Fingerprint spectrum construction method and application of volatile components in apium graveolens
The fingerprint map of the volatile components of Xianghanqin was established through gas chromatography, which solved the problem of difficulty in detecting and controlling the quality of Xianghanqin medicinal materials in the prior art, and achieved rapid and accurate analysis and quality control.
Patent Information
- Application Number
- CN202510378030.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-28
- Publication Date
- 2025-06-17
AI Technical Summary
The prior art is difficult to effectively detect and control the volatile components of the celery medicinal materials, which makes it difficult to guarantee its quality.
The fingerprint map of the volatile components of celery chlorophyllium was established by gas chromatography (GC) method. The volatile components of celery chlorophyllium from different origins were compared using calery chlorophyllium as a reference, providing a basis for the quality control of medicinal materials and their preparations.
It realizes rapid and accurate analysis of volatile ingredients of celery, reduces detection costs, improves the control level of medicinal materials, and can effectively distinguish between different origins and quality of celery.
Smart Images
Figure CN120161144A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a method for detecting volatile components in Apium graveolens L. var. dulce DC. and constructing a fingerprint, and particularly relates to a method for establishing and applying a gas chromatography - fingerprint of volatile components in Apium graveolens L. var. dulce DC., belonging to the technical field of traditional Chinese medicine analysis. Background Art
[0002] The Twenty-five Flavor Pearl Pills are a commonly used Tibetan patent medicine of the precious category in Tibetan medicine, and are the main products of Tibet Zangnuo Pharmaceutical Co., Ltd. It is a pill prepared from 27 medicinal materials including pearl, mother-of-pearl, nutmeg, safflower, Terminalia chebula Retz., Apium graveolens L. var. dulce DC., Zhidasa Zeng, clove, dalbergia wood, etc., and has the efficacy of calming the mind and opening the orifices. It is commonly used in the treatment of diseases such as stroke, hemiplegia, deviation of the mouth and eyes, coma, mental disorder, delirium and mania, and has remarkable curative effects. Improving the quality standard is particularly important for enhancing the quality control level of Tibetan patent medicines, and most preparations of Tibetan medicines have increased the internal control chemical composition indexes. However, there is relatively little research on the quality control of the Twenty-five Flavor Pearl Pills and their related medicinal materials. As a traditional Chinese medicine, Apium graveolens L. var. dulce DC. is not currently included in the Chinese Pharmacopoeia, and its quality standard was only simply controlled in the 1985 edition of the Tibetan Pharmacopoeia, the 2010 edition of the Tibetan Medicine Processing Specification of Qinghai Province, and the 2020 edition of the Inner Mongolia Mongolian Medicine Decoction Pieces Processing Specification, and its quality cannot be effectively guaranteed. In order to ensure the internal quality of the raw materials of Apium graveolens L. var. dulce DC. and clarify its origin, our company has carried out relevant research on the quality standard of Apium graveolens L. var. dulce DC.
[0003] Apium graveolens L. var. dulce DC., also known as Cuminum cyminum L., is a plant of the Umbelliferae family, and is an annual or biennial herb. The fruit is oblong, with a strong fragrance, slightly sweet and spicy. The dried and mature fruit has the efficacy of clearing lung heat, promoting gastric fire, and promoting digestion, and is one of the important raw materials of traditional Tibetan medicine. It is mainly produced in the Mediterranean and Central Asian regions, and is also planted in Xinjiang, Inner Mongolia and other places in China. Due to the strong aromatic smell of Apium graveolens L. var. dulce DC., it has always been used as a natural plant spice and medicinal plant. Its volatile components have important medicinal values. As an important raw material of traditional Tibetan medicine, confirming its volatile components and establishing a detection method for its volatile components play an important role in ensuring the quality of medicinal materials.
[0004] In view of the fact that there is no standard for Apium graveolens L. var. dulce DC. in the Pharmacopoeia (2020 Edition, Volume I); in the 1985 Edition of the Tibetan Pharmacopoeia, only a simple identification of Apium graveolens L. var. dulce DC. was carried out. At present, there are more component analyses of the volatile components in Apium graveolens L. var. dulce DC., and the components are mostly confirmed by gas chromatography-mass spectrometry (GC-MS). Due to the relatively high purchase cost of the detection equipment, it is difficult to be widely promoted. As a common device for determining volatile substances, the gas chromatograph (equipped with an FID detector) has a lower equipment cost. At present, there is no literature report on the gas chromatography (GC) analysis of the volatile components of Apium graveolens L. var. dulce DC. Therefore, it is of great academic research value and practical application value to invent a method that can quickly and accurately analyze the volatile components of Apium graveolens L. var. dulce DC., with a lower detection cost, and can reflect the quality of the medicinal materials to a certain extent. Summary of the Invention
[0005] In order to solve the technical problems existing in the identification and component comparison of Apium graveolens L. var. dulce DC. medicinal materials, one object of the present invention is to provide a method for establishing a gas chromatography (GC) fingerprint of the volatile components of Apium graveolens L. var. dulce DC. Another object of the present invention is to provide the identification and content difference analysis of Apium graveolens L. var. dulce DC. medicinal materials and their preparations by using the above gas chromatography (GC) fingerprint. The fingerprint method of the present invention analyzes the volatile components in Apium graveolens L. var. dulce DC., uses cumin aldehyde as a reference substance, adopts the GC method to establish a gas chromatography fingerprint of the volatile components of Apium graveolens L. var. dulce DC., and compares the volatile components of Apium graveolens L. var. dulce DC. from different origins, so as to provide a basis for the quality control of Apium graveolens L. var. dulce DC. medicinal materials and their preparations, and achieve the purpose of accurately using the medicinal materials and strictly distinguishing other confused products.
[0006] In order to achieve the above first object, the present invention adopts the following technical solutions:
[0007] A method for establishing a gas chromatography-fingerprint of the volatile components of Apium graveolens L. var. dulce DC., the method comprising the following steps:
[0008] 1) Preparation of the reference substance solution
[0009] Precisely pipette the reference substances of α-pinene, myrcene, α-terpinene, thymol, γ-terpinene, and cumin aldehyde, and prepare a reference substance solution;
[0010] 2) Preparation of the test sample solution
[0011] After collecting the Apium graveolens L. var. dulce DC. medicinal materials, according to the volatile extraction method, heat and distill, cool, separate the volatile layer, extract with petroleum ether multiple times, dehydrate, concentrate with a rotary evaporator, make up the volume, and shake well to obtain the test sample solution;
[0012] 3) Gas chromatography analysis
[0013] Collect multiple batches of celery medicinal materials from different origins, prepare test sample solutions according to step 2), inject samples respectively, perform chromatographic analysis, and obtain gas chromatograms;
[0014] 4) Calibration of common peaks in the volatile gas phase fingerprint of celery
[0015] The gas chromatogram obtained in step 4) was analyzed by gas chromatography of the above-mentioned celery test sample solutions from different origins to obtain a gas chromatogram; the results showed that the chromatographic peaks of most components in each sample appeared between the retention time of 3 and 35 minutes, and the chromatographic peaks were well separated, of which 14 chromatographic peaks were characteristic peaks common to all the test samples, of which No. 12 was cuminaldehyde, which was used as a reference peak;
[0016] 5) Gas phase fingerprint analysis results of volatile components of celery
[0017] Through gas chromatography analysis of the celery samples from different origins mentioned above, the fingerprint characteristics of celery are as follows: peaks 1-14 appear in the chromatograms of the above samples, among which peak 12 has the same retention time as the cuminaldehyde in the chromatogram of the cuminaldehyde reference; the relative retention time of each common fingerprint peak is calculated with the retention time of the cuminaldehyde reference as a reference, and the RSD of the relative retention time is less than 1.0%. In all samples, the ratio of the sum of the peak areas of the common fingerprint peaks to the total peak area is greater than 91%.
[0018] As a preferred method, in the above step 1), the cuminaldehyde reference substance is first placed in a 5 mL volumetric flask, n-hexane is added to the scale, and shaken to prepare a cuminaldehyde stock solution, and then the above cuminaldehyde stock solution is accurately pipetted and placed in a 5 mL volumetric flask, n-hexane is added to the scale, and shaken to prepare a cuminaldehyde reference substance solution with a mass concentration of 1.00 mg / mL.
[0019] As a preferred method, after collecting the celery in the above step 2), it is cut into small pieces of 2 cm long, dried in the shade to a water content of less than 9%, and sealed for storage. The small pieces of celery are accurately weighed, placed in a distillation bottle, 0.5L of water is added, soaked for 10 hours, and then distilled and extracted.
[0020] As a preferred method, in the above step 2), dehydration is performed using anhydrous sodium sulfate, and n-hexane is used to dissolve and fix the volatile components.
[0021] As a preferred method, the gas chromatography conditions in the above step 3) are as follows:
[0022] Hydrogen flame ionization detector FID, HP-5 capillary column (0.25μm×0.32mm×30m); injection port temperature is 300℃; FID detector temperature is 300℃; carrier gas is constant flow rate; high-purity nitrogen: 0.5mL / min; fuel gas hydrogen: 30mL / min; auxiliary gas is air: 250mL / min; tail gas flow rate: 20mL / min;
[0023] The split ratio was 100:1; the injection volume was 1.0 μL;
[0024] Heating program: initial temperature is 50℃, hold for 3 minutes, increase to 100℃ at 0.6℃ / min, hold for 5 minutes, and increase to 250℃ at 50℃ / min.
[0025] As a preferred method, 8 to 15 batches of celery medicinal materials from different origins are used in the above step 3). As a most preferred method, 15 batches of celery medicinal materials from different origins are used in the above step 3).
[0026] As a preferred method, in the above step 4), chromatographic peaks 1-14 are chromatographic peaks with retention times of 3.603min, 3.746min, 5.007min, 5.727min, 7.104min, 9.028min, 9.663min, 11.859min, 17.959min, 18.955min, 20.817min, 26.777min, 32.352min, and 34.263min in the chromatogram, respectively.
[0027] In order to achieve the above-mentioned second purpose, the present invention adopts the following technical solutions:
[0028] The invention discloses an identification method for the medicinal material of celery and its preparation, wherein the method adopts a gas chromatography-fingerprint established by the method described in any one of the above technical solutions.
[0029] A method for analyzing the content difference of celery medicinal materials and preparations thereof, the method adopts a gas chromatography-fingerprint established by the method described in any one of the above technical solutions.
[0030] The present invention has the advantages of adopting the above technical solution:
[0031] 1. Ocimum americanum L. is not included in the first volume of the Chinese Pharmacopoeia 2020 edition, and there is no content on the medicinal material standard of Ocimum americanum L. in other national and local standards. As a medicinal material resource widely used in recent years, it is necessary to study and formulate its quality standard. The volatile GC fingerprint of Ocimum americanum L., as a method for the identification and content difference analysis of Ocimum americanum L., is an important part of its quality standard. Compared with the methods in the literature, the method of the present invention is rapid, simple, has good reproducibility and stability, strong characteristics, can be used as the basis for the identification of Ocimum americanum L., and provides a basis for the quality control of Ocimum americanum L. medicinal materials and their preparations.
[0032] 2. The method of the present invention is advanced. By using the GC method, a gas chromatographic fingerprint of the volatility of Ocimum americanum L. is established.
[0033] 3. The method of the present invention is simple to operate, and the instruments, equipment and materials used have good popularity, which is convenient for the promotion and application by medicinal material planting, processing and sales enterprises. Description of the Drawings
[0034] Figure 1 It is the volatile chromatogram of Ocimum americanum L. produced in MN01 (Datong, Shanxi). The 12th peak in the figure is cumin aldehyde.
[0035] Figure 2 It is the chromatogram of reference substances of α-pinene (1), myrcene (2), α-terpinene (3), thymol (4), γ-terpinene (5), cumin aldehyde (6).
[0036] Figure 3 It is the GC chromatogram superposition diagram of the volatility of 15 batches of Ocimum americanum L. samples from different producing areas. Detailed Embodiments
[0037] Example 1
[0038] A method for establishing a gas chromatographic-fingerprint of the volatile components of Ocimum americanum L., the method comprising the following steps:
[0039] 1) Accurately weigh 5 mg of cumin aldehyde reference substance, place it in a 5 mL volumetric flask, add n-hexane to the scale, shake well to prepare a cumin aldehyde stock solution. Then accurately pipette 0.5 mL of the cumin aldehyde stock solution, place it in a 5 mL volumetric flask, add n-hexane to the scale, shake well to prepare a cumin aldehyde reference substance solution with a mass concentration of 10 μg / mL.
[0040] 2) Collect Ocimum americanum from different producing areas, crush and sieve it. Weigh precisely 50 g of the Ocimum americanum powder, place it in a distillation flask, add 0.5 L of water, soak for 10 h, and extract for 1 h using steam distillation method in a distillation apparatus. Place the distillate in a separating funnel, add 50 mL of petroleum ether, shake for 3 min, repeat this operation three times, combine the organic phases, remove the moisture from the organic phases through anhydrous sodium sulfate, then remove the petroleum ether from the obtained extract in a rotary evaporator, and finally dissolve it with n-hexane and make the volume up to 10 mL, shake well to obtain the test solution;
[0041] 3) GC conditions
[0042] Hydrogen flame ionization detector FID, HP-5 capillary column (0.25 μm × 0.32 mm × 30 m); injection port temperature is 300 °C; FID detector temperature is 300 °C; the carrier gas is at a constant flow rate; high-purity nitrogen: 0.5 mL / min; fuel gas hydrogen: 30 mL / min; combustion-supporting gas air: 250 mL / min; tail blow flow rate: 20 mL / min;
[0043] Split ratio is 100:1; injection volume is 1.0 μL;
[0044] Temperature programming: initial temperature is 50 °C, hold for 3 min, rise to 100 °C at a rate of 0.6 °C / min, hold for 5 min, and then rise to 250 °C at a rate of 50 °C / min.
[0045] 4) GC analysis
[0046] Take the test solution in step 2) and measure it according to the conditions in step 3). The obtained GC chromatogram is shown in Figure 1 ; among which the 1-14th chromatographic peaks are the chromatographic peaks with retention times of 3.601 min, 3.744 min, 5.002 min, 5.723 min, 7.092 min, 9.018 min, 9.654 min, 11.869 min, 17.963 min, 18.951 min, 20.808 min, 26.561 min, 32.289 min, 34.102 min in the chromatogram respectively; the ratio of the sum of the peak areas of the 14 characteristic peaks to the total peak area is 94.1%.
[0047] Figure 2 It is the chromatogram of reference substances of α-pinene (1), myrcene (2), α-terpinene (3), thymol (4), γ-terpinene (5), cumin aldehyde (6).
[0048] Example 2
[0049] As Figure 3As shown, 15 batches of Ocimum basilicum L. medicinal materials from different producing areas were collected, and gas chromatograms were obtained by the above method, and then GC analysis was carried out. Table 1 shows the relative retention times of the common fingerprint peaks of 15 batches of Ocimum basilicum L.
[0050] Table 1 Relative retention times of the common fingerprint peaks of 15 batches of Ocimum basilicum L.
[0051] MN01 MN02 MN03 MN04 MN05 MN06 MN07 MN08 MN09 MN10 MN11 MN12 MN13 MN14 MN15 01 3.601 3.604 3.603 3.603 3.603 3.603 3.603 3.604 3.602 3.616 3.610 3.608 3.605 3.600 3.602 02 3.744 3.746 3.745 3.746 3.746 3.746 3.745 3.747 3.745 3.760 3.753 3.751 3.749 3.743 3.745 03 5.002 5.007 4.998 4.997 5.007 5.012 4.998 5.008 5.019 5.033 5.019 5.032 5.028 5.004 5.030 04 5.723 5.728 5.726 5.724 5.727 5.726 5.725 5.728 5.726 5.749 5.738 5.738 5.733 5.723 5.729 05 7.092 7.100 7.094 7.095 7.104 7.104 7.098 7.105 7.112 7.128 7.122 7.133 7.122 7.096 7.114 06 9.018 9.030 9.008 9.006 9.028 9.020 8.999 9.034 9.029 9.061 9.061 9.097 9.041 9.008 9.055 07 9.654 9.670 9.658 9.662 9.663 9.662 9.675 9.678 9.666 9.693 9.686 9.690 9.671 9.663 9.667 08 11.869 11.865 11.856 11.854 11.859 11.852 11.849 11.867 11.859 11.896 11.884 11.880 11.858 11.854 11.863 09 17.963 17.972 17.965 17.969 17.959 17.956 17.955 17.951 17.960 18.023 17.992 17.989 17.974 17.951 17.961 10 18.951 18.955 18.943 18.945 18.955 18.937 18.951 18.956 18.948 19.008 18.992 18.983 18.958 18.944 18.955 11 20.808 20.829 20.811 20.806 20.817 20.828 20.794 20.829 20.807 20.878 20.845 20.870 20.823 20.796 20.824 12 26.561 26.764 26.563 26.615 26.777 26.742 26.600 26.676 26.869 26.726 26.784 27.052 26.940 26.632 26.937 13 32.289 32.366 32.276 32.250 32.352 32.315 32.246 32.324 32.410 32.404 32.398 32.631 32.440 32.310 32.517 14 34.102 34.195 34.937 33.996 34.263 34.266 34.009 34.159 34.423 34.254 34.281 34.669 34.485 34.196 34.643
[0052] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific embodiments described. Obviously, many modifications and variations can be made according to the content of this specification. These embodiments are selected and specifically described in this specification to better explain the principle and practical application of the present invention, so that those skilled in the art can well understand and utilize the present invention. The present invention is only limited by the claims and their full scope and equivalents.
Claims
1. A method for constructing a fingerprint of volatile components in celery, characterized in that: The method comprises the following steps: 1) Preparation of reference solution Accurately aspirate cuminaldehyde reference substance, dissolve it in n-hexane, and prepare cuminaldehyde reference substance solution; 2) Preparation of test solution After the celery is collected, it is crushed and sieved, and an appropriate amount of celery powder is weighed, added to distilled water, soaked overnight, distilled, the effective components are extracted, evaporated to dryness, dissolved, fixed to volume, filtered, and a sample of the test solution is obtained; 3) Gas chromatography analysis Collect multiple batches of celery medicinal materials from different origins, prepare test sample solutions according to step 2), inject samples respectively, perform chromatographic analysis, and obtain gas chromatograms; 4) Calibration of common peaks in the gas phase fingerprint of volatile components of celery The gas chromatogram obtained in step 4) was analyzed by gas chromatography of the above-mentioned celery test sample solutions from different origins to obtain a gas chromatogram; the results showed that the chromatographic peaks of most components in each sample appeared between the retention time of 3min and 35min, and the chromatographic peaks were well separated, of which 14 chromatographic peaks were characteristic peaks common to all the test samples, and species No. 12 was cuminaldehyde, which was used as a reference peak; 5) Gas phase fingerprint analysis results of volatile components of celery Through gas chromatography analysis of the celery samples from different origins mentioned above, the fingerprint characteristics of the volatile components of celery are as follows: peaks 1-14 appear in the chromatograms of the above samples, among which peak 12 has the same retention time as the cuminaldehyde in the chromatogram of the cuminaldehyde reference; the relative retention time of each common fingerprint peak is calculated with the retention time of the cuminaldehyde reference as a reference, and the RSD of the relative retention time is less than 1.0%. In all samples, the ratio of the sum of the peak areas of the common fingerprint peaks to the total peak area is greater than 91%.
2. The method according to claim 1, characterized in that In step 1), the cuminaldehyde reference substance is first placed in a 5 mL volumetric flask, n-hexane is added to the mark, and shaken to prepare a cuminaldehyde stock solution. The cuminaldehyde stock solution is then accurately pipetted and placed in a 5 mL volumetric flask, n-hexane is added to the mark, and shaken to prepare a reference substance solution with a mass concentration of 1.00 mg / mL.
3. The method according to claim 1, characterized in that In step 2), after collecting the celery, it is crushed to make celery powder, which is sealed and stored. The celery powder is accurately weighed and placed in a distillation bottle. 0.5 L of water is added, and the powder is soaked for 10 hours, distilled, and then extracted.
4. The method according to claim 1, characterized in that In step 2), anhydrous sodium sulfate is used for dehydration, and n-hexane is used for volatile dissolution and constant volume.
5. The method according to claim 1, characterized in that: In step 3), the gas chromatography conditions are as follows: HP-5 capillary column 0.25μm×0.32mm×30m; injection port temperature 300℃; FID detector temperature 300℃; carrier gas constant flow rate; high-purity nitrogen: 0.5mL / min; fuel gas hydrogen: 30mL / min; auxiliary gas air: 250mL / min; tail gas flow rate: 20mL / min; The split ratio was 100:1; the injection volume was 1.0 μL; Heating program: initial temperature is 50℃, hold for 3 minutes, increase to 100℃ at 0.6℃ / min, hold for 5 minutes, and increase to 250℃ at 50℃ / min.
6. The method according to claim 1, characterized in that In step 3), 15 batches of celery medicinal materials from different origins are used.
7. The method according to claim 1, characterized in that In step 4), chromatographic peaks 1 to 14 are respectively chromatographic peaks with retention times in the chromatogram: 3.603 min, 3.746 min, 5.007 min, 5.727 min, 7.104 min, 9.028 min, 9.663 min, 11.859 min, 17.959 min, 18.955 min, 20.817 min, 26.777 min, 32.352 min, and 34.263 min.
8. A method for identifying the herbal material of Celery and its powder, characterized in that: The method adopts the gas chromatography-fingerprint established by the method described in any one of claims 1 to 7.
9. A method for analyzing the content difference of the herbal material of Celery and its powder, characterized in that: The method adopts the gas chromatography-fingerprint established by the method described in any one of claims 1 to 7.