Method for detecting alkaloids in coptis deltoidea

Abstract

The present application relates to a kind of detection methods of alkaloids in Coptis, the detection method includes the following steps: with embedding agent infiltrates to be measured Coptis sample, freeze, sectioning;The slice is pasted on the slide, and the alkaloids in the slice are detected using secondary ion time-of-flight mass spectrometry;Wherein, the Coptis sample is Coptis fresh product or Coptis dry product after wet treatment, and the solvent used in wet treatment includes water;The embedding agent includes the water-soluble mixture of polyethylene glycol and polyvinyl alcohol.This application introduces secondary ion time-of-flight mass spectrometry into the detection of alkaloids in Coptis for the first time, without first enriching / extracting alkaloid components in Coptis sample, simple operation, less interference, can effectively avoid the loss of alkaloids, detection precision, accuracy, reproducibility and stability are good, and the detection result can be intuitively presented, and the detection efficiency is high.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a detection method of alkaloids in Coptis chinensis. BACKGROUND

[0002] Coptis chinensis Franch, Coptis deltoidea C.Y.Cheng et Hsiao and Coptis teeta Wall. In clinical applications, Coptis chinensis Franch and its chemical components are often used for treating dysentery, diabetes and its complications, insomnia and other diseases. Modern research shows that the pharmacological effects of Coptis chinensis Franch are mainly related to the alkaloids contained therein. Therefore, the detection of alkaloids in Coptis chinensis Franch is crucial for the quality control of Coptis chinensis Franch.

[0003] Traditionally, the alkaloid content in each cell or tissue is detected by using spectroscopy, high performance liquid chromatography or liquid-mass spectrometry. Although these traditional methods can realize the detection of alkaloids in Coptis chinensis Franch, there are many interference factors (including alkaloid enrichment, extraction and other operations and the interference of solvents involved therein) and alkaloid loss cannot be avoided, and the obtained results are difficult to objectively reflect the real situation of alkaloids in Coptis chinensis Franch. Therefore, how to effectively reduce the interference and avoid the loss of alkaloids in the process of detecting alkaloids in Coptis chinensis Franch is a technical problem to be solved. SUMMARY

[0004] Based on this, the purposes of the present application include providing a detection method of alkaloids in Coptis chinensis Franch, which can effectively avoid the loss of alkaloids with less interference.

[0005] The purposes of the present application can be realized by the following technical solutions:

[0006] A detection method of alkaloids in Coptis chinensis Franch, the detection method comprising the following steps:

[0007] infiltrating a to-be-detected Coptis chinensis Franch sample with an embedding agent, freezing and sectioning;

[0008] attaching the section on a slide and detecting the alkaloids in the section by using secondary ion time-of-flight mass spectrometry;

[0009] wherein,

[0010] the Coptis chinensis Franch sample is fresh Coptis chinensis Franch or Coptis chinensis Franch dry product treated by wetting, and the solvent used for wetting treatment comprises water;

[0011] the embedding agent comprises a water-soluble mixture of polyethylene glycol and polyvinyl alcohol.

[0012] In some embodiments of the present invention, the embedding agent is an OCT embedding agent or a mixture of the OCT embedding agent and water.

[0013] In some embodiments of the present invention, the alkaloid is selected from one, two, three, four, five or six of the following: berberine, coptisine, palmatine, oxidized berberine and tetrandrine.

[0014] In some embodiments of the present invention, the mass spectrometry conditions for the secondary ion time-of-flight mass spectrometry include:

[0015] The mode is a clustering mode;

[0016] The primary ion is Bi1 + Bi3 + Or Bi3 2+ ;

[0017] The energy is 20keV to 30keV;

[0018] The ion beam current is 0.3 pA to 1 pA.

[0019] In some embodiments of the present invention, the mass spectrometry conditions for the secondary ion time-of-flight mass spectrometry method further include:

[0020] The sampling area is 1×1mm 2 ~10×10mm 2 ; or / and,

[0021] The captured pixels are 32×32 pixels. 2 ~1024×1024 pixels 2 ; or / and,

[0022] Lateral resolution is 3μm to 8μm; or / and,

[0023] The secondary ions are positive ions; or / and,

[0024] The mass of the secondary ions ranges from 0 amu to 2058 amu; or / and,

[0025] The incident angle of the beam is 40°–50°; or / and,

[0026] The primary ion dose was maintained at 1.25 × 10⁻⁶. 9 ions / cm 2 the following.

[0027] In some embodiments of the present invention, the temperature for preparing the slices is -20°C to -24°C.

[0028] In some embodiments of the present invention, the freezing conditions include: a temperature of -15°C to -25°C and a time of 20 min to 40 min.

[0029] In some embodiments of the present invention, the thickness of the slice is 5 μm to 20 μm.

[0030] In some embodiments of the present invention, the Coptis chinensis sample to be tested is a cross-section of Coptis chinensis 0.4cm to 0.6cm in length.

[0031] In some embodiments of the present invention, during the slicing process, the slices are made radially along the transverse section of the Coptis chinensis.

[0032] In some embodiments of the present invention, the carrier is a silicon wafer or a mica sheet.

[0033] Compared with traditional technologies, the present invention has the following advantages:

[0034] This invention employs a suitable embedding agent to impregnate a Coptis chinensis sample, followed by cryosectioning. The resulting sections are then mounted on a silicon wafer for secondary ion time-of-flight mass spectrometry (TIF-MS). The sections remain attached to the substrate (e.g., silicon wafer) throughout the detection process to ensure successful analysis, thus enabling the successful application of TIF-MS in the detection of Coptis chinensis alkaloids. This invention is the first to introduce TIF-MS into the detection of Coptis chinensis alkaloids, eliminating the need for prior enrichment / extraction of alkaloids from the sample. The method is simple to operate, minimizes interference, effectively avoids alkaloid loss, and offers good precision, accuracy, reproducibility, and stability. The results are presented intuitively, resulting in high detection efficiency. Furthermore, the composition of the sections remains essentially unchanged before and after detection, allowing for reuse of the sections or remaining samples, avoiding waste and reducing quality control costs. Additionally, this method can detect the distribution of alkaloids in Coptis chinensis, providing a basis for its cultivation. Overall, this invention provides a new method and scientific basis for the quality evaluation of Coptis chinensis. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 This is a cross-sectional view of the root of Coptis chinensis (also known as Coptis chinensis var. spp.) in Example 1 (microscope 40×).

[0037] Figure 2This is a time-of-flight mass spectrometry image (positive ion) of the main alkaloids in a cross-section of Coptis chinensis (Weilian) in Example 1. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the invention. The purpose of providing these embodiments and examples is to enable a more thorough and complete understanding of the disclosure of the present invention. It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present invention, and the equivalent forms obtained also fall within the protection scope of this application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for descriptive purposes only and is not intended to be limiting of the invention.

[0040] Terminology

[0041] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0042] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0043] In this invention, terms such as "multiple", "various", "multiple times", and "multi-source" are used, and unless otherwise specified, they refer to a quantity greater than or equal to 2. For example, "one or more" means one or more types.

[0044] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0045] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this invention, solve the technical problem of this invention, and achieve the expected technical effect of this invention.

[0046] In this article, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this invention.

[0047] In this invention, terms such as "further," "even more," and "particularly" are used for descriptive purposes and to indicate differences in content, but should not be construed as limiting the scope of protection of this invention.

[0048] In this invention, "optionally," "optionally," and "optional" mean that they are optional, that is, they are selected from either "with" or "without." If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "option" is independent.

[0049] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0050] In this invention, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0051] Unless otherwise specified, the temperature parameters in this invention can be either constant temperature treatment or variations within a certain temperature range. It should be understood that the constant temperature treatment allows temperature fluctuations within the precision range controlled by the instrument. Fluctuations are permitted within ranges such as ±5℃, ±4℃, ±3℃, ±2℃, and ±1℃.

[0052] In this invention, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0053] All references to this invention are incorporated herein by reference as if each document were individually incorporated by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, the referenced documents involved in this invention are incorporated in their entirety and for all purposes. When references are made in this invention, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When references are made in this invention, examples and preferred embodiments of the relevant technical features cited may also be incorporated herein by reference, but only to the extent that they enable the implementation of this invention. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptively based on the description in this application.

[0054] Mass spectrometry imaging technology obtains metabolite signals from the surface of biological or tissue samples through mass spectrometry detection. Computer imaging then provides a visualized spatial distribution map of the overall chemical components within the organism, offering a method for simultaneously acquiring information on the morphology of biological samples and their multiple chemical components. Applying mass spectrometry molecular imaging technology to the rapid location of specific chemical components in medicinal plants holds promise for breakthroughs in the evaluation of the properties of traditional Chinese medicine.

[0055] Secondary ion mass spectrometry (TOF-SIMS) offers label-free, highly sensitive, and multi-component analysis capabilities, along with high spatial resolution imaging at the sub-micron and even nanometer scales. The basic principle of TOF-SIMS detection is based on the phenomenon where a primary ion beam carrying energies of several thousand electron volts bombards the surface of a solid sample, resulting in the generation of secondary ions on the sample surface through physical interactions.

[0056] Early secondary ion mass spectrometry (TIMS) was primarily used for materials analysis in industry. With the advent of the biological age, it has been applied to research in numerous life science fields. TIMS is mainly used in the detection of animal tissues such as the brain, liver, small intestine, and skeletal muscle, comparing the imaging changes of lipids, cholesterol, and other endogenous substances in healthy individuals and those with related diseases, providing a direct reflection of disease characteristics. In plant tissues, TIMS has been successfully used to study the spatial distribution of metal ions and active ingredients in plants such as beans and poplars, providing information on the dynamics and structural-functional relationships during plant nutrient transport. The application characteristics of TIMS can compensate for the lack of accurate localization of chemical components and the absence of spatial distribution descriptions of chemical components in tissues and cells during traditional Chinese medicine research. However, to date, there are no research reports on the application of TIMS in medicinal plants used in traditional Chinese medicine.

[0057] This invention is the first to introduce secondary ion time-of-flight mass spectrometry into the detection of the traditional Chinese medicine Coptis chinensis, providing a new detection method for the quality control of Chinese medicinal materials.

[0058] This invention provides a method for detecting alkaloids in Coptis chinensis, the method comprising the following steps:

[0059] The sample of Coptis chinensis to be tested was impregnated with embedding agent, frozen, and sectioned.

[0060] The slices were attached to a silicon wafer, and the alkaloids in the slices were detected by secondary ion time-of-flight mass spectrometry.

[0061] in,

[0062] The Coptis chinensis sample is either fresh Coptis chinensis or dried Coptis chinensis that has been moistened, and the solvent used for moistening includes water.

[0063] The embedding agent comprises a water-soluble mixture of polyethylene glycol and polyvinyl alcohol.

[0064] Chinese medicinal herbs are divided into fresh and dried products. Fresh products refer to medicinal materials that have been freshly picked without undergoing drying or sun-drying processes that cause significant moisture loss. Dried products refer to medicinal materials that have been dried or sun-dried after picking to remove as much moisture as possible, or processed into medicinal slices through heating and processing techniques.

[0065] The inventors discovered that secondary ion time-of-flight mass spectrometry (TIFMS) detection requires special conditions such as vacuum, which can easily cause the slides to warp or break, leading to detection failure. To address this, the inventors selected an embedding agent containing a water-soluble mixture of polyethylene glycol and polyvinyl alcohol, specifically an OCT embedding agent, to wet the sample. This treatment ensures that the slides remain tightly adhered to the slide during subsequent detection, guaranteeing successful analysis.

[0066] In some embodiments of the present invention, the embedding agent is an OCT embedding agent or a mixture of the OCT embedding agent and water.

[0067] In some embodiments of the present invention, the volume percentage of the OCT embedding agent in the mixture is 70%-90%, for example, 70%, 75%, 80%, 85%, or 90%.

[0068] In some embodiments of the present invention, the alkaloid is selected from one, two, three, four, five or six of the following: berberine, coptisine, palmatine, oxidized berberine and tetrandrine.

[0069] The detection method provided by this invention can detect one Coptis chinensis sample or multiple samples simultaneously. During the detection of multiple samples, the relative levels of the corresponding alkaloid content can be determined by comparing the brightness of the detection images.

[0070] In some embodiments of the present invention, the mass spectrometry conditions for the secondary ion time-of-flight mass spectrometry include:

[0071] The mode is a clustering mode;

[0072] The primary ion is Bi1 + Bi3 + Or Bi3 2+ ;

[0073] The energy is 20keV to 30keV;

[0074] The ion beam current is 0.3 pA to 1 pA.

[0075] In the detection method provided by this invention, the energy is, for example, 20keV, 21keV, 22keV, 23keV, 24keV, 25keV, 26keV, 27keV, 28keV, 29keV, or 30keV.

[0076] In the detection method provided by this invention, the ion beam current is, for example, 0.3 pA, 0.4 pA, 0.5 pA, 0.6 pA, 0.7 pA, 0.8 pA, 0.9 pA, or 1 pA.

[0077] In some embodiments of the present invention, the mass spectrometry conditions for the secondary ion time-of-flight mass spectrometry further include: a collection area of ​​1×1 mm. 2 ~10×10mm 2 For example, 1×1mm 2 2×2mm 2 3×3mm 2 4×4mm 25×5mm 2 6×6mm 2 7×7mm 2 8×8mm 2 9×9mm 2 10×10mm 2 .

[0078] In some embodiments of the present invention, the mass spectrometry conditions for the secondary ion time-of-flight mass spectrometry method further include: the image pixel range is 32×32 pixels. 2 ~1024×1024 pixels 2 For example, 32×32 pixels 2 64×64 pixels 2 128×128 pixels 2 256×256 pixels 2 512×512 pixels 2 1024×1024 pixels 2 .

[0079] In some embodiments of the present invention, the mass spectrometry conditions of the secondary ion time-of-flight mass spectrometry method further include: a lateral resolution of 3 μm to 8 μm, for example, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, or 8 μm.

[0080] In some embodiments of the present invention, the mass of the secondary ion is 0 amu to 2058 amu, for example 0 amu, 3 amu, 9 amu, 15 amu, 20 amu, 30 amu, 50 amu, 100 amu, 500 amu, 686 amu, 2058 amu.

[0081] In some embodiments of the present invention, the incident angle of the beam is 40° to 50°, for example, 40°, 41°, 42°, 43°, 44°, 45°, 46°, 47°, 48°, 49°, and 50°.

[0082] In some embodiments of the present invention, the primary ion dose is maintained at 1.25 × 10⁻⁶. 9 ions / cm 2 For example, 1.25 × 10 9 ions / cm 2 1×10 9 ions / cm 2 0.5×10 9 ions / cm 2 0.1×10 9 ions / cm 2 9×10 8 ions / cm2 5×10 8 ions / cm 2 2×10 8 ions / cm 2 .

[0083] Preferably, the mass spectrometry conditions for the secondary ion time-of-flight mass spectrometry include:

[0084] The mode is a clustering mode;

[0085] The primary ion is Bi1 + Bi3 + Or Bi3 2+ ;

[0086] The energy is 20keV to 30keV;

[0087] The ion beam current is 0.3 pA to 1 pA;

[0088] The sampling area is 1×1mm 2 ~10×10mm 2 ;

[0089] The image pixel range is 32×32 pixels. 2 ~1024×1024 pixels 2 ;

[0090] The lateral resolution is 3μm to 8μm;

[0091] The secondary ions are positive ions;

[0092] The mass of the secondary ions ranges from 0 amu to 2058 amu;

[0093] The incident angle of the beam is 40° to 50°;

[0094] The primary ion dose is 1×10 9 ions / cm 2 -1.25×10 9 ions / cm 2 .

[0095] In some embodiments of the present invention, the temperature for preparing the slices is -20°C to -24°C (e.g., -20°C, -20.5°C, -21°C, -21.5°C, -22°C, -24°C).

[0096] In some embodiments of the present invention, the freezing conditions include: a temperature of -15°C to -25°C (e.g., -15°C, -16°C, -17°C, -18°C, -19°C, -20°C, -21°C, -22°C, -23°C, -24°C, -25°C), and a time of 20 min to 40 min (e.g., 20 min, 25 min, 30 min, 35 min, 40 min).

[0097] In some embodiments of the present invention, the thickness of the slice is 5 μm to 20 μm, for example, 5 μm, 8 μm, 10 μm, 12 μm, 15 μm, 17 μm, 19 μm, or 20 μm.

[0098] In some embodiments of the present invention, the specifications of the Coptis chinensis sample to be tested are transverse segments with a length of 0.4cm to 0.6cm (e.g., 0.4cm, 0.45cm, 0.5cm, 0.55cm, 0.6cm).

[0099] In some embodiments of the present invention, during the slicing process, slicing is performed radially along the transverse section. This method of slicing along short radial sections during the slicing process facilitates observation of the distribution of alkaloids, providing a basis for improving the cultivation process of Coptis chinensis.

[0100] In some embodiments of the present invention, the number of detections is not less than 3 times. For example, 4 times, 5 times, 6 times, etc. Specific Implementation

[0102] The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this invention, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0103] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0104] Example 1: Detection method of alkaloids in Coptis chinensis

[0105] 1. Instruments and Materials

[0106] 1.1 Instruments

[0107] The ToF-SIMS IV instrument (Ion-Tof GmbH, Münster, Germany) is equipped with a bismuth liquid metal ion source and a reflective electron analyzer with a multi-channel detector. A cryostat (Leica CM1950, Germany) is also included.

[0108] 1.2 Medicinal Materials and Test Drugs

[0109] The fresh Coptis chinensis was harvested from Mianyang City, Sichuan Province, and identified by Associate Professor Zhang Zhifeng of the School of Chinese Medicine, Macau University of Science and Technology as Coptis chinensis Franch. (Weilian), a plant of the Ranunculaceae family, and as an OCT (Leica Microsystems, Wetzlar, Germany) embedding agent.

[0110] 2. Sample Preparation

[0111] Cut the washed fresh Coptis chinensis (Wei Lian) into 0.5cm long pieces, put them in a suitable box, pour in OCT (Leica Microsystems, Wetzlar, Germany) embedding medium, soak for 20 minutes, and then put them in a -20℃ freezer for 30 minutes.

[0112] The frozen sample was removed and placed on the sample stage, then placed on the stage of the cryostat. After fixation, it was sectioned to a thickness of 10 μm at -22°C. The sections were then directly mounted onto silicon wafers at room temperature and dried under vacuum.

[0113] 3. Selection of the compound to be tested

[0114] The distribution of the main active ingredient alkaloids in Coptis chinensis was observed by analyzing the cross-section of fresh Coptis chinensis rhizomes using TOF-SIMS imaging. The names and m / z values ​​of the ions corresponding to the specified compounds in Coptis chinensis are listed in Table 1.

[0115] Table 1. Names and m / z values ​​of ions corresponding to specified compounds in Coptis chinensis.

[0116]

[0117]

[0118] 4. Sample testing

[0119] The silicon wafer is fixed on the stage and fed into the machine, where it is exposed to 30keV Bi light at a 45° incident angle. 3+ Primary ions ( The spectrum was acquired using pulsed current, along with low-energy electrons for charge compensation. Operation was performed under high-current beaming conditions with cycle times of 100-200 μs. A coverage area of ​​9.50 × 7.00 mm was obtained. 2A random raster pattern of 128×128 pixels in the region. Ion dose maintained at 1.00–1.50×10⁻⁶ pixels. 9 ions / cm 2 This is to limit sample damage.

[0120] The Coptis chinensis samples were analyzed three times using TOF-SIMS to confirm analytical repeatability and bioreproducibility.

[0121] 5. Experimental Results

[0122] The spatial distribution of berberine (epiberberine), coptisine, palmatine, oxidized berberine, and stephania tetrandra (herbaceous root alkaloid) in the rhizome of Coptis chinensis was mapped using TOF-SIMs imaging technology. Intensities were represented by color scales, with bright colors corresponding to high intensity and dark colors to low intensity. For each image, the color scale was normalized to the brightest pixel.

[0123] See results Figure 1 and Figure 2 The image shows that berberine (epberberine), berberine, palmatine, and stephania tetrandra (herbaceous root alkaloid) are dispersed throughout the cross-section. Observing the depth of the spots, it can be seen that berberine (epberberine) has the highest content, followed by berberine, palmatine, and stephania tetrandra (herbaceous root alkaloid). Magnolia alkaloid is only faintly visible. Palmatine is more abundant in the pith than in other parts.

[0124] The sample was measured three times, and there was no significant difference in the brightness of the three images obtained for each alkaloid, indicating that the experimental method has good accuracy, repeatability and reproducibility.

[0125] Example 2: Detection method of alkaloids in Coptis chinensis

[0126] 1. Instruments and Materials

[0127] 1.1 Instruments

[0128] The ToF-SIMS IV instrument (Ion-Tof GmbH, Münster, Germany) is equipped with a bismuth liquid metal ion source and a reflective electron analyzer with a multi-channel detector. A cryostat (Leica CM1950, Germany) is also included.

[0129] 1.2 Medicinal Materials and Test Drugs

[0130] The fresh Coptis chinensis was harvested from Mianyang City, Sichuan Province, and identified by Associate Professor Zhang Zhifeng of the School of Chinese Materia Medica, Macau University of Science and Technology as Coptis chinensis Franch. (Weilian), a plant of the Ranunculaceae family. The embedding agent was OCT embedding agent.

[0131] 2. Sample Preparation

[0132] Cut the dried Coptis chinensis (Wei Lian) into 0.4cm long pieces, put them in a suitable box, pour in distilled water to soak them, take them out, put them in another small box, pour in the embedding agent, soak for 20 minutes, and then put them in a -15℃ freezer for 40 minutes.

[0133] Remove the frozen sample and place it on the sample stage, then place it on the stage of the cryostat, fix it, and slice it to a thickness of 15 μm at a temperature of -24℃.

[0134] The slices were directly bonded to silicon wafers at room temperature and dried under vacuum.

[0135] 3. Selection of the compound to be tested

[0136] The distribution of the main active ingredient alkaloids in Coptis chinensis was observed by analyzing the cross-section of fresh Coptis chinensis rhizomes using TOF-SIMS imaging. The names and m / z values ​​of the ions corresponding to the specified compounds in Coptis chinensis are listed in Table 1.

[0137] 4. Sample testing

[0138] After fixing the mica sheet onto the stage and feeding it into the machine, use 20keV Bi light incident at 48°. 3+ Primary ions ( The spectrum was acquired using pulsed current, along with low-energy electrons for charge compensation. The system operated under high-current beaming conditions with a cycle time of 100 μs. A coverage area of ​​5 × 5 mm was obtained. 2 1024×1024 pixels of the region 2 Random raster pattern for pixels. Ion dose maintained at 1.25 × 10⁻⁶. 9 ions / cm 2 The following measures are taken to limit sample damage.

[0139] Three TOF-SIMS analyses were performed on Coptis chinensis samples to confirm analytical repeatability and bioreproducibility.

[0140] 5. Experimental Results

[0141] Results: The spatial distribution of berberine (epiberberine), coptisine, palmatine, oxidized berberine, and stephania tetrandra (herbaceous root alkaloid) in the rhizome of Coptis chinensis was mapped using TOF-SIMs imaging technology. Intensity was represented by color scales, where bright colors corresponded to high intensity and dark colors to low intensity. For each image, the color scale was normalized to the brightest pixel.

[0142] The results are consistent with those in Example 1. Figure 1 and Figure 2The results were largely consistent: berberine (epberberine), berberine, palmatine, and stephania tetrandra (herb alkaloid) were dispersed throughout the cross-section. Berberine (epberberine) had the highest content, followed by berberine, palmatine, and stephania tetrandra (herb alkaloid). Magnolia alkaloid was only faintly visible. Palmatine was more abundant in the pith than in other parts. The samples were measured three times repeatedly, and the brightness of the three images obtained for each alkaloid showed no significant difference, indicating that the experimental method had good accuracy, repeatability, and reproducibility.

[0143] Comparative Example 1

[0144] This comparative example is a comparative example of Example 1. The difference from Comparative Example 1 is that a carboxymethyl cellulose (CMC) water-soluble vitamin was used instead of the OCT embedding agent at a mass percentage of 0.3%. Results: The sample embedded with carboxymethyl cellulose (CMC) instead of the OCT cryo-embedding agent could not be detected, i.e., no detection results were obtained.

[0145] The above results demonstrate that this invention develops a novel and stable ToF-SIMs method for the rapid and solvent-saving determination of Coptis chinensis, providing a scientific method for studying the quality and tissue distribution of other traditional Chinese medicines.

[0146] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments 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 this specification.

[0147] The above-described embodiments are merely illustrative of several implementation methods of the present invention, facilitating a detailed and specific understanding of the technical solutions of the present invention. However, they should not be construed as limiting the scope of protection of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Furthermore, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided by the present invention through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this invention patent should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for detecting alkaloids in Coptis chinensis, characterized in that, The detection method consists of the following steps: The sample of Coptis chinensis to be tested was impregnated with embedding agent, frozen, and sectioned. The slices were attached to a slide, vacuum dried, and the alkaloids in the slices were detected by secondary ion time-of-flight mass spectrometry. in, The Coptis chinensis sample is either fresh Coptis chinensis or dried Coptis chinensis that has been moistened, and the solvent used for moistening includes water. The embedding agent is an OCT embedding agent; The temperature for preparing the sections is -20℃ to -24℃; The carrier is a silicon wafer; The temperature of the carrier slide is room temperature; The thickness of the slice is 10μm to 15μm; The Coptis chinensis sample to be tested was a cross-section of Coptis chinensis 0.4cm to 0.5cm in length; During the slicing process, slices are made radially along the transverse section of Coptis chinensis; The alkaloids include: berberine, coptisine, palmatine, oxidized berberine, and tetrandrine; The mass spectrometry conditions for the secondary ion time-of-flight mass spectrometry include: The mode is a clustering mode; The primary ion is Bi3. + ; The energy is 20keV to 30keV; The ion beam current is 0.3 pA to 0.8 pA; The sampling area is 1×1mm 2 ~10×10mm 2 ; The captured pixels are 32×32 pixels. 2 ~1024×1024 pixels 2 ; The lateral resolution is 3μm to 8μm; The secondary ions are positive ions; The mass of the secondary ions ranges from 0 amu to 2058 amu; The incident angle of the beam is 45° to 48°; The primary ion dose was maintained at 1–1.5 × 10⁻⁶. 9 ions / cm 2 ; Freezing conditions include: temperature of -15℃ to -20℃, and time of 30 min to 40 min.

2. The method for detecting alkaloids in Coptis chinensis according to claim 1, characterized in that, The number of tests should be no less than 3.

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