Dendrobium nobile medicinal material as well as fingerprint spectrum construction method of decoction pieces, standard decoction and formula granules of dendrobium nobile medicinal material

Abstract

The invention discloses a dendrobium nobile medicinal material and a fingerprint spectrum construction method of decoction pieces, standard decoction and formula granules thereof, and the method comprises the following steps: extracting the dendrobium nobile medicinal material or the decoction pieces, the standard decoction and the formula granules thereof with 30-100 vol% methanol or 30-100 vol% ethanol to obtain a test solution; the method comprises the following steps: dissolving a dendrobine G reference substance, a costus root glycoside D reference substance, a costus root glycoside C reference substance and a costus root glycoside A reference substance with 30-100vol% methanol to obtain a reference substance solution; and determining the test solution and the reference solution by using a liquid chromatograph to obtain the fingerprints of the dendrobium nobile medicinal material or the decoction pieces, the standard decoction and the formula granules of the dendrobium nobile medicinal material. The fingerprint spectrum constructed by the invention has strong specificity, can better reflect the medicinal components of the dendrobium nobile, can effectively identify common counterfeit products, and provides a scientific basis for formulating the quality standard of the dendrobium nobile.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine identification, and particularly to a method for constructing fingerprint spectra of Dendrobium officinale Kimura et Migo herbs, their cut pieces, standard decoctions, and formula granules. Background Art

[0002] The 2020 edition of the Chinese Pharmacopoeia records that Dendrobium is the fresh or dried stem of Dendrobium officinale Kimura et Migo, an orchid plant, which belongs to traditional tonic medicines and mainly has effects such as nourishing yin and clearing heat, promoting the production of body fluid and benefiting the stomach. Modern research shows that Dendrobium officinale contains components such as alkaloids, sesquiterpenes, phenanthrenes, and flavonoids, and alkaloids and sesquiterpenes are its main active components. Its alkaloids mainly show anti-tumor, hypoglycemic, and antiviral effects, and sesquiterpenes have significant anti-tumor effects. Due to the good hypoglycemic, hypolipidemic, antioxidant, nerve cell protection, and anti-tumor activities of Dendrobium officinale, it is commonly used in clinical practice to treat diabetes, hyperlipidemia, and malignant tumors, etc.

[0003] There are a total of 74 species and 2 varieties in the genus Dendrobium of the orchid family to which Dendrobium officinale belongs, and about 51 species of Dendrobium that can be used medicinally have been found among all Dendrobiums. The 2020 edition of the Chinese Pharmacopoeia includes traditional Chinese medicines Dendrobium such as Dendrobium officinale, Dendrobium huoshanense, Dendrobium chrysotoxum, and Dendrobium fimbriatum. The medicinal effects of different Dendrobium with different original sources have their emphases. In order to ensure the safety of medication, it is necessary to construct an effective fingerprint spectrum method to distinguish common counterfeits and easily confused products of Dendrobium officinale, but there is less research in this regard at present. The quality control item of Dendrobium officinale in the 2020 edition of the Chinese Pharmacopoeia only stipulates its dendrobine. Chen Zhihui et al. studied Dendrobium officinale based on fingerprint spectrum technology. In their research, ultraviolet method was mainly used for detection. Through ESI-MS speculation and identification, the common peaks were flavonoid components, while the active components of Dendrobium officinale are alkaloids and sesquiterpenes. The ultraviolet absorption of these components is weak, and the inspection effect under ultraviolet light is poor. Based on this, the current method cannot evaluate the quality of Dendrobium officinale well. 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 officinale herbs, their cut pieces, standard decoctions, and formula granules, which is stable and highly specific and can be used for the identification of common counterfeits.

[0005] To solve the above technical problem, the present invention provides a method for constructing fingerprint spectra of Dendrobium officinale herbs, their cut pieces, standard decoctions, and formula granules, which includes:

[0006] Extract the Dendrobium officinale Kimura et Migo herbs or their cut pieces, standard decoctions, and formula granules with an extraction solvent to obtain a test solution; wherein, the extraction solvent is 30 vol% - 100 vol% methanol or 30 vol% - 100 vol% ethanol, and exemplary ones 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 thereto.

[0007] Dissolve the dendrobioside G reference substance, costuside D reference substance, costuside C reference substance, and costuside A reference substance with 30 vol% - 100 vol% methanol to obtain a reference solution; exemplary, the concentration of methanol (methanol aqueous solution) is 35 vol%, 40 vol%, 45 vol%, 50 vol%, 55 vol%, 60 vol%, 70 vol%, 75 vol%, 80 vol%, or 90 vol%, but not limited thereto; preferably 50 vol% - 80 vol%, more preferably 70 vol%;

[0008] Determine the test solution and the reference solution with a liquid chromatograph to obtain the fingerprint of the Dendrobium officinale Kimura et Migo herbs or their cut pieces, standard decoctions, and formula granules;

[0009] Wherein, the chromatographic column of the liquid chromatograph uses octadecylsilane-bonded silica gel 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, and the gradient elution curve is:

[0010] 0 min - 20 min, mobile phase A changes from 16% → 17%, and mobile phase B changes from 84% → 83%;

[0011] 20 min - 25 min, mobile phase A changes from 17% → 25%, and mobile phase B changes from 83% → 75%;

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

[0013] 30 min - 40 min, mobile phase A changes from 25% → 30%, and mobile phase B changes from 75% → 70%;

[0014] 40 min - 50 min, mobile phase A changes from 30% → 35%, and mobile phase B changes from 70% → 65%;

[0015] From 50 min to 55 min, mobile phase A changes from 35% to 36%, and mobile phase B changes from 65% to 64%.

[0016] From 55 min to 60 min, mobile phase A changes from 36% to 40%, and mobile phase B changes from 64% to 60%.

[0017] From 60 min to 65 min, mobile phase A changes from 40% to 45%, and mobile phase B changes from 60% to 55%.

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

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

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

[0021] The flow rate of the liquid chromatograph is 0.25 mL / min to 0.4 mL / min, and 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 not limited thereto. Preferably, it is 0.28 mL / min to 0.37 mL / min.

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

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

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

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

[0027] The carrier gas used by the evaporative light scattering detector is air, but is not limited thereto.

[0028] The carrier gas flow rate is 2 L / min to 5 L / min, and exemplary are 2.1 L / min, 2.5 L / min, 2.9 L / min, 3.3 L / min, 3.7 L / min, 4.1 L / min, 4.5 L / min or 4.9 L / min, but not limited thereto.

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

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

[0031] As an improvement of the above technical solution, in the step of extracting the Dendrobium nobile medicinal material or its decoction pieces, standard decoction, or formula granules with an extraction solvent to obtain a test solution, an ultrasonic extraction process is used for extraction, wherein the ultrasonic power is 100W to 500W, and exemplary ones are 120W, 160W, 200W, 240W, 280W, 320W, 360W, 400W, 440W or 480W, but not limited thereto.

[0032] The ultrasonic frequency is 20 kHz to 50 kHz, and exemplarily is 22 kHz, 28 kHz, 30 kHz, 34 kHz, 39 kHz, 42 kHz, 45 kHz or 48 kHz, but is not limited thereto.

[0033] The extraction time is 30 min to 60 min, exemplified by 34 min, 38 min, 42 min, 46 min, 50 min, 54 min or 58 min, but not limited thereto.

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

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

[0036] As an improvement of the above technical solution, the fingerprint includes 10 common peaks. Among them, peak 2 is dendrobioside G, peak 6 is costuside D, peak 9 is costuside C, and peak 10 is costuside A;

[0037] Taking peak 2 as S1 peak, calculate the relative retention times of peaks 1, 3-5. The relative retention times of each peak are within ±10% of the specified values. Among them, the specified values of peaks 1, 3-5 are: 0.68, 1.22, 1.85, 2.29;

[0038] Taking peak 6 as S2 peak, calculate the relative retention times of peaks 7-10. The relative retention times of each peak are within ±10% of the specified values. Among them, the specified values of peaks 7-10 are: 1.05, 1.09, 1.14, 1.28.

[0039] Correspondingly, the present invention also discloses a method for identifying Dendrobium nobile, which is used to identify Dendrobium nobile and its counterfeits, and it includes:

[0040] Provide the substance to be identified;

[0041] Construct the fingerprint of the substance to be identified by the fingerprint construction method of the above-mentioned Dendrobium nobile medicinal materials, their decoction pieces, standard decoctions, and formula granules;

[0042] If only peaks 1-10 appear in the fingerprint, the substance to be identified includes or is Dendrobium nobile; otherwise, it is a counterfeit;

[0043] Among them, the counterfeits include Dendrobium wardianum, Dendrobium officinale, Dendrobium chrysanthum, Pholidota chinensis, Dendrobium thyrsiflorum, Dendrobium fimbriatum, Dendrobium fimbriatum var. oculatum, Dendrobium chrysotoxum, Dendrobium erythraeum, Dendrobium wardianum, Dendrobium chrysotoxum, and Dendrobium aduncum.

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

[0045] According to the properties of Dendrobium nobile Lindl. medicinal materials, the present invention reasonably controls the mobile phase conditions of liquid chromatography, constructs the fingerprint of Dendrobium nobile Lindl., effectively shows the poly sesquiterpenoid components of Dendrobium nobile Lindl., provides a scientific experimental basis for the research on the quality evaluation system of Dendrobium nobile Lindl., and the method of the present invention has strong stability and specificity. The present invention also differentiates and identifies the fingerprints of the adulterants and counterfeits of Dendrobium nobile Lindl. such as Dendrobium fimbriatum Hook., Dendrobium chrysanthum Wall., Dendrobium aduncum Wall. ex Lindl., Dendrobium fimbriatum Hook. var. oculatum Hook., Dendrobium officinale Kimura et Migo and Pholidota chinensis Lindl. medicinal materials, providing a new scientific method for the quality control of Dendrobium nobile Lindl. Description of the Drawings

[0046] Figure 1 is the fingerprint of Dendrobium nobile Lindl. medicinal materials when methanol - acetonitrile (1:1) is used as mobile phase A and gradient elution program 2 is adopted in Example 1;

[0047] Figure 2 is the fingerprint of Dendrobium nobile Lindl. medicinal materials when methanol - acetonitrile (3:1) is used as mobile phase A and gradient elution program 2 is adopted in Example 1;

[0048] Figure 3 is the fingerprint of Dendrobium nobile Lindl. medicinal materials when methanol - acetonitrile (3:1) is used as mobile phase A and gradient elution program 1 is adopted in Example 1;

[0049] Figure 4 is the fingerprint of Dendrobium nobile Lindl. medicinal materials when the flow rate of 0.3 mL / min is adopted in the flow rate investigation in Example 1;

[0050] Figure 5 is the fingerprint of Dendrobium nobile Lindl. medicinal materials when the flow rate of 0.28 mL / min is adopted in the flow rate investigation in Example 1;

[0051] Figure 6 is the fingerprint of Dendrobium nobile Lindl. medicinal materials when the flow rate of 0.32 mL / min is adopted in the flow rate investigation in Example 1;

[0052] Figure 7 is the fingerprint of Dendrobium nobile Lindl. medicinal materials when the flow rate of 0.35 mL / min is adopted in the flow rate investigation in Example 1;

[0053] Figure 8 is the fingerprint of Dendrobium nobile Lindl. medicinal materials when the flow rate of 0.37 mL / min is adopted in the flow rate investigation in Example 1;

[0054] Figure 9 is the fingerprint of Dendrobium nobile Lindl. medicinal materials when different extraction solvents are adopted in Example 1;

[0055] Figure 10 is the fingerprint of Dendrobium nobile Lindl. medicinal materials when different extraction methods are adopted in Example 1;

[0056] Figure 11 It is the fingerprint of Dendrobium nobile Lindl. medicinal materials with different extraction times in Example 1;

[0057] Figure 12 It is the result chart of the specificity investigation of the fingerprint of Dendrobium nobile Lindl. medicinal materials in Example 1;

[0058] Figure 13 It is the superimposed chart of the fingerprints of 22 batches of Dendrobium nobile Lindl. medicinal materials in Example 1;

[0059] Figure 14 It is the superimposed chart of the fingerprints of 22 batches of Dendrobium nobile Lindl. decoction pieces in Example 1;

[0060] Figure 15 It is the superimposed chart of the fingerprints of 22 batches of Dendrobium nobile Lindl. standard decoctions in Example 1;

[0061] Figure 16 It is the superimposed chart of the fingerprints of 3 batches of Dendrobium nobile Lindl. formula granules in Example 1;

[0062] Figure 17 It is the fingerprint of the control medicinal materials of Dendrobium nobile Lindl. in Example 1;

[0063] Figure 18 It is the comparison chart of the fingerprints of Dendrobium chrysanthum Wall., Dendrobium wardianum Rchb.f., Dendrobium pendulum Roxb., Dendrobium aduncum Lindl. and Dendrobium nobile Lindl. in Example 2;

[0064] Figure 19 It is the comparison chart of the fingerprints of Pholidota chinensis Lindl., Dendrobium thyrsiflorum Rchb.f., Dendrobium fimbriatum Hook. var. oculatum Hook. and Dendrobium nobile Lindl. in Example 2;

[0065] Figure 20 It is the comparison chart of the fingerprints of Dendrobium erianthum Lindl., Dendrobium officinale Kimura et Migo and Dendrobium wardianum Rchb.f. and Dendrobium nobile Lindl.;

[0066] Among them: Peak 2 is dendrobioside G, Peak 6 is costuside D, Peak 9 is costuside C, and Peak 10 is costuside A. Detailed implementation manners

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

[0068] In the prior art, the research on the fingerprint of Dendrobium nobile Lindl. mostly focuses on flavonoid components, while the effective components of Dendrobium nobile Lindl. are alkaloids and sesquiterpenes, which makes it difficult for the prior art to effectively reflect the quality of Dendrobium nobile Lindl. In addition, there are more than 10 kinds of easily confused and fake products of Dendrobium nobile Lindl., such as Dendrobium fimbriatum Hook., Dendrobium chrysanthum Wall., Dendrobium aduncum Lindl., Dendrobium fimbriatum Hook., Dendrobium officinale Kimura et Migo and Pholidota chinensis Lindl. etc. It is often difficult to effectively identify them in the prior art, especially when they are processed into standard decoctions and traditional Chinese medicine formula granules. For this reason, the present invention is proposed.

[0069] It should be noted that the Dendrobium nobile Lindl. slices in the present invention refer to fresh or dried Dendrobium nobile Lindl. specified in the Chinese Pharmacopoeia (2020 Edition), but are not limited thereto. The standard decoction of Dendrobium nobile Lindl. refers to the preparation obtained by decocting, concentrating and freeze-drying at low temperature of the above-mentioned slices with reference to the Technical Requirements for Quality Control and Standard Setting of Traditional Chinese Medicine Formula Granules. The Dendrobium nobile Lindl. formula granules refer to the preparation obtained by water heating extraction, separation, concentration, drying and granulation of the above-mentioned slices with reference to the Technical Requirements for Quality Control and Standard Setting of Traditional Chinese Medicine Formula Granules, but are not limited thereto.

[0070] Specifically, the instruments, materials and sources of medicinal materials used in the following examples 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 (2.1 mm × 150 mm, 1.7 μm), evaporative light scattering detector (Alltech 2000, Alltech Technologies Co., Ltd.), ten-thousandth balance (ME204E, Mettler Toledo), millionth 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.) are both of analytical grade; acetonitrile (Merck KGaA), methanol (Merck KGaA) and formic acid (Merck KGaA) are all of chromatographic grade, and water is ultrapure water (self-made in the laboratory).

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

[0074] Table 1 Information Table of Medicinal Material Sources

[0075]

[0076] Example 1 Construction of the Fingerprint of Dendrobium officinale

[0077] 1. Preparation of the Test Solution

[0078] Medicinal Material: About 2.0 g of the powder of Dendrobium officinale (passing through No. 3 sieve) was accurately weighed, placed in a stoppered conical flask, 50 mL of 70% methanol was added, weighed, ultrasonically treated (power 300 W, frequency 40 kHz) for 45 minutes, centrifuged, the supernatant was evaporated to dryness, the residue was dissolved in 70% methanol and transferred to a 5 mL volumetric flask, made up to the mark with 70% methanol, shaken well, filtered, and the subsequent filtrate was taken to obtain the test solution.

[0079] Sliced Medicinal Material: About 2.0 g of the powder of sliced Dendrobium officinale (passing through No. 3 sieve) was accurately weighed, placed in a stoppered conical flask, 50 mL of 70% methanol was added, weighed, ultrasonically treated (power 300 W, frequency 40 kHz) for 45 minutes, centrifuged, the supernatant was evaporated to dryness, the residue was dissolved in 70% methanol and transferred to a 5 mL volumetric flask, made up to the mark with 70% methanol, shaken well, filtered, and the subsequent filtrate was taken to obtain the test solution.

[0080] Standard Decoction: About 0.5 g of the powder of the standard decoction of Dendrobium officinale was accurately weighed, placed in a stoppered conical flask, 25 mL of 70% methanol was added, weighed, ultrasonically treated (power 300 W, frequency 40 kHz) for 45 minutes, centrifuged, the supernatant was evaporated to dryness, the residue was dissolved in 70% methanol and transferred to a 5 mL volumetric flask, made up to the mark with 70% methanol, shaken well, filtered, and the subsequent filtrate was taken to obtain the test solution.

[0081] Formula Granules: About 1.0 g of the formula granules of Dendrobium officinale was accurately weighed, placed in a stoppered conical flask, 25 mL of 70% methanol was added, weighed, ultrasonically treated (power 300 W, frequency 40 kHz) for 45 minutes, centrifuged, the supernatant was evaporated to dryness, the residue was dissolved in 70% methanol and transferred to a 5 mL volumetric flask, made up to the mark with 70% methanol, shaken well, filtered, and the subsequent filtrate was taken to obtain the test solution.

[0082] 2. Preparation of the Reference Solution

[0083] Accurately weigh 1.255 mg of the reference substance of dendrobioside G, 1.364 mg of the reference substance of costuside D, 1.309 mg of costuside C, and 0.986 mg of costuside A, place them in a 5 mL volumetric flask, add methanol to make a mixed solution containing 245.980 μg of dendrobioside G, 267.344 μg of costuside D, 256.564 μg of costuside C, and 193.256 μg of costuside A per 1 mL as the reference solution.

[0084]

[0085] ​3. Chromatographic conditions

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

[0087] Table 2 Gradient elution program 1

[0088]

[0089] 4. Determination

[0090] Accurately pipette 2 μL of reference solution and 2 μL of test solution respectively, inject into liquid chromatograph, and measure to obtain the result.

[0091] 5. Methodological investigation

[0092] 5.1 Investigation of mobile phase conditions

[0093] This section investigates the effect of mobile phase conditions on fingerprints. Specifically, take two parallel portions of Dendrobium nobile and prepare the test solution according to the method under "1", and then test it according to the following chromatographic conditions.

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

[0098] The experimental results are as follows Figures 1 - 2As shown in the figure, the results show that when the volume ratio of methanol to acetonitrile is 1:1 and 3:1, the separation of each peak can be achieved and the baseline is stable. However, when methanol-acetonitrile (3:1) is used, the separation effect of the chromatographic peaks is better.

[0099] 5.2 Investigation of gradient elution program

[0100] In this part, the influence of the gradient elution program on the fingerprint was investigated. Specifically, Dendrobium officinale Kimura et Migo was taken, and two parallel samples were prepared into test solution according to the method under item "1", and then tested according to the following chromatographic conditions.

[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 carried out according to the regulations 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 carried out according to the regulations in Table 2; the flow rate was 0.3 mL per minute; the column temperature was 40 °C.

[0103] The experimental results are as Figures 2 - 3 shown. It can be seen from the figure that when the gradient elution program specified in Table 4 is used, some chromatographic peaks are difficult to separate. However, when the gradient elution program specified in Table 2 is used, the separation effect of the chromatographic peaks is good.

[0104] 5.3 Investigation of flow rate

[0105] In this part, the influence of the flow rate on the fingerprint was investigated. Specifically, Dendrobium officinale Kimura et Migo was taken, and five parallel samples were prepared into test solution according to the method under item "1", and then tested according to the following chromatographic conditions.

[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 carried out according to the regulations 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 carried out according to the regulations 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, perform gradient elution according to the regulations in Table 2; flow rate is 0.32 mL per minute; column temperature is 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, perform gradient elution according to the regulations in Table 2; flow rate is 0.35 mL per minute; column temperature is 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, perform gradient elution according to the regulations in Table 2; flow rate is 0.37 mL per minute; column temperature is 40°C.

[0111] The experimental results are as Figures 4 to 8 shown. It can be seen from the figure that when the flow rates are 0.28 mL / min, 0.3 mL / min, 0.32 mL / min, 0.35 mL / min, and 0.37 mL / min, the separation of each chromatographic peak can be achieved. Further, when the flow rate is 0.35 mL / min, the separation effect of the chromatographic peak is better and the peak shape is better.

[0112] 5.4 Investigation on the preparation method of test samples

[0113] (1) Investigation on extraction solvents

[0114] Take about 2.0 g of the same batch of Dendrobium officinale Kimura et Migo medicinal material powder (number: G1, passing through No. 3 sieve), in parallel for 6 groups, with 2 portions in each group, accurately weigh, place in a stoppered conical flask, accurately add 50 mL of 100% methanol, 70% methanol, 30% methanol, 100% ethanol, 70% ethanol, and 30% ethanol respectively, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 45 minutes, let it cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, evaporate the filtrate to dryness, dissolve the residue with 70% methanol, transfer to a 5 mL volumetric flask, add 70% methanol to the scale, shake well, filter, and take the subsequent filtrate to obtain. Determine according to the chromatographic conditions specified in item "3". The results are as Figure 9As shown, it can be seen from the figure that when different extraction solvents are used, the fingerprint can show peaks numbered 1 - 10, and the resolution of each peak is good. Further, when 30% ethanol, 100% methanol, 70% methanol, and 30% methanol are used, the response values of each chromatographic peak are relatively high. Especially when 70% methanol is used, the chromatographic peak response is the highest and the peak shape is the best.

[0115] (2) Investigation of extraction methods

[0116] About 2.0 g of the same batch of Dendrobium nobile Lindl. medicinal material powder (No.: G1, passing through No. 3 sieve) was taken, in duplicate groups with 2 portions in each group, accurately weighed, placed in a stoppered conical flask, accurately added 50 mL of 70% methanol, weighed, ultrasonically treated (power 250 W, frequency 40 kHz) for 45 minutes and refluxed for 45 minutes respectively, cooled, weighed again, made up the lost weight with 70% methanol, shaken well, filtered, the filtrate was evaporated to dryness, the residue was dissolved with 70% methanol, transferred to a 5 mL volumetric flask, made up to the mark with 70% methanol, shaken well, filtered, and the subsequent filtrate was taken to obtain the solution. Determination was carried out according to the chromatographic conditions specified in item "3". The results are as Figure 10 shown. It can be seen from the figure that there are no obvious differences in the peak shapes and separation effects of each characteristic peak with different extraction methods. Considering the simplicity of operation, ultrasonic extraction was determined as the extraction method.

[0117] (3) Investigation of extraction time

[0118] About 2.0 g of the same batch of Dendrobium nobile Lindl. medicinal material powder (No.: G1, passing through No. 3 sieve) was taken, accurately weighed, placed in a stoppered conical flask, added 50 mL of 70% methanol, weighed, ultrasonically treated (power 300 W, frequency 40 kHz) for 30 minutes, 45 minutes, and 60 minutes respectively, cooled, weighed again, made up the lost weight with 70% methanol, shaken well, filtered, the filtrate was evaporated to dryness, the residue was dissolved with 70% methanol, transferred to a 5 mL volumetric flask, made up to the mark with 70% methanol, shaken well, filtered, and the subsequent filtrate was taken to obtain the solution. Determination was carried out according to the chromatographic conditions specified in item "3". The results are as Figure 11 shown. It can be seen from the figure that different extraction times have little effect on the fingerprint of Dendrobium nobile Lindl. medicinal materials. To ensure complete extraction, the ultrasonic extraction time was selected as 45 minutes.

[0119] 6 Methodology investigation

[0120] 6.1 Specificity investigation

[0121] About 2.0 g of Dendrobium nobile Lindl. medicinal material powder (No.: G1, passing through No. 3 sieve) was taken, and the test solution was prepared according to the method in item "1". 5 μL of the test solution, the reference solution in item "2", and the blank solvent were accurately pipetted and injected for analysis according to the chromatographic conditions in item "3". The results are as Figure 12As shown, it can be seen from the figure that the test sample chromatogram has the same chromatographic peaks at the retention times corresponding to the reference substance chromatogram, and the blank solvent has no interference, indicating that the method has good specificity.

[0122] 6.2 Precision investigation

[0123] Take about 2.0 g of the powder of Dendrobium nobile Lindl. (serial number: G1, passing through No. 3 sieve), prepare the test sample solution according to the method under item "1", and inject samples repeatedly 6 times according to the chromatographic conditions under item "3". Using peak 2 - dendrobioside G as the reference peak S1 and peak 6 - costuside D as the reference peak S2, the RSD values of the relative retention times of each characteristic peak are in the range of 0.00% - 0.24%, and the RSD values of the relative peak areas are in the range of 0.63% - 4.01%, both less than 5.0%, indicating good precision of the instrument.

[0124] 6.3 Repeatability investigation

[0125] Take about 2.0 g of the powder of Dendrobium nobile Lindl. (serial number: G1, passing through No. 3 sieve), prepare 6 test sample solutions according to the method under item "1", and inject samples for determination according to the chromatographic conditions under item "3". The results show that for the same batch of samples, when determined repeatedly 6 times, using peak 2 - dendrobioside G as the reference peak S1 and peak 6 - costuside D as the reference peak S2, the RSD values of the relative retention times of each characteristic peak are in the range of 0.00% - 0.24%, and the RSD values of the relative peak areas are in the range of 1.51% - 3.80%, both less than 5.0%, indicating good repeatability of the method.

[0126] 6.4 Stability investigation

[0127] Take about 2.0 g of the powder of Dendrobium nobile Lindl. (serial number: G1, passing through No. 3 sieve), prepare the test sample solution according to the method under item "1", and inject samples for determination at 0, 2, 5, 8, 20, and 26 hours respectively according to the chromatographic conditions under item "3". The same test sample solution is analyzed at 0, 2, 5, 8, 20, and 26 hours respectively. Using peak 2 - dendrobioside G as the reference peak S1 and peak 6 - costuside D as the reference peak S2, the RSD values of the relative retention times of each characteristic peak are in the range of 0.00% - 0.28%, and the RSD of the relative peak area is in the range of 0.73% - 4.14%, both less than 5.0%. The test sample solution is stable within 26 hours.

[0128] 7 Establishment of the fingerprint of Dendrobium nobile Lindl.

[0129] 7.1 Determination results of the fingerprint of Dendrobium nobile Lindl.

[0130] Take 22 batches of Dendrobium nobile Lindl., according to the chromatographic conditions under item "3" and the test sample solution preparation method determined under item "1", using peak 2 - dendrobioside G as the reference peak S1, calculate the relative retention times of peaks 1 - 5 and peak S1, and the fingerprints of 22 batches of medicinal materials are shown inFigure 13 as shown

[0131] 7.2 Determination Results of the Fingerprint of Dendrobium nobile Lindl. Decoction Pieces

[0132] Take the Dendrobium nobile Lindl. medicinal materials, prepare the decoction pieces according to the pharmacopoeia, numbered GP1 - GP22. According to the chromatographic conditions in item "3" and the preparation method of the test solution determined in item "1", using peak 2 - dendrobioside G as the reference peak S1, calculate the relative retention times of peaks 1 - 5 and the S1 peak. The fingerprints of 22 batches of decoction pieces are shown Figure 14 as shown

[0133] 7.3 Determination Results of the Fingerprint of Standard Decoction of Dendrobium nobile Lindl.

[0134] Take the Dendrobium nobile Lindl. decoction pieces, refer to the regulations of "Technical Requirements for Quality Control and Standard Setting of Traditional Chinese Medicine Formula Granules", conduct the decoction of the standard decoction, concentrate and freeze - dry after decoction to obtain the freeze - dried powder of the standard decoction. The freeze - dried powder of the standard decoction is numbered GT1 - GT22. According to the chromatographic conditions in item "3" and the preparation method of the test solution determined in item "1", using peak 2 - dendrobioside G as the reference peak S1, calculate the relative retention times of peaks 1 - 5 and the S1 peak. The fingerprints of 22 batches of standard decoctions are shown Figure 15 as shown

[0135] 7.4 Determination Results of the Fingerprint of Dendrobium nobile Lindl. Formula Granules

[0136] Take the Dendrobium nobile Lindl. decoction pieces, decoct with water, filter, concentrate the filtrate into a clear paste, add appropriate excipients, dry (or dry and pulverize), then add appropriate excipients, mix well, and granulate to obtain. The formula granules are numbered CG1 - CG3. According to the chromatographic conditions in item "3" and the preparation method of the test solution determined in item "1", using peak 2 - dendrobioside G as the reference peak S1, calculate the relative retention times of peaks 1 - 5 and the S1 peak. The fingerprints of 3 batches of formula granules are shown Figure 16 as shown

[0137] 7.5 Establishment of the Common Pattern of the Fingerprint of Dendrobium nobile Lindl. Medicinal Materials

[0138] Take the reference Dendrobium nobile Lindl. medicinal materials (Guangdong Yifang Pharmaceutical Co., Ltd.), prepare the reference substance solution of the reference medicinal materials according to the method in item "1". For 22 batches of Dendrobium nobile Lindl. medicinal materials, prepare the test solutions according to the method in item "1", and inject samples for determination according to the chromatographic conditions in item "3" respectively to obtain the fingerprint of the reference Dendrobium nobile Lindl. medicinal materials; use the "Similarity Evaluation Software for Traditional Chinese Medicine Chromatographic Fingerprints" to generate the reference chromatogram by the average method to establish the reference fingerprint of Dendrobium nobile Lindl. medicinal materials( Figure 17 ).

[0139] The test sample chromatogram should exhibit 10 characteristic peaks, and the retention times of these 10 characteristic peaks should correspond to those in the reference chromatogram of the reference medicinal material; the peak corresponding to the dendrobioside G reference is peak S1, and calculate the relative retention times of peaks 1, 3-5; the peak corresponding to the costuside D reference is peak S2, and calculate the relative retention times of peaks 7-10 and peak S, and the relative retention times should be within the range of ±10% of the specified values. The specified values 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), 1.28 (peak 10).

[0140] Example 2: Application of the fingerprint of Dendrobium nobile Lindl. medicinal material

[0141] This example provides a method for identifying Dendrobium nobile Lindl. medicinal material. Specifically, take Dendrobium nobile Lindl., Dendrobium chrysotoxum Lindl., Dendrobium officinale Kimura et Migo, Dendrobium wardianum, Pholidota chinensis Lindl., Dendrobium thyrsiflorum Rchb. f., Dendrobium fimbriatum Hook., Dendrobium fimbriatum Hook. var. oculatum Hook., Dendrobium chrysanthum Wall., Dendrobium wardianum, Dendrobium pendulum Roxb. and Dendrobium aduncum Lindl., and prepare the test sample solution according to the conditions under item "1" in Example 1, and inject the sample for testing according to the chromatographic conditions under item "3". The results are shown in the figure ( Figures 18 - 20 ). The results show that there are significant differences between the fingerprint chromatograms of Dendrobium fimbriatum Hook., Dendrobium chrysanthum Wall., Dendrobium aduncum Lindl., Dendrobium fimbriatum Hook. var. oculatum Hook., Dendrobium officinale Kimura et Migo and Pholidota chinensis Lindl. and that of Dendrobium nobile Lindl. Among them, the fingerprint chromatograms of Dendrobium fimbriatum Hook., Dendrobium chrysanthum Wall., Dendrobium aduncum Lindl., Dendrobium fimbriatum Hook. var. oculatum Hook., Dendrobium officinale Kimura et Migo and Pholidota chinensis Lindl. do not have the 10 characteristic peaks in the fingerprint chromatogram of Dendrobium nobile Lindl. Dendrobium chrysanthum Wall. has a unique peak 11 between 45-50 minutes, Pholidota chinensis Lindl. has a specific peak 18 between 25-30 minutes, Dendrobium thyrsiflorum Rchb. f. has specific peaks 13, 14, 15 between 10-15 minutes, and Dendrobium chrysotoxum Lindl. has a unique peak 20 between 45-50 minutes. To sum up, the fingerprint method established by the present invention can effectively distinguish various adulterants of Dendrobium nobile Lindl.

[0142] The above is the preferred implementation mode of the invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements are also regarded as the protection scope of the present invention.

Claims

1. A method for constructing the fingerprint of Dendrobium nobile Lindl. medicinal materials, their decoction pieces, standard decoctions, and formula granules, characterized in that, it includes: Extracting Dendrobium nobile Lindl. medicinal materials, their decoction pieces, standard decoctions, or formula granules with an extraction solvent to obtain a test solution; Dissolving the dendrobioside G reference substance, costuside D reference substance, costuside C reference substance, and costuside A reference substance in methanol with a volume fraction of 30 vol% - 100 vol% to obtain a reference substance solution; Measuring the test solution and the reference substance solution with a liquid chromatograph to obtain the fingerprint of Dendrobium nobile Lindl. medicinal materials, their decoction pieces, standard decoctions, or formula granules; Among them, the chromatographic column of the liquid chromatograph uses octadecylsilane-bonded silica gel 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: 0 min - 20 min, mobile phase A changes from 16% → 17%, mobile phase B changes from 84% → 83%; 20 min - 25 min, mobile phase A changes from 17% → 25%, mobile phase B changes from 83% → 75%; 25 min - 30 min, mobile phase A is 25%, mobile phase B is 75%; 30 min - 40 min, mobile phase A changes from 25% → 30%, mobile phase B changes from 75% → 70%; 40 min - 50 min, mobile phase A changes from 30% → 35%, mobile phase B changes from 70% → 65%; 50 min - 55 min, mobile phase A changes from 35% → 36%, mobile phase B changes from 65% → 64%; 55 min - 60 min, mobile phase A changes from 36% → 40%, mobile phase B changes from 64% → 60%; 60 min - 65 min, mobile phase A changes from 40% → 45%, mobile phase B changes from 60% → 55%; Among them, the extraction solvent is methanol with a volume fraction of 30 vol% - 100 vol% or ethanol with a volume fraction of 30 vol% - 100 vol%; in mobile phase A, the volume ratio of methanol to acetonitrile is 1:1 - 3:

1.

2. The method for constructing the fingerprint of Dendrobium nobile Lindl. medicinal materials, their decoction pieces, standard decoctions, and formula granules according to claim 1, characterized in that, the volume ratio of methanol to acetonitrile in mobile phase A is 3:1; mobile phase B is a 0.1 vol% formic acid aqueous solution.

3. The method for constructing the fingerprint of Dendrobium nobile Lindl. medicinal materials, their decoction pieces, standard decoctions, and formula granules according to claim 1, characterized in that, the column length of the chromatographic column is 100 mm - 150 mm, the column diameter is 2 mm - 3 mm, the column temperature is 20 °C - 45 °C, and the particle size of the stationary phase is 1.5 μm - 2.1 μm; the flow rate of the liquid chromatograph is 0.25 mL / min - 0.4 mL / min, the injection volume of the test solution is 1 μL - 3 μL, and the injection volume of the reference substance solution is 1 μL - 3 μL.

4. The method for constructing the fingerprint of Dendrobium nobile Lindl. medicinal materials, their decoction pieces, standard decoctions, and formula granules according to claim 1 or 3, characterized in that, The column length of the chromatographic column is 150 mm, the column diameter is 2.1 mm, the column temperature is 40 °C, and the particle size of the stationary phase is 1.7 μm; 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 the fingerprint of the Dendrobium officinale Kimura et Migo medicinal materials, their cut pieces, standard decoctions, and formula granules as described in claim 1, characterized in that, It is detected by an evaporative light scattering detector, the drift tube temperature is 100 °C to 120 °C, the carrier gas is air, and the carrier gas flow rate is 2 L / min to 5 L / min.

6. The method for constructing the fingerprint of the Dendrobium officinale Kimura et Migo medicinal materials, their cut pieces, standard decoctions, and formula granules as described in claim 5, characterized in that, The drift tube temperature is 110 °C, the carrier gas is air, and the carrier gas flow rate is 3.2 L / min.

7. The method for constructing the fingerprint of the Dendrobium officinale Kimura et Migo medicinal materials, their cut pieces, standard decoctions, and formula granules as described in claim 1, characterized in that, In the step of extracting the Dendrobium officinale Kimura et Migo medicinal materials, their cut pieces, 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 for extraction.

8. The method for constructing the fingerprint of the Dendrobium officinale Kimura et Migo medicinal materials, their cut pieces, standard decoctions, and formula granules as described in claim 1 or 7, characterized in that, In the step of extracting the Dendrobium officinale Kimura et Migo medicinal materials, their cut pieces, standard decoctions, or formula granules with an extraction solvent to obtain a test solution, an ultrasonic extraction process is used for extraction, wherein the ultrasonic power is 100 W to 500 W, the frequency is 20 kHz to 50 kHz, and the extraction time is 30 min to 60 min.

9. The method for constructing the fingerprint of the Dendrobium officinale Kimura et Migo medicinal materials, their cut pieces, standard decoctions, and formula granules as described in claim 1, characterized in that, The extraction solvent is 70 vol% methanol.

10. The method for constructing the fingerprint of the Dendrobium officinale Kimura et Migo medicinal materials, their cut pieces, standard decoctions, and formula granules as described in claim 1, characterized in that, In the reference solution, the concentration of dendrobioside G reference substance is 220 μg / mL to 260 μg / mL, the concentration of costuside D reference substance is 250 μg / mL to 300 μg / mL, the concentration of costuside C reference substance is 250 μg / mL to 300 μg / mL, and the concentration of costuside A reference substance is 150 μg / mL to 220 μg / mL.

11. The method for constructing the fingerprint of the Dendrobium officinale Kimura et Migo medicinal materials, their cut pieces, standard decoctions, and formula granules as described in claim 1, characterized in that, The fingerprint includes 10 common peaks, among which, peak 2 is dendrobioside G, peak 6 is costuside D, peak 9 is costuside C, and peak 10 is costuside A; Taking peak 2 as S1 peak, calculate the relative retention times of peaks 1, 3 - 5, and the relative retention times of each peak are within ±10% of the specified value, wherein the specified values of peaks 1, 3 - 5 are respectively: 0.68、1.22、1.85、2.29； Taking peak 6 as S2 peak, calculate the relative retention times of peaks 7 - 10, and the relative retention times of each peak are within ±10% of the specified value, wherein, The specified values for peaks 7 to 10 are respectively: 1.05、1.09、1.14、1.28。 12. A method for identifying Dendrobium nobile Lindl., used to distinguish Dendrobium nobile Lindl. from its counterfeits, characterized in that, it includes: providing the substance to be identified; constructing the fingerprint of the substance to be identified by the method for constructing the fingerprint of the medicinal materials and decoction pieces, standard decoctions, and formula granules of Dendrobium nobile Lindl. described in any one of claims 1 to 11; if only peaks 1 to 10 are presented in the fingerprint, the substance to be identified includes or is Dendrobium nobile Lindl.; otherwise, it is a counterfeit; wherein, the counterfeits include Dendrobium wardianum, Dendrobium officinale, Dendrobium chrysanthum, Pholidota chinensis, Dendrobium thyrsiflorum, Dendrobium fimbriatum, Dendrobium pendulum, Dendrobium chrysotoxum, Dendrobium erythraeum, Dendrobium wardianum, Dendrobium chrysotoxum, and Dendrobium aduncum.

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