Method for constructing characteristic map of Acorus calamus, and method for identifying Acorus calamus and its counterfeit products

Abstract

The present application relates to the technical field of identification of traditional Chinese medicine, and in particular to a method for constructing a characteristic spectrum of Acorus calamus and a method for identifying Acorus calamus. The method for constructing the characteristic spectrum of Acorus calamus comprises: taking a test sample of Acorus calamus, adding water for extraction to obtain an extract, and then extracting the extract with petroleum ether to prepare a test solution; subjecting the test solution to liquid chromatography detection, and constructing a characteristic spectrum of Acorus calamus based on the measured liquid chromatogram; the conditions for liquid chromatography detection include: using octadecylsilane bonded silica gel as a filler, using a mixture of methanol and acetonitrile as mobile phase A, and water as mobile phase B for gradient elution. The method for constructing the characteristic spectrum of Acorus calamus of the present application can more comprehensively reflect the characteristic components of Acorus calamus, compare the characteristic spectrum of the medicinal material to be tested with that of Acorus calamus, and achieve identification based on the presence or absence of characteristic peaks. The identification method is intuitive, simple, and efficient.

Description

Technical Field

[0001] The present application relates to the technical field of identification of traditional Chinese medicines, and in particular to a method for constructing a characteristic spectrum of Acorus calamus and a method for identifying Acorus calamus and its counterfeits. Background Art

[0002] Acorus tatarinowii Schott is the dried rhizome of Acorus tatarinowii Schott, a plant of the Araceae family. It is dug in autumn and winter, the fibrous roots and mud are removed, and it is sun-dried. It is oblate-cylindrical, mostly curved, often branched, 3cm to 20cm long, 0.3cm to 1cm in diameter, the surface is brown or gray-brown, rough, with unevenly dense segments, the internodes are 0.2 to 0.8cm long, with fine longitudinal lines, fibrous roots or dot-shaped root scars remaining on one side, and the leaf scars are triangular, arranged alternately on the left and right, some with hairy scale-like leaf base remnants on them, hard in texture, fibrous in cross section, off-white or slightly reddish, with obvious endodermal rings, and numerous vascular bundle dots and brown oil cells visible.

[0003] Acorus calamus has an aromatic aroma and a bitter, slightly pungent flavor. It has the effects of opening the orifices, clearing phlegm, refreshing the mind, improving intelligence, and stimulating the appetite. It is used to treat coma, epilepsy, forgetfulness, insomnia, tinnitus, deafness, abdominal distension without hunger, and dysentery caused by muteness. The chemical components of Acorus calamus can be broadly divided into volatile and non-volatile components. Volatile components are numerous, primarily including phenylpropanoids (simple phenylpropanoids, lignans, and coumarins) and terpenes (monoterpenes, sesquiterpenes, diterpenes, and triterpenes). Non-volatile components primarily include alkaloids, aldehydes and acids, quinones and ketones, sterols, amino acids, and carbohydrates.

[0004] Currently, there are few studies on the high performance liquid chromatography fingerprint or characteristic spectrum for the identification of authentic Acorus calamus. Only one method has been found to report the use of sub-ultra-high performance liquid chromatography to identify Acorus calamus from counterfeits. This method uses the peak area size of the characteristic peak for distinction, and the detection results are not intuitive enough.

[0005] Therefore, it is necessary to develop a convenient and efficient identification method for Acorus tatarinowii. Summary of the Invention

[0006] Based on this, one or more embodiments of the present application provide a method for constructing a characteristic map of Acorus calamus and a method for identifying Acorus calamus and its counterfeits.

[0007] The technical solution of this application includes the following:

[0008] A method for constructing a characteristic spectrum of Acorus tatarinowii, comprising:

[0009] A test sample of Acorus calamus is extracted with water to obtain an extract, and the extract is then extracted with petroleum ether to prepare a test sample solution;

[0010] The test solution is subjected to liquid chromatography to prepare a characteristic spectrum of the Acorus tatarinowii;

[0011] The liquid chromatography detection conditions include: using octadecylsilane bonded silica gel as a filler, using a mixture of methanol and acetonitrile as mobile phase A, and water as mobile phase B for gradient elution.

[0012] Preferably, the gradient elution procedure comprises:

[0013] From 0 to 5 min, the volume percentage of the mobile phase A increased from 10% to 34%;

[0014] From 5 to 13 minutes, the volume percentage of the mobile phase A increased from 34% to 35%;

[0015] From 13 to 13.5 min, the volume percentage of the mobile phase A increased from 35% to 50%;

[0016] From 13.5 to 20 minutes, the volume percentage of the mobile phase A increased from 50% to 62%;

[0017] From 20 to 25 minutes, the volume percentage of the mobile phase A increased from 62% to 90%;

[0018] 25-26 min, the volume percentage of the mobile phase A decreased from 90% to 10%;

[0019] From 26 to 30 minutes, the volume percentage of the mobile phase A was maintained at 10%.

[0020] Preferably, the conditions for liquid chromatography detection further include at least one of the following:

[0021] (1) The detection wavelength is 270~280nm;

[0022] (2) Column temperature is 35-45°C;

[0023] (3) Flow rate: 0.18–0.22 mL / min;

[0024] (4) In the mobile phase A, the volume ratio of methanol to acetonitrile is (0.8-1.2):1;

[0025] (5) The injection volume is 1~3μL.

[0026] Preferably, the Acorus calamus sample is extracted with water, comprising:

[0027] The Acorus calamus test sample and the water were mixed in a mass volume ratio of 1 g: 15-25 mL, and heated under reflux for 50-70 min to obtain the extract;

[0028] Furthermore, the heating reflux temperature is 95-105°C.

[0029] A method for identifying Acorus calamus and its counterfeits, comprising:

[0030] Take the medicinal material to be tested, add water to extract to obtain an extract, and then extract the extract with petroleum ether to prepare a test solution;

[0031] Performing liquid chromatography on the test solution to obtain a chromatogram of the test medicinal material;

[0032] The characteristic spectrum of Acorus tatarinowii prepared according to the characteristic spectrum construction method described above is used as the reference spectrum of Acorus tatarinowii;

[0033] Comparing the chromatogram of the medicinal material to be tested with the chromatogram of the Acorus tatarinowii reference to identify whether the medicinal material to be tested is Acorus tatarinowii;

[0034] The liquid chromatography detection conditions include: using octadecylsilane bonded silica gel as a filler, using a mixture of methanol and acetonitrile as mobile phase A, and water as mobile phase B for gradient elution.

[0035] Preferably, the gradient elution procedure comprises:

[0036] From 0 to 5 min, the volume percentage of the mobile phase A increased from 10% to 34%;

[0037] From 5 to 13 minutes, the volume percentage of the mobile phase A increased from 34% to 35%;

[0038] From 13 to 13.5 min, the volume percentage of the mobile phase A increased from 35% to 50%;

[0039] From 13.5 to 20 minutes, the volume percentage of the mobile phase A increased from 50% to 62%;

[0040] From 20 to 25 minutes, the volume percentage of the mobile phase A increased from 62% to 90%;

[0041] 25-26 min, the volume percentage of the mobile phase A decreased from 90% to 10%;

[0042] From 26 to 30 minutes, the volume percentage of the mobile phase A was maintained at 10%.

[0043] Preferably, the conditions for liquid chromatography detection further include at least one of the following:

[0044] (1) The detection wavelength is 270~280nm;

[0045] (2) Column temperature is 35-45°C;

[0046] (3) Flow rate: 0.18–0.22 mL / min;

[0047] (4) In the mobile phase A, the volume ratio of methanol to acetonitrile is (0.8-1.2):1;

[0048] (5) The injection volume is 1~3μL.

[0049] Preferably, the medicinal material to be tested is taken and extracted with water, including:

[0050] The medicinal material to be tested and the water are mixed in a mass volume ratio of 1 g: 15-25 mL, and heated under reflux for 50-70 min to obtain the extract;

[0051] Furthermore, the heating reflux temperature is 95-105°C.

[0052] Preferably, if the chromatogram of the medicinal material to be tested has the following characteristics, the medicinal material to be tested is Acorus tatarinowii:

[0053] Presenting at least 4 characteristic peaks: peak 1, peak 2, peak 3 and peak 4, wherein peak 3 is the characteristic peak of β-asarone;

[0054] Taking peak 3 as the reference peak, the relative retention times of the remaining peaks are: peak 1: 0.525 ± 10%; peak 2: 0.766 ± 10%; peak 4: 1.016 ± 10%;

[0055] Otherwise it is a fake.

[0056] Preferably, the counterfeit product is selected from Acorus calamus, Acorus foeniculum, Bergenia or Acorus calamus.

[0057] The method for constructing the characteristic spectrum of Acorus calamus of the present application can more intuitively reflect the characteristic components of Acorus calamus. In addition, the construction method is time-saving and requires less reagents, which can improve detection efficiency.

[0058] The identification method of the present application compares the characteristic spectra of the medicinal material to be tested with that of Acorus calamus, and identification can be achieved based on the presence or absence of characteristic peaks. The identification method is intuitive, simple, and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the specific implementation methods or the description of the prior art. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0060] Figure 1The comparison results of chromatographic detection of different types of mobile phase A in Example 1 of the present application are shown.

[0061] Figure 2 The comparison results of chromatographic detection of different types of mobile phase B in Example 1 of the present application are shown.

[0062] Figure 3 This is the specificity investigation result in the methodological test of Example 1 of this application.

[0063] Figure 4 This is the result of the repeatability study in the methodological test of Example 1 of the present application.

[0064] Figure 5 This is the result of the precision investigation in the methodological test of Example 1 of this application.

[0065] Figure 6 This is the intermediate precision investigation result in the methodological test of Example 1 of this application.

[0066] Figure 7 This is the durability investigation result in the methodological test of Example 1 of this application.

[0067] Figure 8 This is the result of the 0-24h sample stability test in the methodological test of Example 1 of this application.

[0068] Figure 9 This is an overlay of the liquid chromatograms of 20 batches of Acorus tatarinowii tested in Example 2 of the present application.

[0069] Figure 10 This is an overlay of the liquid chromatograms of three batches of Acorus calamus tested in Example 3 of the present application.

[0070] Figure 11 This is an overlay of the liquid chromatograms of four batches of Acorus calamus tested in Example 3 of the present application.

[0071] Figure 12 This is an overlay of the liquid chromatograms of four batches of Acorus calamus tested in Example 3 of the present application.

[0072] Figure 13 This is an overlay of the liquid chromatograms of three batches of Bergenia tested in Example 3 of the present application.

[0073] Figure 14 This is a comparison result of the reference atlas of Acorus calamus, Acorus foeniculum, Acorus 9-section and Bergenia obtained in Example 3 of the present application and the reference atlas of Acorus calamus in Example 2. DETAILED DESCRIPTION

[0074] Below in conjunction with embodiment and example, further elaborate the application.It should be understood that these examples are only used to illustrate the application and are not used to limit the scope of the application.In addition, it should be understood that after reading the content taught in this application, those skilled in the art can make various changes or modifications to the application, and these equivalent forms also fall within the protection scope of the claims appended hereto.

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0076] In this application, "further", "preferably" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0077] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0078] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution within the numerical interval is considered continuous and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as every numerical value between these two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two numerical endpoints of the numerical range, as well as every integer between the two endpoints. In addition, when multiple ranges are provided to describe a feature or characteristic, these ranges can be combined. In other words, unless otherwise specified, ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0079] In this application, weight can be mass units known in the chemical industry, such as μg, mg, g, and kg.

[0080] In the present application, in the chromatogram of liquid phase detection, the horizontal axis is the retention time in minutes (min), and the vertical axis is the signal intensity (AU).

[0081] Acorus calamus is a traditional aromatic Chinese medicine for invigorating the mind. It can invigorate the mind, soothe the spirit, improve intelligence, and eliminate dampness and expectoration. It is commonly used for epilepsy, phlegm syncope, fever coma, amnesia, insomnia, aphasia due to stroke, etc., and is widely used clinically. At present, the identification or detection methods for Acorus calamus are mostly aimed at the control of water content and extracts. The control of the index components mainly reflects the content of volatile oil. In order to further improve and optimize the identification or detection methods for Acorus calamus, one aspect of the present application provides a method for constructing a characteristic spectrum of Acorus calamus, which can be used to more characteristically reflect the intrinsic chemical components of the water extract of Acorus calamus, and can be used to provide a reference spectrum required for identification, so as to achieve intuitive and rapid identification of Acorus calamus.

[0082] The method for constructing a characteristic spectrum of Acorus tatarinowii according to one embodiment of the present application includes:

[0083] Take the Acorus calamus test sample, add water to extract, obtain an extract, and then extract the extract with petroleum ether to prepare a test sample solution;

[0084] The test solution is subjected to liquid chromatography detection, and a characteristic spectrum of Acorus tatarinowii is constructed according to the measured liquid chromatogram;

[0085] The conditions for liquid chromatography detection include: using octadecylsilane bonded silica gel as a filler, a mixture of methanol and acetonitrile as mobile phase A, and water as mobile phase B for gradient elution.

[0086] Preferably, the gradient elution procedure includes:

[0087] From 0 to 5 min, the volume ratio of mobile phase A increased from 10% to 34%;

[0088] From 5 to 13 min, the volume ratio of mobile phase A increased from 34% to 35%;

[0089] From 13 to 13.5 min, the volume percentage of mobile phase A increased from 35% to 50%;

[0090] From 13.5 to 20 min, the volume percentage of mobile phase A increased from 50% to 62%;

[0091] From 20 to 25 min, the volume percentage of mobile phase A increased from 62% to 90%;

[0092] From 25 to 26 min, the volume percentage of mobile phase A decreased from 90% to 10%;

[0093] From 26 to 30 min, the volume percentage of mobile phase A was maintained at 10%.

[0094] Preferably, the detection wavelength is 270-280 nm.

[0095] Preferably, the column temperature is 35-45°C.

[0096] Preferably, the flow rate is 0.18~0.22mL / min.

[0097] Preferably, in mobile phase A, the volume ratio of methanol to acetonitrile is (0.8-1.2):1.

[0098] Preferably, the injection volume is 1-3 μL.

[0099] In one embodiment, a sample of Acorus calamus is taken and water is added for extraction, comprising:

[0100] Mix the Acorus calamus sample and water in a mass volume ratio of 1 g: 15-25 mL, and heat under reflux for 50-70 min to obtain an extract;

[0101] Furthermore, the heating reflux temperature is 95-105°C.

[0102] As a commonly used Chinese herbal medicine, Acorus calamus is often confused with its counterfeit counterparts in actual use. Therefore, identifying Acorus calamus is one of the purposes of this application. By comparing the characteristic spectra of the tested medicinal material with the standard product constructed using the construction method of this application, the present application can visually identify Acorus calamus and its counterfeits (including identifying Acorus calamus with the common confused products Acorus tatarinowii, Acorus foeniculum, Bergenia chinensis, and Acorus tatarinowii) by the presence or absence of characteristic peaks. This method is accurate, rapid, and efficient, providing an important reference for the quality control of Acorus calamus.

[0103] The method for identifying Acorus calamus and its counterfeit products according to one embodiment of the present application includes:

[0104] Take the medicinal material to be tested, add water to extract, obtain an extract, and then extract the extract with petroleum ether to prepare a solution of the product to be tested;

[0105] The solution of the test product is subjected to liquid chromatography to obtain a chromatogram of the test medicinal material;

[0106] A characteristic spectrum of Acorus tatarinowii prepared by the method for constructing a characteristic spectrum described in any of the above technical solutions is used as a reference spectrum of Acorus tatarinowii;

[0107] Comparing the chromatogram of the tested medicinal material with the reference chromatogram of Acorus tatarinowii to identify whether the tested medicinal material is Acorus tatarinowii;

[0108] The conditions for liquid chromatography detection include: using octadecylsilane bonded silica gel as a filler, a mixture of acetonitrile and methanol as mobile phase A, and water as mobile phase B for gradient elution.

[0109] The medicinal materials to be tested can be Acorus calamus and common Acorus calamus counterfeits, such as Acorus tatarinowii, Acorus foeniculum, Acorus bergenia and Acorus tatarinowii.

[0110] Preferably, the gradient elution procedure includes:

[0111] From 0 to 5 min, the volume percentage of mobile phase A increased from 10% to 34%;

[0112] From 5 to 13 min, the volume percentage of mobile phase A increased from 34% to 35%;

[0113] From 13 to 13.5 min, the volume percentage of mobile phase A increased from 35% to 50%;

[0114] From 13.5 to 20 min, the volume percentage of mobile phase A increased from 50% to 62%;

[0115] From 20 to 25 min, the volume percentage of mobile phase A increased from 62% to 90%;

[0116] From 25 to 26 min, the volume percentage of mobile phase A decreased from 90% to 10%;

[0117] From 26 to 30 min, the volume percentage of mobile phase A was maintained at 10%.

[0118] Preferably, the detection wavelength is 270-280 nm.

[0119] Preferably, the column temperature is 35-45°C.

[0120] Preferably, the flow rate is 0.18~0.22mL / min.

[0121] Preferably, in mobile phase A, the volume ratio of methanol to acetonitrile is (0.8-1.2):1.

[0122] Preferably, the injection volume is 1-3 μL.

[0123] In one embodiment, the medicinal material to be tested is taken and water is added for extraction, including:

[0124] Mix the medicinal material to be tested and water in a mass volume ratio of 1 g: 15-25 mL, heat and reflux for 50-70 minutes to obtain an extract;

[0125] Furthermore, the heating reflux temperature is 95-105°C.

[0126] Preferably, the chromatogram of the medicinal material to be tested has the following characteristics, and the medicinal material to be tested is Acorus tatarinowii:

[0127] Presenting at least 4 characteristic peaks: peak 1, peak 2, peak 3 and peak 4, wherein peak 3 is the characteristic peak of β-asarone;

[0128] Taking peak 3 as the reference peak, the relative retention times of the remaining peaks are: peak 1: 0.525 ± 10%; peak 2: 0.766 ± 10%; peak 4: 1.016 ± 10%;

[0129] Otherwise it is a fake.

[0130] Preferably, the counterfeit product is selected from Acorus calamus, Acorus foeniculum, Bergenia or Acorus calamus.

[0131] The following are some specific examples.

[0132] The sources of the medicinal materials used in the following examples are shown in Table 1. Other raw materials and reagents not marked can be obtained from the market or can be prepared by those skilled in the art according to known methods.

[0133] Table 1 Sources of Acorus calamus and its counterfeit medicinal materials

[0134]

[0135] Example 1

[0136] This example describes the construction process of the characteristic spectrum of Acorus tatarinowii and the methodological verification of its liquid phase analysis method.

[0137] 1. Constructing a characteristic map of Acorus tatarinowii

[0138] 1.1 Determine the preparation method of the test solution

[0139] 1.1.1 Preliminary preparation method

[0140] Take the powder of Acorus tatarinowii (the source of the medicinal material is shown in Table 1), add water to decoct, let cool, centrifuge at 4000 rpm for 10 min, take the supernatant, place it in a separatory funnel, extract with petroleum ether, combine the petroleum ether solutions, and recover the solution under reduced pressure to dryness. Dissolve the residue in methanol solution, transfer it to a 5 mL volumetric flask, add 70% methanol to the scale, shake well, filter, and take the filtrate to obtain the product.

[0141] 1.1.2 Investigation of the preparation method of the test solution

[0142] In the following (1) to (5), different water addition amounts, different decoction times, different petroleum ether extraction times, different petroleum ether extraction volumes, and different concentrations of methanol as re-dissolution solvents were investigated respectively.

[0143] (1) Investigation of water addition amount

[0144] Take 3 batches of Acorus tatarinowii medicinal materials (see Table 1 for medicinal material sources) powder, pass through No. 3 sieve, take 1g of each into a stoppered conical flask, add 10mL, 20mL, and 30mL of water respectively, decoct for 60min, let cool, centrifuge at 4000rpm for 10min, take the supernatant, put it into a separatory funnel, extract with petroleum ether 3 times, 20mL each time, combine the petroleum ether solutions, recover the solution under reduced pressure to dryness, dissolve the residue in 70% methanol, transfer to a 5mL volumetric flask, add 70% methanol to the scale, shake well, filter, and take the filtrate to prepare the test solution.

[0145] (2) Investigation of decoction time

[0146] Take 4 batches of Acorus tatarinowii medicinal materials (see Table 1 for medicinal material sources) powder, pass through No. 3 sieve, take 1g of each into a stoppered conical flask, add 20mL of water, decoct for 15min, 30min, 60min, and 90min respectively, let cool, centrifuge at 4000rpm for 10min, take the supernatant, put it into a separatory funnel, extract with petroleum ether 3 times, 20mL each time, combine the petroleum ether solutions, reduce pressure and recover the solution to dryness, add 70% methanol to dissolve the residue, transfer it to a 5mL volumetric flask, add 70% methanol to the scale, shake well, filter, and take the filtrate to prepare the test solution.

[0147] (3) Investigation of extraction times

[0148] Take 3 batches of Acorus tatarinowii medicinal materials (see Table 1 for medicinal material sources) powder, pass through No. 3 sieve, take 1g of each into a stoppered conical flask, add 20mL of water respectively, decoct for 60min, let cool, centrifuge at 4000rpm for 10min, take the supernatant, put it into a separatory funnel, extract with petroleum ether 2 times, 3 times and 4 times, 20mL each time, combine the petroleum ether solutions, recover the solution under reduced pressure to dryness, dissolve the residue in 70% methanol, transfer to a 5mL volumetric flask, add 70% methanol to the scale, shake well, filter, and take the filtrate to prepare the test solution.

[0149] (4) Investigation of extraction volume

[0150] Take 3 batches of Acorus tatarinowii medicinal materials (see Table 1 for medicinal material sources) powder, pass through No. 3 sieve, take 1g of each into a stoppered conical flask, add 20mL of water respectively, decoct for 60min, let cool, centrifuge at 4000rpm for 10min, take the supernatant, put it into a separatory funnel, and extract with petroleum ether three times, each time with 10mL, 20mL and 30mL respectively. Combine the petroleum ether solutions, recover the solution under reduced pressure to dryness, dissolve the residue in 70% methanol, transfer it to a 5mL volumetric flask, add 70% methanol to the scale, shake well, filter, and take the filtrate to prepare the test solution.

[0151] (5) Investigation of reconstitution solvent

[0152] Take 4 batches of Acorus tatarinowii medicinal materials (see Table 1 for medicinal material sources) powder, pass through No. 3 sieve, take 1g and put it into a stoppered conical flask, add 20mL of water respectively, decoct for 60min, cool, centrifuge at 4000rpm for 10min, take the supernatant, put it into a separatory funnel, extract with petroleum ether 3 times, 20mL each time, combine the petroleum ether solution, reduce pressure and recover the solution to dryness, add pure water (0% methanol methanol solution), 30% methanol solution, 50% methanol solution, 70% methanol solution and pure methanol (100% methanol solution) to dissolve the residue, transfer it to a 5mL volumetric flask, add 70% methanol to the scale, shake well, filter, and take the filtrate to prepare the test solution.

[0153] Take the test solution prepared in (1) to (5) above, and inject and measure it according to the chromatographic conditions under "1.3". Record the results of the chromatographic test and calculate the "total peak area to sample weight ratio" and "peak 3 area percentage". The results are shown in Table 2.

[0154] The main characteristic component of Acorus calamus is Peak 3 (β-asarone). Considering the stability and intuitiveness of the chromatogram, sample preparation conditions with a large ratio of total peak area to sample weight and a small percentage of Peak 3 area were selected. In addition, when extracting with different volumes of extraction solvent, an extraction volume of about 20 mL per time is more appropriate. Too little will easily cause emulsification, requiring long-term heating to achieve emulsification and stratification, while too much will prevent sufficient contact between the two phases.

[0155] Table 2 Results of sample preparation conditions

[0156]

[0157] 1.1.3 Established method for preparing test solution

[0158] Based on the investigation process in 1.1.2 and the calculation results in Table 2, and considering factors such as energy, time cost, and economic benefits, the following conditions were determined for the preparation of the test solution: Take the powdered Acorus tatarinowii, pass it through a No. 3 sieve, take 1 g, place it in a stoppered conical flask, add 20 mL of water, and boil for 60 minutes. Let it cool, centrifuge it at 4000 rpm for 10 minutes, remove the supernatant, place it in a separatory funnel, and extract it three times with 20 mL of petroleum ether. Combine the petroleum ether solutions, recover the solution under reduced pressure 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. In this article, "decoction" in the aqueous extraction process of Chinese medicinal materials is equivalent to "heating under reflux," that is, adding 20 mL of water and heating under reflux at 120-130°C for 50-70 minutes. The instrument for heating reflux can be an electric heating mantle or an instrument with equivalent heating function, which continuously provides the temperature for reflux of the solvent. There is no restriction on the model of the instrument, for example, it can be a TC-15 jacketed thermostat (electric heating mantle).

[0159] 1.2 Preparation of reference solution

[0160] Take an appropriate amount of β-asarone reference substance (batch number: 112018-201601, China Food and Drug Inspection Institute), accurately weigh it, and add methanol to make a solution containing 50 μg of β-asarone per 1 mL, which is used as the reference substance solution.

[0161] 1.3 Determine the liquid chromatography detection method

[0162] 1.3.1 Preliminary liquid chromatography detection method

[0163] Use octadecylsilane bonded silica gel as the packing (column length, 150 mm, inner diameter, 2.1 mm, particle size, 1.6 μm); use an organic solvent as mobile phase A and water as mobile phase B, using a gradient elution as specified in Table 3; flow rate, 0.2 mL / min; column temperature, 40°C; detection wavelength, 275 nm. The number of theoretical plates, calculated based on β-asarone, should be no less than 5000.

[0164] Table 3 Gradient elution table

[0165]

[0166] 1.3.2 Investigation of liquid chromatography detection method

[0167] (1) Investigation of mobile phase A

[0168] Octadecylsilane bonded silica gel was used as the packing (column length, 150 mm, inner diameter, 2.1 mm, particle size, 1.6 μm). Gradient elution was performed using various organic solvents as mobile phase A and water as mobile phase B, as specified in Table 3. The flow rate was 0.2 mL / min, the column temperature was 40°C, and the detection wavelength was 275 nm. Mobile phase A options included: pure methanol; methanol:acetonitrile = 3:1; methanol:acetonitrile = 2:1; methanol:acetonitrile = 1:1; methanol:acetonitrile = 1:2; methanol:acetonitrile = 1:3; and pure acetonitrile.

[0169] The comparison results of chromatographic detection of different types of mobile phase A are as follows Figure 1 ,according to Figure 1 The higher the methanol ratio, the greater the separation between peak 3 and peak 4. However, if the methanol ratio is too high, the peak shape of peak 2 will deteriorate. Therefore, methanol: acetonitrile = 1:1 is selected as mobile phase A, and the peak shape of peak 2 is better and the peak is more stable.

[0170] (2) Investigation of mobile phase B

[0171] Octadecylsilane bonded silica gel was used as the packing (column length, 150 mm, inner diameter, 2.1 mm, particle size, 1.6 μm); mobile phase A consisted of methanol:acetonitrile in a 1:1 ratio, and mobile phase B consisted of various aqueous solutions, with gradient elution as specified in Table 3. The flow rate was 0.2 mL / min, the column temperature was 40°C, and the detection wavelength was 275 nm. Mobile phase B options included 0.1% phosphoric acid, 0.1% formic acid, 0.1% glacial acetic acid, and ultrapure water.

[0172] The comparison results of chromatographic detection of different types of mobile phase B are as follows Figure 2 ,according to Figure 2 The type of mobile phase B has little effect on the characteristic spectrum. From the perspective of cost saving and ease of operation, ultrapure water is selected as the water phase.

[0173] 1.3.3 Established liquid chromatography detection method

[0174] Based on the results of the investigation in 1.3.2, and taking into account factors such as energy, time cost, and economic benefits, the liquid chromatography detection method was determined as follows: using octadecylsilane bonded silica gel as the filler (column length 150 mm, inner diameter 2.1 mm, particle size 1.6 μm); using methanol:acetonitrile = 1:1 as mobile phase A, and water as mobile phase B, with gradient elution as specified in Table 3; a flow rate of 0.2 mL / min; a column temperature of 40°C; and a detection wavelength of 275 nm.

[0175] 1.4 Determination method

[0176] Accurately pipette 3 μL of the sample (the test solution or reference solution prepared in the above steps), inject it into the liquid chromatograph, and measure it.

[0177] 1.5 Methodological Review

[0178] 1.5.1 Specificity Investigation

[0179] Take 70% methanol blank solvent, β-asarone reference solution and Acorus tatarinowii test solution prepared according to the method in "1.1.3" respectively, and inject and test according to the detection method in "1.3.3". The obtained chromatogram is shown in Figure 3 The chromatographic data are shown in Table 4. After comparison with the reference substance, peak 3 was β-asarone, which was selected as the reference peak S. The specificity investigation showed that the peaks were well separated and there was no solvent interference.

[0180] Table 4 Results of specificity investigation

[0181]

[0182] 1.5.2 Repeatability Study

[0183] Accurately weigh the same batch of Acorus calamus test sample and prepare Acorus calamus test sample solution according to the method under "1.1.3", 6 portions each, and inject and test according to the detection method under "1.3.3". The obtained chromatogram is shown in Figure 4 The obtained chromatographic data are shown in Table 5. The RSD of the peak area of ​​β-asarone was calculated to be 0.82%. Taking it as the reference peak (S), the RSDs of the relative retention time and relative peak area of ​​each common peak were calculated to be 0.01% ~ 0.10% and 0.71% ~ 2.79%, respectively, indicating that the method has good repeatability.

[0184] Table 5 Repeatability test results

[0185]

[0186] 1.5.3 Precision investigation

[0187] Accurately weigh one sample of Acorus tatarinowii, prepare the sample solution according to the method in "1.1.3", repeat the injection 6 times according to the detection method in "1.3.3", and the obtained chromatogram is shown in Figure 5 The obtained chromatographic data are shown in Table 6. The RSD of the peak area of ​​β-asarone was calculated to be 0.82%. Taking it as the reference peak (S), the RSDs of the relative retention time and relative peak area of ​​each common peak were calculated to be 0.01%~0.08% and 0.06%~0.27%, respectively, indicating that the instrument had good precision.

[0188] Table 6 Precision inspection results

[0189]

[0190] 1.5.4 Intermediate precision study

[0191] Accurately weigh a sample of Acorus calamus, prepare the sample solution according to the method under "1.1.3", and repeat the injection 6 times using another instrument of the same model compared to the detection equipment in Section 1.5.3. The chromatogram is shown in Figure 6 The obtained chromatographic data are shown in Table 7. The RSD of the β-asarone peak area measured under 1.5.3 and the β-asarone peak area measured under this item was calculated to be 1.93%. Taking it as the reference peak (S), the RSDs of the relative retention time and relative peak area of ​​each common peak were calculated to be 0.02% ~ 1.23% and 1.74% ~ 3.32%, respectively, indicating good intermediate precision.

[0192] Table 7 Intermediate precision inspection results

[0193]

[0194] 1.5.5 Durability Assessment

[0195] The effects of different flow rates (0.18 mL / min, 0.20 mL / min, 0.22 mL / min), injection volumes (1 μL, 2 μL, 3 μL), column temperatures (35°C, 40°C, 45°C), and different batches of Waters CORTECS T3 (2.1 mm × 150 mm, 1.6 μm) chromatographic columns (numbered BH-375, JC-U34, and JC-U2, respectively) on the method were investigated. In addition to the changes in the investigated conditions, the chromatographic injection conditions under "1.3" were followed. The resulting chromatograms are shown in the figure. Figure 7 The obtained chromatographic data are shown in Table 8. Taking β-asarone as the reference peak (S), the relative retention time RSDs of the common peaks under different flow rates, injection volumes, column temperatures, and column batches were calculated to be 0.01% to 3.34%, 0.03% to 0.14%, 0.01% to 1.39%, and 0.01% to 0.21%, respectively; the relative peak area RSDs of the common peaks were 0.48% to 2.09%, 0.25% to 1.28%, 0.17% to 2.43%, and 0.19% to 0.62%, respectively. This shows that this method has good durability and can stably produce peaks under slight changes in conditions.

[0196] Table 8 Durability test results

[0197]

[0198] 1.5.6 Sample stability assessment

[0199] Accurately aspirate the Acorus calamus test solution and inject the sample at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 18 h, and 24 h according to the chromatographic conditions under "1.3.3". The chromatograms are shown in the figure. Figure 8 The chromatographic data are shown in Table 9. The RSD of the β-asarone peak area was calculated to be 0.20%. Taking it as the reference peak (S), the RSDs of the relative retention time and relative peak area of ​​each common peak were calculated to be 0.01% ~ 0.24% and 0.03% ~ 0.47%, respectively, indicating that the Acorus tatarinowii test solution was very stable within 24 h.

[0200] Table 9 Sample stability test results

[0201]

[0202] Example 2

[0203] This example uses the method established in Example 1 to process and analyze multiple batches of Acorus tatarinowii medicinal materials (see Table 1 for medicinal material sources) using liquid chromatography to construct a characteristic spectrum of Acorus tatarinowii, including the following steps:

[0204] Take 20 batches of Acorus tatarinowii medicinal materials, and prepare 20 batches of test solutions according to the method established in the subsection "1.1 Preparation of Test Solution" in Example 1; perform liquid chromatography analysis according to the method established in the subsection "1.3 Liquid Phase Analysis Method" in Example 1, and perform sample injection and determination according to the method established in the subsection "1.4 Determination Method" in Example 1 to obtain liquid chromatograms of 20 batches of Acorus tatarinowii medicinal materials. Figure 9 This is an overlay of 20 batches of liquid chromatograms. These chromatograms were used to identify common peaks using the "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation Software". Figure 9 The relative peak areas of the common peaks for each batch of samples are shown in Table 10, with Peak 3 as the reference. The similarity evaluation results are shown in Table 11. The similarity of the characteristic spectra of the Acorus tatarinowii samples ranged from 0.892 to 0.996.

[0205] Table 10 Relative peak areas of common peaks in 20 batches of Acorus tatarinowii

[0206]

[0207] Table 11 Similarity evaluation results of characteristic spectra of Acorus calamus, Acorus foeniculum, Acorus calamus, Acorus tatarinowii, and Bergenia

[0208]

[0209] Example 3

[0210] This example uses the method established in Example 1 to process and perform liquid chromatography analysis on multiple batches of adulterated Acorus tatarinowii medicinal materials (see Table 1 for medicinal material sources) to construct characteristic spectra of the adulterated Acorus tatarinowii medicinal materials. The characteristic spectra of the adulterated Acorus tatarinowii medicinal materials are then compared with the characteristic spectra of Acorus tatarinowii constructed in Example 2 to verify whether the characteristic spectra obtained by the construction method of this application can be used for the identification of Acorus tatarinowii. The steps include:

[0211] Take 3 batches of Acorus calamus, 4 batches of Acorus fennel, 3 batches of Bergenia, and 4 batches of Acorus calamus medicinal materials (see Table 1 for the source of medicinal materials), and prepare 3 batches of Acorus calamus, 4 batches of Acorus fennel, 3 batches of Bergenia, and 4 batches of Acorus calamus test solution according to the method established in the subsection "1.1 Preparation of Test Solution" in Example 1; liquid chromatography analysis was performed according to the method established in the subsection "1.3 Liquid Phase Analysis Method" in Example 1, and the sample was injected and determined according to the method established in the subsection "1.4 Determination Method" in Example 1. The chromatographic fingerprints of the 3 batches of Acorus calamus, 4 batches of Acorus fennel, 3 batches of Bergenia, and 4 batches of Acorus calamus medicinal materials were marked with common peaks using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software" and superimposed to obtain Figure 10 The chromatograms of the three batches of Acorus calamus are shown as overlays. Figure 11 The chromatogram overlay of 4 batches of Fennel and Acorus calamus is shown. Figure 12 The chromatogram overlay of the four batches of Acorus calamus shown in FIG. Figure 13 The chromatograms of the three batches of Bergenia shown in FIG were overlaid. Based on the overlaid graphs, the corresponding reference spectra of Acorus calamus, Acorus foeniculum, Acorus 9-section, and Bergenia were generated. The reference spectra of these adulterants were compared with the reference spectra of Acorus calamus in Example 2. The comparison results are shown in FIG. Figure 14 The relative peak area and peak area data are shown in Tables 12 to 14.

[0212] Table 12 Common peak relative peak areas of characteristic spectrum of Acorus calamus

[0213]

[0214] Table 13 Common peak relative peak areas of characteristic spectra of fennel and Acorus calamus

[0215]

[0216] Table 14 Retention time and peak area of ​​common peaks in the characteristic spectra of Acorus calamus and Bergenia

[0217]

[0218] according to Figure 14 , Acorus calamus has two peaks at 12~13 minutes, namely Peak 5 and Peak 6, while Acorus calamus does not have them; Acorus calamus has two characteristic small peaks before Peak 3 (β-asarone), namely Peak 7 and Peak 8, while Acorus calamus does not have them; Bergenia and Acorus calamus lack the four characteristic peaks of Acorus calamus, and have one characteristic peak at 26~27 minutes; it can be seen that the characteristic map construction method of the present application can be used to identify Acorus calamus and Acorus calamus, Acorus calamus, Bergenia, and Acorus calamus.

[0219] In summary, the characteristic spectrum of Acorus calamus obtained by the characteristic spectrum construction method of the present application has the advantages of few impurity peaks, strong specificity, and easy identification. The characteristic spectrum includes at least 4 characteristic peaks that can be used to achieve authenticity identification, and identifies the indicator component β-asarone; and can be used to distinguish common Acorus calamus confusion products, such as Acorus water calamus, Acorus fennel, Bergenia and Acorus 9-section, and the identification method is simple, fast, intuitive and accurate.

[0220] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When cited documents are involved in this application, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be adaptively amended according to the description in this application.

[0221] The various technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the various technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of the description in this specification.

[0222] The embodiments described above only express several implementation methods of the present application, but they should not be understood as limiting the scope of the patent application. It should be pointed out that, for those of ordinary skill in the art, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of the present application shall be based on the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A method for constructing a characteristic spectrum of Acorus tatarinowii, characterized in that: include: A test sample of Acorus calamus is extracted with water to obtain an extract, and the extract is then extracted with petroleum ether to prepare a test sample solution; Take β-asarone reference substance to prepare reference solution; The test solution and the reference solution are subjected to liquid chromatography detection, and a characteristic spectrum of Acorus tatarinowii is constructed according to the measured liquid chromatogram; The liquid chromatography detection conditions include: using octadecylsilane bonded silica gel as a filler, using a mixture of methanol and acetonitrile as mobile phase A and water as mobile phase B for gradient elution; the volume ratio of methanol to acetonitrile in the mobile phase A is 1:1; the detection wavelength is 270-280 nm; the chromatographic column is a Waters CORTECS T3, with specifications of 2.1 mm × 150 mm, 1.6 μm; The gradient elution procedure includes: From 0 to 5 min, the volume percentage of the mobile phase A increased from 10% to 34%; From 5 to 13 minutes, the volume percentage of the mobile phase A increased from 34% to 35%; 13-13.5 min, the volume percentage of the mobile phase A increased from 35% to 50%; From 13.5 to 20 minutes, the volume percentage of the mobile phase A increased from 50% to 62%; 20-25 min, the volume percentage of the mobile phase A increased from 62% to 90%; 25-26 min, the volume percentage of the mobile phase A decreased from 90% to 10%; From 26 to 30 minutes, the volume percentage of the mobile phase A was maintained at 10%.

2. The method for constructing a characteristic map according to claim 1, wherein: The conditions for the liquid chromatography detection also include at least one of the following: (1) Column temperature is 35-45°C; (2) Flow rate: 0.18–0.22 mL / min; (3) The injection volume is 1 to 3 μL.

3. The method for constructing a characteristic map according to claim 1, wherein: Take the Acorus calamus test sample and add water to extract, including: The Acorus calamus test sample and the water are mixed in a mass volume ratio of 1 g:15-25 mL, and heated under reflux for 50-70 minutes to obtain the extract.

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