A method for specific detection of medicinal stems of dendrobium candidum from sichuan origin
The fingerprint spectrum of Dendrobium nobile was constructed by high performance liquid chromatography, which solved the problem of difficulty in identifying Dendrobium nobile from Sichuan in the existing technology, and realized the specific identification and quality control of Dendrobium nobile from Sichuan.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CHENGDU INSTITUTE OF BIOLOGY CHINESE ACADEMY OF SCIENCES
- Filing Date
- 2023-10-16
- Publication Date
- 2026-06-23
AI Technical Summary
Existing technologies are insufficient for the targeted identification of Dendrobium nobile from specific production areas, harvesting seasons, and varieties. Furthermore, existing fingerprinting methods cannot effectively distinguish Dendrobium nobile from Sichuan from other regions or varieties, leading to problems of mixed seed sources and the substitution of inferior products in the Dendrobium nobile medicinal herb industry.
A high-performance liquid chromatography (HPLC) method was used to construct a fingerprint spectrum of Dendrobium nobile. The test solution was prepared by mixing Dendrobium nobile powder with methanol and sonicating it. The solution was then detected on a C18 reversed-phase column. A specific mobile phase and elution program were set, the fingerprint spectrum was recorded, 15 characteristic peaks were identified, and an HPLC standard fingerprint spectrum of Dendrobium nobile from Sichuan was established.
This method enables the specific identification of Dendrobium nobile from Sichuan, distinguishing Dendrobium nobile from different production areas and harvesting seasons. It provides a simple, characteristic, precise, and reproducible detection method.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of fingerprinting, specifically relating to a method for targeted detection of medicinal stems of Dendrobium nobile from Sichuan. Background Technology
[0002] Dendrobium nobile Lindl. is a perennial epiphytic herb with extensive and outstanding medicinal value. Clinically, it is widely used to treat chronic pharyngitis, gastrointestinal diseases, ophthalmic diseases, thrombotic obliterative diseases, diabetes, arthritis, and cancer. Dendrobium nobile is mainly distributed in Chishui and Xishui in Guizhou Province, and Luzhou in Sichuan Province, with the Dendrobium nobile from Hejiang County, Luzhou, Sichuan Province, being particularly prized and protected as a national geographical indication product.
[0003] However, in recent years, the Dendrobium medicinal herb industry has been plagued by mixed seed sources and the substitution of inferior products for superior ones, making it difficult to specifically identify Dendrobium from specific production areas, harvesting seasons, and varieties.
[0004] The fingerprint spectrum of Chinese medicinal materials refers to the spectrum obtained by using specific analytical methods and instruments after appropriate processing of Chinese medicinal materials, which can indicate the characteristics of the medicinal materials. Currently, there is no research specifically on the stems of *Dendrobium nobile* from the traditional producing area of Sichuan. While some existing studies have established fingerprint spectra for *Dendrobium* from other regions or varieties, they generally suffer from the following shortcomings: 1) Insufficient common peaks in the chromatograms, making it impossible to establish a characteristic fingerprint spectrum for *Dendrobium nobile*, and it is easily confused with *Dendrobium officinale*, *Dendrobium chrysanthum*, etc.; 2) Differences between *Dendrobium nobile* from the traditional producing area of Sichuan and other producing areas; 3) Inability to distinguish *Dendrobium nobile* harvested during the optimal harvesting season from those harvested in other seasons. Summary of the Invention
[0005] The purpose of this invention is to provide a method for targeted detection of medicinal stems of Dendrobium nobile from Sichuan.
[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is: a method for constructing a Dendrobium fingerprint spectrum, comprising the following steps:
[0007] (1) Mix the Dendrobium powder to be tested with methanol, sonicate, remove, cool and filter; evaporate the filtrate under reduced pressure, dissolve the residue in methanol, filter, shake well to obtain the test solution;
[0008] (2) Take the test solution, perform high performance liquid chromatography detection, and record the fingerprint spectrum;
[0009] The chromatographic conditions for the chromatographic detection were as follows: the chromatographic column was a C18 reversed-phase column; the column temperature was 30℃; the detection wavelength was 270nm; and the total flow rate was 0.8mL / min.
[0010] Preferably, in step (1), after the first filtration and before the filtrate is evaporated under reduced pressure, the dregs and filter paper obtained from the first filtration are cut into small pieces and placed in an Erlenmeyer flask, mixed with 80% methanol, ultrasonically treated, taken out, cooled, filtered, and the two filtrates are combined and then evaporated under reduced pressure.
[0011] Preferably, in the chromatographic conditions of step (2), mobile phase A is acetonitrile and mobile phase B is 0.2% formic acid solution.
[0012] Preferably, in the chromatographic conditions of step (2), the sample loading volume of the test solution is 10 μL.
[0013] Preferably, in step (2), the elution program is as follows: 0-10 min, mobile phase A accounts for 5%-10% of the total mobile phase volume, with the remainder being mobile phase B; 10-35 min, mobile phase A accounts for 10%-20% of the total mobile phase volume, with the remainder being mobile phase B; 35-55 min, mobile phase A accounts for 20%-28% of the total mobile phase volume, with the remainder being mobile phase B; 55-60 min, mobile phase A accounts for 28%-80% of the total mobile phase volume, with the remainder being mobile phase B; 60-75 min, mobile phase A accounts for 80%-85% of the total mobile phase volume, with the remainder being mobile phase B.
[0014] Accordingly, the method is applied to the identification of Dendrobium nobile harvested in winter from Sichuan.
[0015] Accordingly, the standard fingerprint spectrum obtained using the method contains 15 characteristic peaks.
[0016] Accordingly, the application of the standard fingerprint spectrum in identifying Dendrobium nobile harvested in winter from Sichuan involves obtaining a fingerprint spectrum of the Dendrobium nobile to be tested using the method described above, comparing it with the standard fingerprint spectrum, and identifying the Dendrobium nobile with the fingerprint spectrum containing all 15 characteristic peaks of the standard fingerprint spectrum as Dendrobium nobile harvested in winter from Sichuan.
[0017] This invention offers the following advantages: It constructs a novel HPLC fingerprint chromatogram for Dendrobium nobile, identifying 15 key characteristic peaks as common peaks, and establishing for the first time a standard HPLC fingerprint chromatogram for authentic Dendrobium nobile from Sichuan. This chromatogram can separate most chemical components from authentic Dendrobium nobile from Sichuan, exhibiting strong characteristic properties and comprehensively reflecting the chemical composition information of authentic Dendrobium nobile from Sichuan. It can also highly specifically identify the authenticity of authentic Dendrobium nobile from Sichuan. The method provided by this invention is simple, retains characteristic components completely, and provides a stable test solution; it also boasts high precision and good reproducibility. Attached Figure Description
[0018] Figure 1Standard fingerprint spectrum for the medicinal stems of Dendrobium nobile from the traditional producing areas of Sichuan harvested in winter;
[0019] Figure 2 Chromatograms of Dendrobium nobile extracted using different extraction methods;
[0020] Figure 3 HPLC fingerprints under different sample loading conditions;
[0021] Figure 4 To detect HPLC fingerprints at a wavelength of 250 nm;
[0022] Figure 5 To detect HPLC fingerprints at a wavelength of 270 nm;
[0023] Figure 6 To detect HPLC fingerprints at a wavelength of 330 nm;
[0024] Figure 7 The HPLC fingerprint is obtained under methanol-water mobile phase conditions.
[0025] Figure 8 The HPLC fingerprint is obtained under the condition of using acetonitrile-water as the mobile phase.
[0026] Figure 9 The HPLC fingerprint is obtained under the condition of using acetonitrile-0.2% formic acid as the mobile phase.
[0027] Figure 10 The HPLC fingerprint is obtained under the condition of using acetonitrile-0.2% glacial acetic acid as the mobile phase.
[0028] Figure 11 The HPLC fingerprint under elution procedure 1 conditions;
[0029] Figure 12 The HPLC fingerprint under elution procedure 2 conditions;
[0030] Figure 13 The HPLC fingerprint under elution procedure 3 conditions;
[0031] Figure 14 HPLC fingerprints of the medicinal stems of Dendrobium nobile from 10 batches of Sichuan-produced authentic producing areas;
[0032] Figure 15 The original HPLC fingerprint of Dendrobium nobile stems from Hejiang County, Luzhou City, Sichuan Province, a traditional producing area of Sichuan Province, under the conditions of the method of the present invention, during the optimal harvesting period in winter.
[0033] Figure 16The original HPLC fingerprint of Dendrobium nobile stems from Hejiang County, Luzhou City, Sichuan Province, a traditional producing area of Sichuan Province, under the conditions of the method of the present invention;
[0034] Figure 17 The original HPLC fingerprint of Dendrobium nobile stems from Lizhou District, Guangyuan City, Sichuan Province, the introduced production area, under the conditions of the method of this invention;
[0035] Figure 18 The original HPLC fingerprint of Dendrobium nobile stems from Fumin County, Kunming City, Yunnan Province (a non-traditional producing area) under the conditions of the method of the present invention;
[0036] Figure 19 This is the original HPLC fingerprint of Dendrobium nobile stems from Hejiang County, Luzhou City, Sichuan Province, a traditional producing area of Sichuan Province, harvested during the summer at a non-optimal time, under the conditions described in this invention. Detailed Implementation
[0037] I. This invention provides a method for targeted detection of medicinal stems of Dendrobium nobile harvested in winter from Sichuan, specifically including the following steps:
[0038] 1. Preparation of the test solution. Mix Dendrobium nobile powder with methanol, sonicate, remove, cool, and filter. Cut the residue and filter paper into small pieces and place them in an Erlenmeyer flask, mix with 80% methanol, sonicate, remove, cool, and filter. Combine the two filtrates, evaporate to dryness under reduced pressure, dissolve the residue in methanol, filter through a 0.45 μm microporous membrane, and shake well to obtain the test solution.
[0039] 2. HPLC detection. Pipette the test solution and inject it into a high-performance liquid chromatograph for detection, recording the fingerprint chromatogram.
[0040] The chromatographic conditions were as follows: C18 reversed-phase column; column temperature 30℃; detection wavelength 270nm; mobile phase A was acetonitrile, mobile phase B was 0.2% formic acid solution; and total flow rate was 0.8mL / min.
[0041] The preferred elution program is as follows: 0–10 min, mobile phase A accounts for 5%–10% of the total mobile phase volume, with the remainder being mobile phase B; 10–35 min, mobile phase A accounts for 10%–20% of the total mobile phase volume, with the remainder being mobile phase B; 35–55 min, mobile phase A accounts for 20%–28% of the total mobile phase volume, with the remainder being mobile phase B; 55–60 min, mobile phase A accounts for 28%–80% of the total mobile phase volume, with the remainder being mobile phase B; 60–75 min, mobile phase A accounts for 80%–85% of the total mobile phase volume, with the remainder being mobile phase B.
[0042] 3. Record the chromatogram obtained in step 2 and import it into the National Pharmacopoeia Commission's "Similarity Evaluation System for Chromatographic Characteristic Chromatographic Profiles of Traditional Chinese Medicine (2012 Edition)". Select stems from multiple batches of Sichuan-produced Dendrobium nobile medicinal materials, and obtain fingerprint profiles according to steps 1 and 2. Determine the standard fingerprint profile, and calibrate the chromatographic peaks with larger peak areas and better resolution. The standard fingerprint profile is determined to have 15 characteristic peaks. The standard fingerprint profile is as follows: Figure 1 As shown, the retention times (average of multiple batches) of the 15 characteristic peaks are as follows: 6.882 min for peak 1, 8.284 min for peak 2, 10.674 min for peak 3, 11.67 min for peak 4, 18.69 min for peak 5, 28.94 min for peak 6, 33.726 min for peak 7, 37.842 min for peak 8, 47.593 min for peak 9, 48.965 min for peak 10, 53.437 min for peak 11, 54.201 min for peak 12, 61.312 min for peak 13, 68.968 min for peak 14, and 71.671 min for peak 15.
[0043] 4. Grind the Dendrobium to be tested into powder, perform the tests according to steps 1 and 2, and draw a chromatogram. Compare the drawn chromatogram with the standard fingerprint chromatogram prepared in step 3. If both have 15 characteristic peaks, it is Dendrobium nobile from Sichuan and harvested in winter (experiments have shown that the stems of Dendrobium nobile harvested in winter have the best quality).
[0044] Second, this invention also provides a standard fingerprint spectrum that can specifically identify the harvesting time and source of Dendrobium. The standard fingerprint spectrum is as follows: Figure 1 As shown, the sample obtained by following steps 1 to 3 above has 15 characteristic peaks.
[0045] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. All obtained data are average values obtained after at least three repetitions, and each repetition yields valid data.
[0046] Example 1: The effect of different extraction methods on the results of the test solution
[0047] 1. Select Dendrobium nobile from the same batch harvested in December 2020 from Hejiang County, Luzhou City, Sichuan Province, and prepare test solutions according to the following methods:
[0048] Method (1): Weigh 0.4g of Dendrobium nobile powder, add 20mL of methanol, sonicate for 60min, remove, cool, and filter. Cut the residue and filter paper into small pieces and place them in an Erlenmeyer flask, add 20mL of 80% methanol, sonicate for 60min, remove, cool, and filter. Combine the two filtrates, evaporate to dryness under reduced pressure, dissolve the residue in an appropriate amount of methanol, place in a 2mL volumetric flask, add methanol to the mark, filter through a microporous membrane (0.45μm), shake well, and obtain test solution A.
[0049] Method (2): Weigh 0.4 g of Dendrobium nobile powder, place it in a stoppered conical flask, add 50 mL of acetonitrile, sonicate (power 250 W, frequency 40 kHz) for 2.5 h, cool to room temperature, centrifuge, and concentrate the supernatant under reduced pressure to dryness. Dissolve the residue in 20 mL of water, extract three times with 20 mL of ether each time. Combine the ether layers, evaporate to dryness using a rotary evaporator, dissolve the residue in 90% acetonitrile, make up to 1.5 mL, filter through a 0.45 μm microporous membrane to obtain test solution B.
[0050] Method (3): Weigh 1g of Dendrobium nobile powder into a ground glass flask, moisten with an appropriate amount of 10% ammonia, add 50mL of methanol, seal and let stand for 30min, reflux in a water bath at 85℃ for 4h, filter while hot, evaporate the filtrate to dryness at 65℃, dissolve with 5mL of methanol and make up to volume, filter with a 0.45μm microporous membrane to obtain test solution C.
[0051] 2. Chromatographic conditions were set as follows: Agilent ZORBAX SB-C18 column (250×4.6mm, 5μm); mobile phase: acetonitrile (A)-0.2% formic acid (B); gradient elution (0–10 min, 5%–10% A; 10–35 min, 10%–20% A; 35–55 min, 20%–28% A; 55–60 min, 28%–80% A; 60–75 min, 80%–85% A). Detection wavelength: 270 nm; flow rate: 0.6 mL / min; column temperature: 30℃; injection volume: 10 μL.
[0052] 3. Conduct a methodological review.
[0053] 3.1 Precision test: Accurately weigh the same sample powder, prepare the test solution according to step 1 (1), and detect it according to the chromatographic conditions in step 2. Repeat the injection 6 times, with 10 μL injected each time, and record the chromatogram. Using peak 7 as the reference peak, its retention time and peak area are 1. Calculate that the relative retention time of the common chromatographic peak is ≤1.13%, and the RSD value of the relative peak area is ≤2.19%, indicating that the instrument precision is good.
[0054] 3.2 Stability test: The same sample powder was accurately weighed and the test solution was prepared according to the method (1) in step 1. The chromatographic conditions in step 2 were used for detection. The sample was injected at 0h, 2h, 4h, 8h, 12h and 24h respectively, and the chromatograms were recorded. The chromatographic peak with better peak shape and larger peak area was used as the internal reference peak. The RSD of the relative retention time of the common chromatographic peak was calculated to be ≤1.29%, and the RSD of the relative peak area was ≤2.53%, indicating that the method has good stability within 24h.
[0055] 3.3 Repeatability test: Six portions of the same sample powder were accurately weighed and tested according to the chromatographic conditions in step 1 (1). Chromatographic peaks with better peak shape and larger peak area were used as internal reference peaks. The RSD of the relative retention time of the common chromatographic peaks was calculated to be ≤1.27 and the RSD of the relative peak area was ≤1.99%, indicating that the method has good repeatability.
[0056] 4. Fingerprint chromatogram determination: 10 μL of each group of test solutions from step 1 is injected into the high-performance liquid chromatograph (HPLC), and the determination is performed under the chromatographic conditions of step 2. The chromatograms are recorded. The chromatograms of methods (1) to (3) are shown below. Figure 2 As shown in S1 to S3. Figure 2 The baseline of the chromatogram obtained by using the test solution obtained by method (1) is stable, and there are many chromatographic peaks with high peak height and large peak area. Therefore, method (1) is selected for subsequent experiments.
[0057] Example 2: The effect of different chromatographic detection conditions on the results
[0058] 1. Effect of sample loading volume. The test solution was obtained according to step 1 of Example 1, method (1), and the chromatographic conditions were set according to step 2 of Example 1, wherein the sample loading volume was set to 2 μL and 10 μL, respectively, with other conditions remaining unchanged. The chromatograms obtained at 2 μL and 10 μL are shown below. Figure 3 As shown in S1 and S2.
[0059] The results showed that using a 10 μL sample loading volume resulted in more chromatographic peaks in the chromatogram, which was the preferred condition.
[0060] 2. Effect of HPLC Detection Wavelength. The test solution was obtained according to step 1 (1) of Example 1, and the chromatographic conditions were set according to step 2 of Example 1, wherein the detection wavelengths were set to 220 nm, 270 nm, and 330 nm, respectively, while other conditions remained unchanged. The chromatograms obtained at 220 nm, 270 nm, and 330 nm are shown below. Figure 4 , 5 As shown in Figure 6.
[0061] The results showed that more chromatographic peaks appeared at 270 nm, the main chromatographic peaks could be separated and the peak shapes were better, which is a more preferred condition.
[0062] 3. Effect of elution mobile phase. The test solution was obtained according to step 1 of Example 1, method (1), and the chromatographic conditions were set according to step 2 of Example 1, wherein the mobile phases were set as follows: methanol (A)-water (B), acetonitrile (A)-water (B), acetonitrile (A)-0.2% formic acid (B), and acetonitrile (A)-0.2% glacial acetic acid (B); other conditions remained unchanged; the chromatograms obtained for each group are shown below. Figures 7-10 As shown.
[0063] The results showed that using acetonitrile (A)-0.2% formic acid (B) as the mobile phase resulted in a more stable baseline and better resolution in the chromatograms, making it a more preferred condition.
[0064] 4. Effect of elution program. The test solution was obtained according to step 1 of Example 1, and the chromatographic conditions were set according to step 2 of Example 1. The elution program was adjusted as follows (repeated twice):
[0065] Elution program 1: 0–5 min, 0%–5% A; 5–10 min, 5%–10% A; 10–20 min, 10%–60% A; 20–30 min, 60%–80% A; 30–40 min, 80%–100% A. The resulting fingerprint chromatogram is as follows: Figure 11 As shown.
[0066] Elution program 2: 0–5 min, 0%–1% A; 5–15 min, 1%–10% A; 15–55 min, 10%–30% A; 55–65 min, 30%–35% A; 65–75 min, 35%–60% A; 75–85 min, 60%–80% A. The resulting fingerprint chromatogram is as follows: Figure 12 As shown.
[0067] Elution program 3: 0–10 min, 5%–10% A; 10–35 min, 10%–20% A; 35–55 min, 20%–28% A; 55–60 min, 28%–80% A; 60–75 min, 80%–85% A. The resulting fingerprint chromatogram is as follows: Figure 13 As shown.
[0068] In the above elution procedures, 0%-5%A means that mobile phase A accounts for 0%-5% of the total mobile phase volume, and the remainder is mobile phase B, and so on.
[0069] The results showed that the chromatogram obtained by elution program 3 had a stable baseline, a shorter total detection time, was able to separate the main chromatographic peaks with better peak shapes, and had high detection efficiency, making it a better condition.
[0070] Example 3: Repeatability demonstration of optimized fingerprint pattern construction conditions and construction of standard fingerprint patterns
[0071] Ten batches of Dendrobium nobile were selected, and test solutions were obtained according to step 1 of Example 1. Chromatographic conditions were set according to step 2 of Example 1 to obtain fingerprint spectra. The sources of each batch of Dendrobium nobile are shown in Table 1.
[0072] Table 1. Sources of Dendrobium nobile medicinal materials
[0073] serial number Origin Harvesting time Drying method S1 Hejiang County, Luzhou City, Sichuan Province 2020.12 drying S2 Hejiang County, Luzhou City, Sichuan Province 2020.12 drying S3 Hejiang County, Luzhou City, Sichuan Province 2020.12 drying S4 Hejiang County, Luzhou City, Sichuan Province 2020.12 drying S5 Hejiang County, Luzhou City, Sichuan Province 2020.12 drying S6 Hejiang County, Luzhou City, Sichuan Province 2021.12 drying S7 Hejiang County, Luzhou City, Sichuan Province 2021.12 drying S8 Hejiang County, Luzhou City, Sichuan Province 2021.12 drying S9 Hejiang County, Luzhou City, Sichuan Province 2021.12 drying S10 Hejiang County, Luzhou City, Sichuan Province 2021.12 drying
[0074] The chromatograms were imported into the National Pharmacopoeia Commission's "Similarity Evaluation System for Chromatographic Characteristic Chromatograms of Traditional Chinese Medicine (2012 Edition)". The original HPLC fingerprints of the medicinal stems of 10 batches of Dendrobium nobile from Sichuan's traditional producing areas are shown below. Figure 14 As shown in Table 2, the similarity of the fingerprint spectra of each batch reached 0.96 or higher, proving that the overall chemical composition characteristics of the authentic Dendrobium nobile from Sichuan are similar, and the fingerprint spectra construction method provided by this invention has good repeatability.
[0075] Table 2. Similarity evaluation results of fingerprint spectra of 10 batches of Sichuan-produced Dendrobium nobile.
[0076] serial number Similarity serial number Similarity S1 0.994 S6 0.976 S2 0.998 S7 0.967 S3 0.990 S8 0.975 S4 0.999 S9 0.992 S5 0.997 S10 0.991
[0077] Based on the fingerprint profiles of the stems of 10 batches of Sichuan-produced Dendrobium nobile medicinal materials, a standard fingerprint profile was developed, such as... Figure 1 As shown, chromatographic peaks with larger peak areas and better resolution were calibrated, and 15 characteristic peaks were identified in the standard fingerprint spectrum. The retention times (mean values) of the 15 characteristic peaks of the 10 batches of Dendrobium nobile authentic medicinal material test solutions were as follows: No. 1 (6.882 min), No. 2 (8.284 min), No. 3 (10.674 min), No. 4 (11.67 min), No. 5 (18.69 min), No. 6 (28.94 min), No. 7 (33.726 min), No. 8 (37.842 min), No. 9 (47.593 min), No. 10 (48.965 min), No. 11 (53.437 min), No. 12 (54.201 min), No. 13 (61.312 min), No. 14 (68.968 min), and No. 15 (71.671 min).
[0078] Example 4: Demonstration of the specificity of the preferred fingerprint pattern construction conditions
[0079] Different sources / species of Dendrobium were selected, and test solutions were obtained according to step 1 (1) of Example 1. Chromatographic conditions were set according to step 2 of Example 1 to obtain fingerprint spectra. The sources of each batch of Dendrobium and their corresponding fingerprint spectra are shown in Table 3.
[0080] Table 3. Types and sources of Dendrobium
[0081] Group Origin Harvesting time type HPLC chromatogram Group 1 Hejiang County, Luzhou City, Sichuan Province 2020.12 Dendrobium nobile Figure 15 Group 2 Hejiang County, Luzhou City, Sichuan Province 2020.12 Dendrobium chrysanthum Figure 16 Group 3 Lizhou District, Guangyuan City, Sichuan Province 2020.12 Dendrobium nobile Figure 17 Group 4 Fumin County, Kunming City, Yunnan Province 2020.12 Dendrobium nobile Figure 18 Group 5 Hejiang County, Luzhou City, Sichuan Province 2021.06 Dendrobium nobile Figure 19
[0082] The similarity between the fingerprint spectrum and the standard fingerprint spectrum was calculated, and the results are shown in Table 4.
[0083] Table 4. Similarity evaluation results of fingerprint spectra of Dendrobium from different sources
[0084] Group Similarity Group 1 1 Group 2 0.59 Group 3 0.15 Group 4 0.28 Group 5 0.58
[0085] The results showed that the fingerprint spectra obtained by using the method of this invention to detect Dendrobium from other sources, species, and harvesting seasons all had a similarity of less than 60% with Dendrobium nobile samples from Hejiang County, Luzhou City, Sichuan Province, the authentic producing area of Sichuan. This proves that the fingerprint spectra constructed by the method of this invention can specifically identify the medicinal stems of Dendrobium nobile from authentic Sichuan producing areas harvested in winter.
[0086] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.
Claims
1. A method for constructing fingerprint profiles of Dendrobium nobile harvested in winter in Hejiang County, Luzhou City, Sichuan Province, characterized in that: Includes the following steps: (1) Mix the Dendrobium powder to be tested with methanol, sonicate, remove, cool and filter; evaporate the filtrate under reduced pressure, dissolve the residue in methanol, filter, shake well to obtain the test solution; (2) Take the test solution and perform high performance liquid chromatography detection, and record the fingerprint spectrum; The chromatographic conditions for the chromatographic detection are as follows: the chromatographic column is a C18 reversed-phase column; the column temperature is 30℃; the detection wavelength is 270nm; the total flow rate is 0.8mL / min; in the chromatographic conditions of step (2), the mobile phase A is acetonitrile, and the mobile phase B is 0.2% formic acid solution; the elution program is as follows: 0-10min, mobile phase A accounts for 5%-10% of the total mobile phase volume, and the remainder is mobile phase B; 10-35min, mobile phase A accounts for 10%-20% of the total mobile phase volume, and the remainder is mobile phase B; 35-55min, mobile phase A accounts for 20%-28% of the total mobile phase volume, and the remainder is mobile phase B; 55-60min, mobile phase A accounts for 28%-80% of the total mobile phase volume, and the remainder is mobile phase B; 60-75min, mobile phase A accounts for 80%-85% of the total mobile phase volume, and the remainder is mobile phase B.
2. The method according to claim 1, characterized in that: In step (1), after the first filtration and before the filtrate is evaporated under reduced pressure, the dregs and filter paper obtained from the first filtration are cut into small pieces and placed in an Erlenmeyer flask, mixed with 80% methanol, ultrasonically treated, taken out, cooled, filtered, and the two filtrates are combined and then evaporated under reduced pressure.
3. The method according to claim 1, characterized in that: In the chromatographic conditions of step (2), the sample loading volume of the test solution is 10 μL.
4. The application of the method according to any one of claims 1 to 3 in the identification of Dendrobium nobile harvested in winter from Hejiang County, Luzhou City, Sichuan Province.
5. A method for constructing a standard fingerprint spectrum of Dendrobium nobile harvested in winter from Hejiang County, Luzhou City, Sichuan Province using the method described in any one of claims 1 to 3, characterized in that: The standard fingerprint spectrum constructed using the method described above contains 15 characteristic peaks.