, accurately add 25~100mL of purified water, seal tightly, weigh, soak in an 80℃ water bath for 30~90min, then extract by ultrasonication for 30~90min, let it stand at room temperature, make up the weight loss with purified water, and filter through a 0.22µm microporous membrane to obtain the test solution; 3) The test solution and reference solution of spider lily were detected by high performance liquid chromatography to construct the fingerprint spectrum of spider lily. The chromatographic conditions were as follows: a C18 column, 250 mm × 4.6 mm, 5 μm; column temperature of 25–35 °C; gradient elution using acetonitrile-0.1% formic acid solution: 0–30 min, 6%–13% acetonitrile, 30–45 min, 13%–18% acetonitrile, 45–55 min, 18%–23% acetonitrile, 55–60 min, 23%–40% acetonitrile, 60–70 min, 40%–60% acetonitrile; flow rate of 1.0 μL / min; detection wavelength of 327 nm for 0–54 min and 284 nm for 54–70 min; injection volume of 5–20 µL.

2. The method for constructing the fingerprint spectrum of spider lily medicinal material according to claim 1, characterized in that, In step 2), the ultrasonic extraction power is 180W and the frequency is 40KHz.

3. A quality control method for spider lily medicinal material, characterized in that, Follow these steps to achieve the following: 1) Take the *Symplocos edulis* medicinal material to be tested and obtain the fingerprint spectrum of the *Symplocos edulis* medicinal material to be tested according to the method described in claim 1; 2) Compare the fingerprint spectrum obtained in step 1) with the standard fingerprint spectrum, calculate the similarity of each characteristic peak, and use a similarity of ≥0.995 as the quality control standard.

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