Method for constructing fingerprint spectra of Dendrobium nobile medicinal materials, processed slices, standard decoctions, and formulated granules

Abstract

This invention discloses a method for constructing fingerprint spectra of Dendrobium nobile medicinal materials, its processed slices, standard decoctions, and formulated granules. The method includes: extracting Dendrobium nobile medicinal materials, its processed slices, standard decoctions, and formulated granules with 30 vol%–100 vol% methanol or 30 vol%–100 vol% ethanol to obtain a test solution; dissolving dendrobin G, costusside D, costusside C, and costusside A reference standards in 30 vol%–100 vol% methanol to obtain a reference solution; and determining the test solution and reference solution using liquid chromatography to obtain the fingerprint spectra of Dendrobium nobile medicinal materials, its processed slices, standard decoctions, and formulated granules. The fingerprint spectra constructed by this invention are highly specific, can effectively reflect the active ingredients of Dendrobium nobile, and can effectively identify common adulterants, providing a scientific basis for the formulation of quality standards for Dendrobium nobile.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine identification technology, and in particular to a method for constructing fingerprint spectra of Dendrobium nobile medicinal material and its processed slices, standard decoctions, and formulated granules. Background Technology

[0002] According to the 2020 edition of the Chinese Pharmacopoeia, Dendrobium nobile Lindl., a plant in the Orchidaceae family, is a traditional tonic with effects primarily including nourishing yin and clearing heat, benefiting the stomach and promoting the production of body fluids. Modern research shows that Dendrobium nobile contains alkaloids, sesquiterpenes, phenanthrene, and flavonoids, with alkaloids and sesquiterpenes being the main active ingredients. Its alkaloids mainly exhibit antitumor, hypoglycemic, and antiviral effects, while sesquiterpenes have significant antitumor effects. Due to its good hypoglycemic, hypolipidemic, antioxidant, nerve cell protective, and antitumor activities, Dendrobium nobile is often used clinically to treat diabetes, hyperlipidemia, and malignant tumors.

[0003] Dendrobium nobile, belonging to the genus Dendrobium in the Orchidaceae family, comprises 74 species and 2 varieties, of which approximately 51 species have been identified as medicinally usable. The 2020 edition of the Chinese Pharmacopoeia includes Dendrobium nobile, Dendrobium huoshanense, Dendrobium chrysanthum, and Dendrobium fimbriatum, among others. Different Dendrobium species from different origins have varying medicinal efficacies. To ensure safe use, it is necessary to develop effective fingerprinting methods to distinguish common adulterants and easily confused varieties of Dendrobium nobile, but current research in this area is limited. The 2020 edition of the Chinese Pharmacopoeia only specifies the quality control of Dendrobium nobile for its dendrobine. Chen Zhihui et al. conducted research on Dendrobium nobile based on fingerprinting technology. In their study, they mainly used ultraviolet light detection. ESI-MS was used to identify common peaks as flavonoids. However, the active ingredients of Dendrobium nobile are alkaloids and sesquiterpenes. These components have weak ultraviolet absorption and poor visibility under ultraviolet light. Therefore, the current method cannot effectively evaluate the quality of Dendrobium nobile. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for constructing fingerprint spectra of Dendrobium nobile medicinal material and its processed slices, standard decoctions and formula granules, which is stable and highly specific and can be used for the identification of common counterfeit products.

[0005] To address the aforementioned technical problems, this invention provides a method for constructing fingerprint spectra of Dendrobium nobile medicinal materials and their processed slices, standard decoctions, and formulated granules, comprising:

[0006] The medicinal material of Dendrobium nobile or its processed slices, standard decoctions, and formula granules are extracted using an extraction solvent to obtain a test solution. The extraction solvent is 30 vol% to 100 vol% methanol or 30 vol% to 100 vol% ethanol, and exemplary solvents are 34 vol% methanol, 42 vol% methanol, 50 vol% methanol, 58 vol% methanol, 66 vol% methanol, 74 vol% methanol, 82 vol% methanol, 90 vol% methanol, 95 vol% methanol, 35 vol% ethanol, 46 vol% ethanol, 57 vol% ethanol, 68 vol% ethanol, 79 vol% ethanol, or 90 vol% ethanol, but not limited to these.

[0007] Dendrobinin G, costusin D, costusin C, and costusin A reference standards are dissolved in 30 vol% to 100 vol% methanol to obtain a reference solution. Exemplarily, the concentration of methanol (methanol-water solution) is 35 vol%, 40 vol%, 45 vol%, 50 vol%, 55 vol%, 60 vol%, 70 vol%, 75 vol%, 80 vol%, or 90 vol%, but is not limited thereto; preferably 50 vol% to 80 vol%, more preferably 70 vol%.

[0008] The test solution and reference solution were analyzed by liquid chromatography to obtain the fingerprint spectrum of Dendrobium nobile or its processed slices, standard decoctions, and formula granules.

[0009] The liquid chromatograph uses an octadecylsilane-bonded silica gel column as the stationary phase, a mixed solution of methanol and acetonitrile as mobile phase A, and a 0.05 vol%–0.2 vol% formic acid aqueous solution as mobile phase B for gradient elution. The gradient elution curve is as follows:

[0010] From 0 min to 20 min, mobile phase A decreased from 16% to 17%, and mobile phase B decreased from 84% to 83%.

[0011] Between 20 and 25 minutes, mobile phase A decreased from 17% to 25%, and mobile phase B decreased from 83% to 75%.

[0012] 25 min to 30 min, mobile phase A is 25%, mobile phase B is 75%;

[0013] Over 30 to 40 minutes, mobile phase A decreased from 25% to 30%, and mobile phase B decreased from 75% to 70%.

[0014] Over 40-50 minutes, mobile phase A decreased from 30% to 35%, and mobile phase B decreased from 70% to 65%.

[0015] Between 50 and 55 minutes, mobile phase A decreased from 35% to 36%, and mobile phase B decreased from 65% to 64%.

[0016] Between 55 and 60 minutes, mobile phase A decreased from 36% to 40%, and mobile phase B decreased from 64% to 60%.

[0017] Over 60-65 minutes, mobile phase A decreased from 40% to 45%, and mobile phase B decreased from 60% to 55%.

[0018] The volume ratio of methanol to acetonitrile is 1:1 to 3:1, with exemplary ratios of 1:1, 1.4:1, 1.8:1, 2:1, 2.3:1, 2.6:1, or 3:1, but not limited thereto. The concentration of the formic acid aqueous solution in mobile phase B is exemplary, but not limited thereto, at 0.07 vol%, 0.09 vol%, 0.11 vol%, 0.13 vol%, 0.15 vol%, or 0.17 vol%.

[0019] As an improvement to the above technical solution, the volume ratio of methanol to acetonitrile in the mobile phase A is 3:1; and the mobile phase B is a 0.1 vol% aqueous solution of formic acid.

[0020] As an improvement to the above technical solution, the column length of the chromatographic column is 100mm to 150mm, the column diameter is 2mm to 3mm, the particle size of the stationary phase is 1.5μm to 2.1μm, and the column temperature is 20℃ to 45℃; for example, the column temperature is 22℃, 26℃, 30℃, 34℃, 38℃, 42℃ or 44℃, but is not limited thereto.

[0021] The flow rate of the liquid chromatograph is 0.25 mL / min to 0.4 mL / min, exemplary values ​​are 0.27 mL / min, 0.3 mL / min, 0.31 mL / min, 0.33 mL / min, 0.35 mL / min, 0.37 mL / min or 0.39 mL / min, but is not limited thereto. A preferred flow rate is 0.28 mL / min to 0.37 mL / min.

[0022] The injection volume of the test solution is 1 μL to 3 μL, with examples being 1.3 μL, 1.6 μL, 1.9 μL, 2.2 μL, 2.5 μL or 2.8 μL, but not limited thereto.

[0023] The injection volume of the reference solution is 1 μL to 3 μL, with examples being 1.3 μL, 1.6 μL, 1.9 μL, 2.2 μL, 2.5 μL or 2.8 μL, but not limited to these.

[0024] As an improvement to the above technical solution, the chromatographic column has a length of 150 mm, a diameter of 2.1 mm, a column temperature of 40 °C, and a stationary phase particle size of 1.7 μm.

[0025] The flow rate of the liquid chromatograph is 0.35 mL / min, the injection volume of the test solution is 2 μL, and the injection volume of the reference solution is 2 μL.

[0026] As an improvement to the above technical solution, the liquid chromatograph uses an evaporative light scattering detector for detection, and its drift tube temperature is 100℃~120℃, for example 103℃, 106℃, 111℃, 114℃ or 117℃, but is not limited thereto.

[0027] The carrier gas used in the evaporative light scattering detector is air, but it is not limited to this.

[0028] The carrier gas flow rate is 2L / min to 5L / min, with examples being 2.1L / min, 2.5L / min, 2.9L / min, 3.3L / min, 3.7L / min, 4.1L / min, 4.5L / min or 4.9L / min, but not limited to these.

[0029] As an improvement to the above technical solution, the drift tube temperature is 110℃, the carrier gas is air, and the carrier gas flow rate is 3.2L / min.

[0030] As an improvement to the above technical solution, in the step of extracting Dendrobium nobile medicinal material or its processed slices, standard decoctions, or formula granules with an extraction solvent to obtain a test solution, a heating reflux extraction process or an ultrasonic extraction process is used.

[0031] As an improvement to the above technical solution, in the step of extracting Dendrobium nobile medicinal material or its processed slices, standard decoctions, or formula granules with an extraction solvent to obtain a test sample solution, ultrasonic extraction is used. The ultrasonic power is 100W to 500W, and exemplary values ​​are 120W, 160W, 200W, 240W, 280W, 320W, 360W, 400W, 440W, or 480W, but not limited to these.

[0032] The ultrasonic frequency is 20kHz to 50kHz, with examples being 22kHz, 28kHz, 30kHz, 34kHz, 39kHz, 42kHz, 45kHz or 48kHz, but not limited to these.

[0033] The extraction time is 30 min to 60 min, with examples being 34 min, 38 min, 42 min, 46 min, 50 min, 54 min or 58 min, but not limited to these.

[0034] As an improvement to the above technical solution, the extraction solvent is 70 vol% methanol.

[0035] As an improvement to the above technical solution, the concentration of dendrobin G reference standard in the reference solution is 220 μg / mL to 260 μg / mL, the concentration of costoside D reference standard is 250 μg / mL to 300 μg / mL, the concentration of costoside C reference standard is 250 μg / mL to 300 μg / mL, and the concentration of costoside A reference standard is 150 μg / mL to 220 μg / mL.

[0036] As an improvement to the above technical solution, the fingerprint spectrum includes 10 common peaks, wherein peak 2 is dendrobin G, peak 6 is costus glycoside D, peak 9 is costus glycoside C, and peak 10 is costus glycoside A.

[0037] Taking peak 2 as S1 peak, calculate the relative retention times of peaks 1, 3 to 5. The relative retention times of each peak are within ±10% of the specified values. The specified values ​​for peaks 1, 3 to 5 are 0.68, 1.22, 1.85, and 2.29, respectively.

[0038] Taking peak 6 as the S2 peak, the relative retention times of peaks 7 to 10 were calculated. The relative retention times of each peak were within ±10% of the specified values. The specified values ​​for peaks 7 to 10 were 1.05, 1.09, 1.14, and 1.28, respectively.

[0039] Accordingly, this invention also discloses a method for identifying Dendrobium nobile, used to distinguish Dendrobium nobile from its adulterants, comprising:

[0040] Provide the substance to be identified;

[0041] The fingerprint spectrum of the substance to be identified was constructed using the above-mentioned method for constructing fingerprint spectra of Dendrobium nobile medicinal materials and its processed slices, standard decoctions, and formula granules.

[0042] If the fingerprint spectrum only shows peaks 1 to 10, then the substance to be identified includes or is Dendrobium nobile; otherwise, it is a counterfeit.

[0043] The counterfeit products include Dendrobium nobile, Dendrobium officinale, Dendrobium chrysanthum, Dendrobium chrysanthum, Dendrobium globulus, Dendrobium verbenae, Dendrobium fringeii, Dendrobium nobile, Dendrobium chrysanthum, Dendrobium chrysanthum, Dendrobium chrysanthum, Dendrobium chrysanthum, Dendrobium chrysanthum, and Dendrobium chrysanthum.

[0044] Implementing this invention has the following beneficial effects:

[0045] Based on the properties of Dendrobium nobile, this invention constructs a fingerprint spectrum for Dendrobium nobile by rationally controlling the mobile phase conditions of liquid chromatography. This effectively reveals the polysesquiterpenoid components of Dendrobium nobile, providing a scientific experimental basis for the research of Dendrobium nobile quality evaluation systems. Furthermore, the method of this invention exhibits strong stability and specificity. This invention also identifies and distinguishes adulterants and counterfeits of Dendrobium nobile, such as Dendrobium verbenae, Dendrobium nobile var. spp., Dendrobium hookeriana, Dendrobium fimbriatum, Dendrobium officinale, and Dendrobium chrysanthum, providing a new scientific method for the quality control of Dendrobium nobile. Attached Figure Description

[0046] Figure 1 The fingerprint spectrum of Dendrobium nobile medicinal material in Example 1 using methanol-acetonitrile (1:1) as mobile phase A and gradient elution program 2;

[0047] Figure 2 The fingerprint spectrum of Dendrobium nobile medicinal material in Example 1 using methanol-acetonitrile (3:1) as mobile phase A and gradient elution program 2;

[0048] Figure 3 This is the fingerprint spectrum of Dendrobium nobile medicinal material in Example 1 using methanol-acetonitrile (3:1) as mobile phase A and gradient elution program 1;

[0049] Figure 4 This is the fingerprint spectrum of Dendrobium nobile medicinal material when the flow rate was 0.3 mL / min in the flow rate study in Example 1;

[0050] Figure 5 This is the fingerprint spectrum of Dendrobium nobile medicinal material when the flow rate was 0.28 mL / min in the flow rate study in Example 1;

[0051] Figure 6 This is the fingerprint spectrum of Dendrobium nobile medicinal material when the flow rate was 0.32 mL / min in the flow rate study in Example 1;

[0052] Figure 7 This is the fingerprint spectrum of Dendrobium nobile medicinal material when the flow rate was 0.35 mL / min in the flow rate study in Example 1;

[0053] Figure 8 This is the fingerprint spectrum of Dendrobium nobile medicinal material when the flow rate was 0.37 mL / min in the flow rate study in Example 1;

[0054] Figure 9 These are the fingerprint spectra of Dendrobium nobile medicinal materials when using different extraction solvents in Example 1;

[0055] Figure 10 These are the fingerprint spectra of Dendrobium nobile medicinal materials obtained by using different extraction methods in Example 1;

[0056] Figure 11 This is the fingerprint spectrum of Dendrobium nobile medicinal material when different extraction times were used in Example 1;

[0057] Figure 12 This is a graph showing the specificity of the fingerprint spectrum of Dendrobium nobile in Example 1;

[0058] Figure 13 It is a superimposed image of the fingerprint spectra of 22 batches of Dendrobium nobile medicinal materials in Example 1;

[0059] Figure 14 It is a superimposed image of the fingerprint spectra of 22 batches of Dendrobium nobile slices in Example 1;

[0060] Figure 15 It is a superimposed image of the fingerprint spectra of 22 batches of Dendrobium nobile standard decoction in Example 1;

[0061] Figure 16 It is a superimposed image of the fingerprint spectra of the three batches of Dendrobium nobile formula granules in Example 1;

[0062] Figure 17 This is the fingerprint spectrum of the Dendrobium nobile reference material in Example 1;

[0063] Figure 18 This is a comparison chart of fingerprint spectra of Dendrobium nobile, Dendrobium chrysanthum, Dendrobium moniliforme, Dendrobium truncatum, and Dendrobium nobile in Example 2;

[0064] Figure 19 This is a comparison chart of fingerprint spectra of Dendrobium nobile, Dendrobium chrysanthum, Dendrobium verum, Dendrobium fringeii, and Dendrobium nobile in Example 2;

[0065] Figure 20 This is a comparative fingerprint spectrum of *Dendrobium nobile*, *Dendrobium officinale*, *Dendrobium chrysanthum*, and *Dendrobium chrysanthum*.

[0066] Among them: peak 2 is dendrobin G, peak 6 is costus glycoside D, peak 9 is costus glycoside C, and peak 10 is costus glycoside A. Detailed Implementation

[0067] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0068] Existing research on the fingerprint spectrum of Dendrobium nobile mainly focuses on flavonoids, while the active medicinal components of Dendrobium nobile are alkaloids and sesquiterpenes. This makes it difficult for existing technologies to effectively reflect the quality of Dendrobium nobile. Furthermore, there are more than 10 easily confused and adulterated varieties of Dendrobium nobile, including Dendrobium verbenae, Dendrobium nobile var. chinensis, Dendrobium nobile var. chinensis, Dendrobium officinale, and Dendrobium chrysanthum. Existing technologies often struggle to effectively identify these adulterants, especially when they are processed into standard decoctions or traditional Chinese medicine granules. Therefore, this invention is proposed.

[0069] It should be noted that the Dendrobium nobile slices in this invention refer to fresh or dried Dendrobium nobile as defined in the Chinese Pharmacopoeia (2020 edition), but are not limited to these. The standard Dendrobium nobile decoction refers to a preparation made from the aforementioned slices through decoction, concentration, and low-temperature freeze-drying, in accordance with the "Technical Requirements for Quality Control and Standard Formulation of Traditional Chinese Medicine Formula Granules." Dendrobium nobile formula granules refer to preparations made from the aforementioned slices through water extraction, separation, concentration, drying, and granulation, in accordance with the "Technical Requirements for Quality Control and Standard Formulation of Traditional Chinese Medicine Formula Granules," but are not limited to these.

[0070] Specifically, the sources of the instruments, materials, and medicinal herbs used in the following embodiments are as follows:

[0071] (1) Instruments: Agilent ultra-high performance liquid chromatograph (model 1290, Agilent Technologies), Waters ultra-high performance liquid chromatograph (H-Class Plus, Waters Corporation), Waters BEH C18 (2.1mm×150mm, 1.7μm), evaporative light scattering detector (Alltech 2000, Alltech Technology Co., Ltd.), 0.01% balance (ME204E, Mettler Toledo), 0.1% balance (XP26, Mettler Toledo), laboratory ultrapure water system (Milli-Q Direct, Merck KGaA).

[0072] (2) Reagents: Methanol (Xilong Scientific Co., Ltd.) and ethanol (Xilong Scientific Co., Ltd.) were both analytical grade; acetonitrile (Merck Co., Ltd.), methanol (Merck Co., Ltd.) and formic acid (Merck Co., Ltd.) were all chromatographic grade; water was ultrapure water (prepared in the laboratory).

[0073] (3) Test drugs: Dendroside G (batch number: 14013, content: ≥98%, Shanghai Shidander Standard Technical Service Co., Ltd.); Dendronobiloside D (batch number: 14012, content: ≥98%, Shanghai Shidander Standard Technical Service Co., Ltd.); Dendronobiloside C (batch number: 14011, content: ≥98%, Shanghai Shidander Standard Technical Service Co., Ltd.); Dendronobiloside A (batch number: 14010, content: ≥98%, Shanghai Shidander Standard Technical Service Co., Ltd.); Dendrobium (Dendrobium nobile) reference material (Guangdong Yifang Pharmaceutical Co., Ltd.); Batch number information of 22 batches of Dendrobium (Dendrobium nobile) medicinal materials is shown in Table 1.

[0074] Table 1 Information on the Source of Medicinal Materials

[0075]

[0076] Example 1: Construction of fingerprint spectrum of Dendrobium nobile

[0077] 1. Preparation of the test solution

[0078] Medicinal materials: Take about 2.0g of Dendrobium nobile powder (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, add 50mL of 70% methanol, weigh it, sonicate (power 300W, frequency 40kHz) for 45 minutes, centrifuge, take the supernatant and evaporate it to dryness, dissolve the residue in 70% methanol and transfer it to a 5mL volumetric flask, add 70% methanol to the mark, shake well, filter, and take the filtrate to obtain the product.

[0079] Prepared slices: Take about 2.0g of Dendrobium nobile powder (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, add 50mL of 70% methanol, weigh it, sonicate (power 300W, frequency 40kHz) for 45 minutes, centrifuge, take the supernatant and evaporate it to dryness, dissolve the residue in 70% methanol and transfer it to a 5mL volumetric flask, add 70% methanol to the mark, shake well, filter, and take the filtrate to obtain the product.

[0080] Standard decoction: Take about 0.5g of Dendrobium nobile standard decoction powder, accurately weigh it, place it in a stoppered conical flask, add 25mL of 70% methanol, weigh it, sonicate (power 300W, frequency 40kHz) for 45 minutes, centrifuge, take the supernatant and evaporate it to dryness, dissolve the residue in 70% methanol and transfer it to a 5mL volumetric flask, add 70% methanol to the mark, shake well, filter, and take the filtrate to obtain the decoction.

[0081] Formula granules: Take about 1.0g of Dendrobium nobile formula granules, accurately weigh them, place them in a stoppered conical flask, add 25mL of 70% methanol, weigh them, sonicate (power 300W, frequency 40kHz) for 45 minutes, centrifuge, take the supernatant and evaporate to dryness, dissolve the residue in 70% methanol and transfer it to a 5mL volumetric flask, add 70% methanol to the mark, shake well, filter, and take the filtrate to obtain the product.

[0082] 2. Preparation of reference solution

[0083] Accurately weigh 1.255 mg of dendrobinin G reference standard, 1.364 mg of costus glycoside D reference standard, and costus glycoside C.

[0084] 1.309 mg of valerin A and 0.986 mg of costoside A were placed in a 5 mL volumetric flask and methanol was added to prepare a mixed solution containing 245.980 μg of dendrobin G, 267.344 μg of costoside D, 256.564 μg of costoside C, and 193.256 μg of costoside A per mL, which was used as the reference solution.

[0085] 3. Chromatographic conditions

[0086] Chromatographic column: Waters BEH C18 (2.1 mm × 150 mm, 1.7 μm); mobile phase A: methanol-acetonitrile (3:1), mobile phase B: 0.1% formic acid solution; elution mode: gradient elution; flow rate: 0.35 mL / min, column temperature: 40 °C, evaporative light scattering detector; drift tube temperature: 110 °C; carrier gas: air, flow rate: 3.2 L / min. The gradient elution program is shown in Table 2. The theoretical plate number, calculated based on the costoside D peak, should be no less than 5000.

[0087] Table 2 Gradient elution program 1

[0088]

[0089] 4. Measurement

[0090] Accurately pipette 2 μL of the reference solution and 2 μL of the test solution into the liquid chromatograph and determine the result.

[0091] 5. Methodological Examination

[0092] 5.1 Investigation of mobile phase conditions

[0093] This section examines the effect of mobile phase conditions on fingerprint chromatograms. Specifically, two parallel portions of Dendrobium nobile were prepared as test solutions according to the method described in section "1", and then tested under the chromatographic conditions below.

[0094] Mobile phase 1: Waters BEH-C18 (column length 150 mm, inner diameter 2.1 mm, particle size 1.7 μm); using methanol-acetonitrile (1:1) as mobile phase A and 0.1% formic acid solution as mobile phase B, gradient elution was performed according to the specifications in Table 3; the flow rate was 0.3 mL per minute; the column temperature was 40 °C.

[0095] Table 3 Gradient elution program 2

[0096]

[0097] Mobile phase 2: Waters BEH-C18 (column length 150 mm, inner diameter 2.1 mm, particle size 1.7 μm); using methanol-acetonitrile (3:1) as mobile phase A and 0.1% formic acid solution as mobile phase B, gradient elution was performed according to the specifications in Table 3; the flow rate was 0.3 mL per minute; the column temperature was 40 °C.

[0098] Experimental results are as follows Figures 1-2As shown, the results indicate that peak separation was achieved with stable baselines when the volume ratio of methanol to acetonitrile was 1:1 and 3:1. However, the peak separation was better when using a methanol-acetonitrile ratio of 3:1.

[0099] 5.2 Gradient elution procedure investigation

[0100] This section examines the effect of gradient elution procedures on fingerprint chromatograms. Specifically, two parallel portions of Dendrobium nobile were prepared as test solutions according to the method described in section "1", and then tested under the chromatographic conditions below.

[0101] Gradient elution program 1: Waters BEH-C18 (column length 150 mm, inner diameter 2.1 mm, particle size 1.7 μm); using methanol-acetonitrile (3:1) as mobile phase A and 0.1% formic acid solution as mobile phase B, gradient elution was performed according to the specifications in Table 3; the flow rate was 0.3 mL per minute; the column temperature was 40 °C.

[0102] Gradient elution program 2: Waters BEH-C18 (column length 150 mm, inner diameter 2.1 mm, particle size 1.7 μm); using methanol-acetonitrile (3:1) as mobile phase A and 0.1% formic acid solution as mobile phase B, gradient elution was performed according to the specifications in Table 2; the flow rate was 0.3 mL per minute; the column temperature was 40 °C.

[0103] Experimental results are as follows Figures 2-3 As shown in the figure, when using the gradient elution program specified in Table 4, some chromatographic peaks are difficult to separate. However, when using the gradient elution program specified in Table 2, the chromatographic peaks are separated well.

[0104] 5.3 Flow velocity investigation

[0105] This section examines the effect of flow rate on fingerprint chromatograms. Specifically, five parallel portions of Dendrobium nobile were prepared as test solutions according to the method described in section "1", and then tested under the chromatographic conditions below.

[0106] Flow rate 1: Waters BEH-C18 (column length 150 mm, inner diameter 2.1 mm, particle size 1.7 μm); using methanol-acetonitrile (3:1) as mobile phase A and 0.1% formic acid solution as mobile phase B, gradient elution was performed according to the specifications in Table 2; the flow rate was 0.3 mL per minute; the column temperature was 40 °C.

[0107] Flow rate 2: Waters BEH-C18 (column length 150 mm, inner diameter 2.1 mm, particle size 1.7 μm); using methanol-acetonitrile (3:1) as mobile phase A and 0.1% formic acid solution as mobile phase B, gradient elution was performed according to the specifications in Table 2; the flow rate was 0.28 mL per minute; the column temperature was 40 °C.

[0108] Flow rate 3: Waters BEH-C18 (column length 150 mm, inner diameter 2.1 mm, particle size 1.7 μm); using methanol-acetonitrile (3:1) as mobile phase A and 0.1% formic acid solution as mobile phase B, gradient elution was performed according to the specifications in Table 2; the flow rate was 0.32 mL per minute; the column temperature was 40 °C.

[0109] Flow rate 4: Waters BEH-C18 (column length 150 mm, inner diameter 2.1 mm, particle size 1.7 μm); using methanol-acetonitrile (3:1) as mobile phase A and 0.1% formic acid solution as mobile phase B, gradient elution was performed according to the specifications in Table 2; the flow rate was 0.35 mL per minute; the column temperature was 40 °C.

[0110] Flow rate 5: Waters BEH-C18 (column length 150 mm, inner diameter 2.1 mm, particle size 1.7 μm); using methanol-acetonitrile (3:1) as mobile phase A and 0.1% formic acid solution as mobile phase B, gradient elution was performed according to the specifications in Table 2; the flow rate was 0.37 mL per minute; the column temperature was 40 °C.

[0111] Experimental results are as follows Figures 4 to 8 As shown in the figure, it can be seen that the chromatographic peaks can be separated at flow rates of 0.28 mL / min, 0.3 mL / min, 0.32 mL / min, 0.35 mL / min, and 0.37 mL / min. Furthermore, the separation effect and peak shape are better at a flow rate of 0.35 mL / min.

[0112] 5.4 Investigation of the preparation method of the test sample

[0113] (1) Investigation of extraction solvent

[0114] Take approximately 2.0 g of the same batch of Dendrobium nobile powder (No. G1, passed through a No. 3 sieve), in six parallel groups, two portions per group, accurately weigh each portion, and place them in stoppered conical flasks. Accurately add 50 mL each of 100% methanol, 70% methanol, 30% methanol, 100% ethanol, 70% ethanol, and 30% ethanol, respectively, weigh each portion, and sonicate (250 W, 40 kHz) for 45 minutes. After cooling, weigh again, and replenish the lost weight with 70% methanol. Shake well, filter, evaporate the filtrate to dryness, dissolve the residue in 70% methanol, transfer to a 5 mL volumetric flask, add 70% methanol to the mark, shake well, filter, and collect the filtrate. Determine the chromatographic conditions specified in section "3". Results are as follows: Figure 9As shown in the figure, the fingerprint chromatograms all exhibit peaks 1-10 with good separation when using different extraction solvents. Furthermore, the response values ​​of each chromatographic peak are higher when using 30% ethanol, 100% methanol, 70% methanol, and 30% methanol, especially when using 70% methanol, which shows the highest peak response and the best peak shape.

[0115] (2) Examination of extraction methods

[0116] Take approximately 2.0 g of the same batch of Dendrobium nobile powder (No. G1, passed through a No. 3 sieve), in two parallel groups, two portions per group, accurately weighed, and placed in stoppered conical flasks. Accurately add 50 mL of 70% methanol, weigh, and sonicate (250 W, 40 kHz) for 45 minutes, then reflux for 45 minutes. Cool, weigh again, and replenish the lost weight with 70% methanol. Shake well, filter, evaporate the filtrate to dryness, dissolve the residue in 70% methanol, transfer to a 5 mL volumetric flask, add 70% methanol to the mark, shake well, filter, and collect the filtrate. Determine the chromatographic conditions specified in section "3". Results are as follows: Figure 10 As shown in the figure, it can be seen that there is no significant difference in the peak shape and separation effect of each characteristic peak when different extraction methods are used. Considering the simplicity of operation, ultrasonic extraction was selected as the extraction method.

[0117] (3) Examination of extraction time

[0118] Take approximately 2.0 g of the same batch of Dendrobium nobile powder (No. G1, passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, add 50 mL of 70% methanol, weigh it again, and sonicate it (300 W, 40 kHz) for 30 minutes, 45 minutes, and 60 minutes respectively. After cooling, weigh it again, make up the weight loss with 70% methanol, shake well, filter, evaporate the filtrate to dryness, dissolve the residue in 70% methanol, transfer it to a 5 mL volumetric flask, add 70% methanol to the mark, shake well, filter, and collect the filtrate. Determine the chromatographic conditions according to section "3". The results are as follows: Figure 11 As shown in the figure, different extraction times do not significantly affect the fingerprint spectrum of Dendrobium nobile. To ensure complete extraction, an ultrasonic extraction time of 45 minutes was selected.

[0119] 6. Methodological Investigation

[0120] 6.1 Specificity Examination

[0121] Take approximately 2.0 g of Dendrobium nobile powder (No. G1, passed through a No. 3 sieve), prepare the test solution according to the method in section "1", accurately pipette 5 μL each of the test solution, the reference solution in section "2", and the blank solvent, and inject them for analysis according to the chromatographic conditions in section "3". The results are as follows: Figure 12As shown in the figure, the chromatogram of the test sample has the same chromatographic peak at the corresponding retention time as that of the reference sample, and there is no interference from the blank solvent, indicating that the method has good specificity.

[0122] 6.2 Precision test

[0123] Take approximately 2.0 g of Dendrobium nobile powder (No. G1, passed through a No. 3 sieve) and prepare the test solution according to the method in section "1". Inject the sample 6 times repeatedly under the chromatographic conditions in section "3". Using peak 2-dendrobinoside G as the reference peak S1 and peak 6-costunol D as the reference peak S2, the relative retention time RSD values ​​of each characteristic peak are in the range of 0.00% to 0.24%, and the relative peak area RSD values ​​are in the range of 0.63% to 4.01%, all less than 5.0%, indicating good instrument precision.

[0124] 6.3 Repeatability Test

[0125] Approximately 2.0 g of Dendrobium nobile powder (No. G1, passed through a No. 3 sieve) was used to prepare six test solutions according to the method described in section "1". The solutions were then injected and analyzed under the chromatographic conditions described in section "3". The results showed that when the same batch of samples was repeatedly measured six times, with peak 2-dendrobinoside G as the reference peak S1 and peak 6-costunol D as the reference peak S2, the relative retention time RSD values ​​of each characteristic peak were in the range of 0.00%–0.24%, and the relative peak area RSD values ​​were in the range of 1.51%–3.80%, all less than 5.0%, indicating good repeatability of the method.

[0126] 6.4 Stability Test

[0127] Take approximately 2.0 g of Dendrobium nobile powder (No. G1, passed through a No. 3 sieve) and prepare a test solution according to the method described in section "1". Inject the sample at 0, 2, 5, 8, 20, and 26 hours according to the chromatographic conditions described in section "3". Analyze the same test solution at 0, 2, 5, 8, 20, and 26 hours. Use peak 2-dendrobinoside G as reference peak S1 and peak 6-costunosin D as reference peak S2. The relative retention time (RSD) of each characteristic peak is in the range of 0.00%–0.28%, and the relative peak area (RSD) is in the range of 0.73%–4.14%, all less than 5.0%. The test solution is stable within 26 hours.

[0128] 7. Establishment of fingerprint spectrum of Dendrobium nobile medicinal material

[0129] 7.1 Results of fingerprint analysis of Dendrobium nobile medicinal material

[0130] Twenty-two batches of Dendrobium nobile medicinal materials were collected and prepared according to the chromatographic conditions under section "3" and the test solution preparation method determined under section "1". Using peak 2-dendrobinoside G as the reference peak S1, the relative retention times of peaks 1-5 and peak S1 were calculated. The fingerprint chromatograms of the 22 batches of medicinal materials are shown below. Figure 13 As shown.

[0131] 7.2 Results of fingerprint analysis of Dendrobium nobile slices

[0132] Dendrobium nobile medicinal material was processed according to the Pharmacopoeia to obtain decoction pieces, numbered GP1 to GP22. Following the chromatographic conditions in section "3" and the test solution preparation method determined in section "1", with peak 2-dendrobinoside G as the reference peak S1, the relative retention times of peaks 1-5 and peak S1 were calculated. The fingerprint chromatograms of 22 batches of decoction pieces are shown below. Figure 14 As shown.

[0133] 7.3 Results of fingerprint analysis of standard decoction of Dendrobium nobile

[0134] Dendrobium nobile slices were prepared and decocted according to the "Technical Requirements for Quality Control and Standard Formulation of Traditional Chinese Medicine Formula Granules". After decoction, the powder was concentrated and freeze-dried to obtain the freeze-dried standard decoction powder. The freeze-dried standard decoction powders were numbered GT1 to GT22. Following the chromatographic conditions in section "3" and the preparation method of the test solution determined in section "1", the relative retention times of peaks 1-5 and peak S1 were calculated using peak 2-dendrobinoside G as the reference peak S1. The fingerprint chromatograms of the 22 batches of standard decoctions are shown below. Figure 15 As shown.

[0135] 7.4 Results of fingerprint analysis of Dendrobium nobile formula granules

[0136] Take Dendrobium nobile slices, decoct with water, filter, concentrate the filtrate into a clear extract, add appropriate amount of excipients, dry (or dry and pulverize), add appropriate amount of excipients again, mix well, granulate, and the product is obtained. The formulation granules are numbered CG1~CG3. Following the chromatographic conditions under section "3" and the test solution preparation method determined under section "1", using peak 2-dendrobinoside G as reference peak S1, calculate the relative retention times of peaks 1-5 with peak S1. The fingerprint spectra of the three batches of formulation granules are shown below. Figure 16 As shown.

[0137] 7.5 Establishment of a common pattern for the fingerprint spectrum of Dendrobium nobile medicinal materials

[0138] A reference solution of *Dendrobium nobile* (Guangdong Yifang Pharmaceutical Co., Ltd.) was prepared according to the method described in section "1". Twenty-two batches of *Dendrobium nobile* were used to prepare test solutions according to the method described in section "1". The solutions were then injected and analyzed under the chromatographic conditions described in section "3" to obtain the fingerprint chromatogram of the *Dendrobium nobile* reference material. A reference chromatogram was generated using the "Software for Similarity Evaluation of Chromatographic Fingerprints of Traditional Chinese Medicine" using the average method, thus establishing a reference fingerprint chromatogram for *Dendrobium nobile*. Figure 17 ).

[0139] The chromatogram of the test sample should show 10 characteristic peaks, and the retention times should correspond to the 10 characteristic peaks in the chromatogram of the reference medicinal material. The peak corresponding to the dendrobinin G reference standard is designated as peak S1, and the relative retention times of peaks 1, 3-5 are calculated. The peak corresponding to the costoside D reference standard is designated as peak S2, and the relative retention times of peaks 7-10 with peak S are calculated. The relative retention times should be within ±10% of the specified values, which are: 0.68 (peak 1), 1.22 (peak 3), 1.85 (peak 4), 2.29 (peak 5), 1.05 (peak 7), 1.09 (peak 8), 1.14 (peak 9), and 1.28 (peak 10).

[0140] Example 2: Application of fingerprint spectrum of Dendrobium nobile medicinal material

[0141] This embodiment provides a method for identifying Dendrobium nobile medicinal materials. Specifically, Dendrobium nobile, Dendrobium chrysanthum, Dendrobium officinale, Dendrobium nobile var. truncatum, Dendrobium nobile var. truncatum, Dendrobium nobile var. truncatum, Dendrobium nobile var. truncatum, Dendrobium nobile var. truncatum, Dendrobium nobile var. truncatum, Dendrobium nobile var. truncatum, and Dendrobium nobile var. truncatum are taken and prepared into a test solution according to the conditions under item "1" in Example 1, and injected for testing according to the chromatographic conditions under item "3". The results are shown in the figure. Figures 18-20 The results showed that the fingerprint spectra of Dendrobium verbenae, Dendrobium nobile, Dendrobium truncatum, Dendrobium fimbriatum, Dendrobium officinale, and Dendrobium nobile were significantly different from those of Dendrobium nobile. Specifically, the fingerprint spectra of Dendrobium verbenae, Dendrobium nobile, Dendrobium truncatum, Dendrobium fimbriatum, and Dendrobium nobile did not possess the 10 characteristic peaks of the Dendrobium nobile fingerprint spectra. Dendrobium nobile had a unique peak (11) between 45 and 50 minutes; Dendrobium nobile had a specific peak (18) between 25 and 30 minutes; Dendrobium nobile had specific peaks (13, 14, and 15) between 10 and 15 minutes; and Dendrobium nobile had a unique peak (20) between 45 and 50 minutes. In conclusion, the fingerprint spectra method established in this invention can effectively distinguish various adulterants of Dendrobium nobile.

[0142] The above description is a preferred embodiment of the 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 invention, and these improvements and modifications are also considered to be within the scope of protection of the invention.

Claims

1. A method for constructing fingerprint spectra of Dendrobium nobile medicinal material and its processed slices, standard decoctions, and formulated granules, characterized in that, include: The Dendrobium nobile medicinal material or its processed slices, standard decoctions, and formula granules were extracted using an extraction solvent to obtain the test solution. Dendrobin G reference standard, dendronobiloside D reference standard, dendronobiloside C reference standard, and dendronobiloside A reference standard were dissolved in 30 vol% to 100 vol% methanol to obtain reference standard solutions; The test solution and reference solution were analyzed by liquid chromatography to obtain the fingerprint spectrum of Dendrobium nobile or its processed slices, standard decoctions, and formula granules. The liquid chromatograph uses a Waters BEH C18 column with a length of 150 mm and a diameter of 2.1 mm. The stationary phase has a particle size of 1.7 μm. A mixed solution of methanol and acetonitrile is used as mobile phase A, and a 0.05 vol%–0.2 vol% formic acid aqueous solution is used as mobile phase B for gradient elution. The gradient elution curve is as follows: From 0 min to 20 min, mobile phase A decreased from 16% to 17%, and mobile phase B decreased from 84% to 83%. Over 20-25 minutes, mobile phase A decreased from 17% to 25%, and mobile phase B decreased from 83% to 75%. 25-30 min, mobile phase A is 25%, mobile phase B is 75%; Over 30-40 minutes, mobile phase A decreased from 25% to 30%, and mobile phase B decreased from 75% to 70%. Over 40-50 minutes, mobile phase A decreased from 30% to 35%, and mobile phase B decreased from 70% to 65%. From 50 to 55 minutes, mobile phase A decreased from 35% to 36%, and mobile phase B decreased from 65% to 64%. From 55 to 60 minutes, mobile phase A decreased from 36% to 40%, and mobile phase B decreased from 64% to 60%. Over 60-65 minutes, mobile phase A decreased from 40% to 45%, and mobile phase B decreased from 60% to 55%. The liquid chromatograph uses an evaporative light scattering detector for detection. The extraction solvent is 30 vol% to 100 vol% methanol or 30 vol% to 100 vol% ethanol; in the mobile phase A, the volume ratio of methanol to acetonitrile is 3:

1.

2. The method for constructing fingerprint spectra of Dendrobium nobile medicinal materials and its processed slices, standard decoctions, and formulated granules as described in claim 1, characterized in that, The mobile phase B is a 0.1 vol% aqueous formic acid solution.

3. The method for constructing fingerprint spectra of Dendrobium nobile medicinal materials and its processed slices, standard decoctions, and formulated granules as described in claim 1, characterized in that, The column temperature of the chromatographic column is 20℃~45℃; The flow rate of the liquid chromatograph is 0.25 mL / min to 0.4 mL / min, the injection volume of the test solution is 1 μL to 3 μL, and the injection volume of the reference solution is 1 μL to 3 μL.

4. The method for constructing fingerprint spectra of Dendrobium nobile medicinal materials and their processed slices, standard decoctions, and formulated granules as described in claim 1 or 3, characterized in that, The column temperature of the chromatographic column is 40℃; The flow rate of the liquid chromatograph is 0.35 mL / min, the injection volume of the test solution is 2 μL, and the injection volume of the reference solution is 2 μL.

5. The method for constructing fingerprint spectra of Dendrobium nobile medicinal materials and its processed slices, standard decoctions, and formulated granules as described in claim 1, characterized in that, The drift tube temperature of the evaporative light scattering detector is 100℃~120℃, the carrier gas is air, and the carrier gas flow rate is 2L / min~5L / min.

6. The method for constructing fingerprint spectra of Dendrobium nobile medicinal materials and its processed slices, standard decoctions, and formulated granules as described in claim 5, characterized in that, The drift tube temperature is 110℃, the carrier gas is air, and the carrier gas flow rate is 3.2L / min.

7. The method for constructing fingerprint spectra of Dendrobium nobile medicinal materials and their processed slices, standard decoctions, and formulated granules as described in claim 1, characterized in that, In the step of extracting Dendrobium nobile medicinal material or its processed slices, standard decoctions, and formula granules with an extraction solvent to obtain a test solution, the extraction process is carried out by heating reflux extraction or ultrasonic extraction.

8. The method for constructing fingerprint spectra of Dendrobium nobile medicinal materials and their processed slices, standard decoctions, and formulated granules as described in claim 1 or 7, characterized in that, In the step of extracting Dendrobium nobile medicinal material or its processed slices, standard decoctions, and formula granules with an extraction solvent to obtain a test solution, ultrasonic extraction is used, wherein the ultrasonic power is 100W~500W, the frequency is 20kHz~50kHz, and the extraction time is 30min~60min.

9. The method for constructing fingerprint spectra of Dendrobium nobile medicinal materials and its processed slices, standard decoctions, and formulated granules as described in claim 1, characterized in that, The extraction solvent is 70 vol% methanol.

10. The method for constructing fingerprint spectra of Dendrobium nobile medicinal materials and their processed slices, standard decoctions, and formulated granules as described in claim 1, characterized in that, The concentrations of dendronobiloside G, dendronobiloside D, dendronobiloside C, and dendronobiloside A in the reference solutions were 220 μg / mL to 260 μg / mL, 250 μg / mL to 300 μg / mL, 250 μg / mL to 300 μg / mL, and 150 μg / mL to 220 μg / mL, respectively.

11. The method for constructing fingerprint spectra of Dendrobium nobile medicinal materials and their processed slices, standard decoctions, and formulated granules as described in claim 1, characterized in that, The fingerprint spectrum includes 10 common peaks, of which peak 2 is dendronobiloside G, peak 6 is dendronobiloside D, peak 9 is dendronobiloside C, and peak 10 is dendronobiloside A; Taking peak 2 as peak S1, calculate the relative retention times of peaks 1, 3-5. The relative retention times of each peak should be within ±10% of the specified value. The specified values ​​for peaks 1, 3-5 are as follows: 0.68、1.22、1.85、2.29； Taking peak 6 as the S2 peak, the relative retention times of peaks 7-10 were calculated. The relative retention times of each peak were within ±10% of the specified value. The specified values ​​for peaks 7 to 10 are as follows: 1.05、1.09、1.14、1.28。 12. A method for identifying Dendrobium nobile, used to distinguish Dendrobium nobile from its adulterants, characterized in that, include: Provide the substance to be identified; The fingerprint spectrum of the substance to be identified is constructed using the fingerprint spectrum construction method of Dendrobium nobile medicinal material and its processed slices, standard decoctions, and formula granules as described in any one of claims 1 to 11; If the fingerprint spectrum only shows peaks 1 to 10, then the substance to be identified is Dendrobium nobile; otherwise, it is a counterfeit. The counterfeit products include Dendrobium nobile, Dendrobium officinale, Dendrobium chrysanthum, Dendrobium chrysanthum, Dendrobium globulus, Dendrobium verbenae, Dendrobium fringeii, Dendrobium nobile, Dendrobium chrysanthum, Dendrobium chrysanthum, Dendrobium chrysanthum, Dendrobium chrysanthum, Dendrobium chrysanthum, and Dendrobium chrysanthum.

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