CN113759008B - Construction method and application of areca or burnt areca characteristic spectrum - Google Patents

Construction method and application of areca or burnt areca characteristic spectrum Download PDF

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CN113759008B
CN113759008B CN202010872389.4A CN202010872389A CN113759008B CN 113759008 B CN113759008 B CN 113759008B CN 202010872389 A CN202010872389 A CN 202010872389A CN 113759008 B CN113759008 B CN 113759008B
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peak
sample
formula
detected
areca
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CN113759008A (en
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张志强
付静
沈建梅
马智玲
万莹莹
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Beijing Tcmages Pharmaceutical Co Ltd
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract

The invention belongs to the technical field of medicine detection, and particularly relates to a construction method and application of a characteristic spectrum of areca nuts or burnt areca nuts. The method adopts high performance liquid chromatography to obtain characteristic chromatogram of Arecae semen or burnt Arecae semen with high accuracy, and can be used for evaluating quality of Arecae semen and burnt Arecae semen, and simultaneously used as evaluation standard for controlling each process link in production of Arecae semen or burnt Arecae semen; and the characteristic map can accurately express the consistency of the drug effect substances of the betel nut and the scorched betel nut under different properties. The characteristic spectrum obtained by the construction method provided by the invention can be used for identifying the betel nut and the burnt betel nut.

Description

Construction method and application of areca or burnt areca characteristic spectrum
Technical Field
The invention belongs to the technical field of medicine detection, and particularly relates to a construction method and application of a characteristic spectrum of areca nuts or burnt areca nuts.
Background
Betel nut is a dry mature seed of Areca catechu L, a plant of Palmae, and is mainly produced in Hainan, guangxi, and other places. The preparation method comprises processing semen Arecae and charred semen Arecae, wherein the semen Arecae has effects of killing parasite, removing food stagnation, promoting qi circulation, promoting diuresis, and preventing malaria; semen Arecae Preareta has effects of resolving food stagnation. The clinical application shows that the property of betel nut can be relieved after being processed and is good at promoting digestion and removing food stagnation, the change of the property is the change of the content of the components, and the content of various components in the betel nut is reduced by being stir-baked to brown.
Because the properties and the components of the traditional Chinese medicine are changed after the traditional Chinese medicine is processed, the decoction pieces can be identified by the traditional experience because the appearance of the decoction pieces is changed, but the traditional Chinese medicine formula granules lose the appearance property after water extraction, and the identification is difficult to realize by the traditional experience of appearance and the like. He Xiaoyan et al, published in the analysis of characteristic patterns of different processed products during the frying process of betel nut, recorded that the content of chemical components of the roasted betel nut is changed, which is mostly the content of index components such as arecoline. The formula particle is prepared by carrying out water extraction, concentration, drying and granulation on single Chinese medicinal decoction pieces, and the chemical component content and the transmission of the Chinese medicinal formula particle are changed after the Chinese medicinal formula particle is prepared by a series of modern preparation processes. The prior art lacks a method for simply and visually identifying betel nut and burnt betel nut formula particles.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to overcome the defect that no identification method for betel nuts and burnt betel nuts exists in the prior art, so that a construction method and application of a characteristic spectrum of betel nuts or burnt betel nuts are provided.
Therefore, the invention provides the following technical scheme.
The invention provides a construction method of a characteristic spectrum of areca or burnt areca, which comprises the steps of obtaining the characteristic spectrum of the areca or the burnt areca, wherein the characteristic peaks of the characteristic spectrum comprise 4 common characteristic peaks which are respectively a No. 1 peak, a No. 2 peak, a No. 3 peak and a No. 4 peak, wherein the No. 2 peak is an arecoline hydrobromide peak, and the No. 4 peak is a catechin peak;
taking the No. 2 peak as a reference peak, and the relative retention time of the No. 1 peak is 0.83; taking the No. 4 peak as a reference peak, and the relative retention time of the No. 3 peak is 0.80;
wherein the relative retention time of the common characteristic peak is within + -10% of the specified value.
The construction method of the characteristic map of the betel nut or the burnt betel nut comprises the following steps,
preparation of reference solutions: preparing arecoline hydrobromide reference substance solution and catechin reference substance solution from arecoline hydrobromide and catechin reference substance;
preparing reference solution of reference medicinal material from Arecae semen;
preparing a test solution: taking a sample to be detected to prepare a test solution;
chromatographic conditions are as follows: measuring by high performance liquid chromatography, and using octadecylsilane chemically bonded silica as filler; taking acetonitrile as a mobile phase A and phosphoric acid as a mobile phase B, and performing gradient elution; the detection wavelength is 200-250nm; the flow rate is 0.5-1.5ml/min;
and (3) determination: respectively sucking reference substance solution and sample solution, injecting into high performance liquid chromatograph, and measuring;
wherein the procedure of gradient elution comprises: 0-5min, mobile phase A: the volume ratio of the mobile phase B is 3%:97 percent; 5-6min, mobile phase A: the volume ratio of the mobile phase B is 3-8%:97-92%;6-45min, mobile phase A: the volume ratio of the mobile phase B is 8%:92 percent.
The chromatographic conditions are as follows: measuring by high performance liquid chromatography, and taking octadecylsilane chemically bonded silica as filler; taking acetonitrile as a mobile phase A and taking phosphoric acid with the volume fraction of 0.1% as a mobile phase B, and carrying out gradient elution; the detection wavelength is 215nm; the flow rate is 1.0ml/min; the theoretical plate number is not less than 3000 calculated according to arecoline peak.
The preparation steps of the reference substance solution of the reference substance comprise: adding arecoline hydrobromide and catechin control into 40-60% alcohol solution;
the preparation method of the reference solution of the reference medicinal materials comprises the following steps: adding 40-60% alcohol solution into Arecae semen control, extracting for 20-30min, filtering, and collecting filtrate;
the preparation method of the test solution comprises precisely weighing a sample to be tested, adding alcohol solution, extracting for 20-40min, adding lost solvent, filtering, and collecting filtrate.
The concentration of the arecoline hydrobromide reference substance solution is 20-70 μ g/ml; the concentration of the catechin reference substance solution is 50-135 mug/ml;
the concentration of the reference solution of the reference medicinal material is 15-30mg/ml;
the concentration of the test solution is 3-30mg/ml;
the sample to be detected is a standard decoction freeze-dried powder sample to be detected, a formula particle sample to be detected, a medicinal material sample to be detected or a decoction piece sample to be detected.
When the sample to be detected is a medicinal material or decoction pieces, the concentration of the sample solution is 15-25mg/ml; when the sample to be tested is standard decoction freeze-dried powder or formula granules, the concentration of the test solution is 3-15mg/ml.
The invention also provides a method for detecting the betel nuts and the burnt betel nuts, the characteristic spectrum of the sample to be detected is obtained according to the construction method and the operation method, and the detection is carried out according to the peak area ratio of the No. 1 peak to the No. 2 peak and the peak area ratio of the No. 4 peak to the No. 2 peak in the characteristic spectrum of the sample to be detected.
When the sample to be detected is a standard decoction freeze-dried powder sample to be detected or a formula particle sample to be detected, the peak height of the No. 1 peak is higher than the No. 2 peak, the sample to be detected is betel nut, and when the peak height of the No. 2 peak is higher than the No. 1 peak, the sample to be detected is burnt betel nut.
Further, when the sample to be detected is a standard decoction freeze-dried powder sample to be detected, the detection is carried out by adopting a formula I and a formula II, and when beta 1 is more than 2.2 and beta 2 is more than 3.6, the sample to be detected is the betel nut standard decoction freeze-dried powder;
when the beta 1 is less than or equal to 2.2 and the beta 2 is less than or equal to 3.6, the sample to be detected is the standard areca catechu decoction freeze-dried powder;
Figure BDA0002651539740000031
wherein S is 1 Is the peak area of peak No. 1, S 2 Is the peak area of peak No. 2, S 4 The peak area of peak No. 4.
Further, when the sample to be detected is a sample to be detected of the formula particle, the formula I and the formula II are adopted for detection, when beta 1 is more than 2.2 and beta 2 is more than 3.6, the sample to be detected is the betel nut formula particle, otherwise, the sample is the burnt betel nut formula particle;
Figure BDA0002651539740000032
wherein S is 1 Is the peak area of peak No. 1, S 2 Is the peak area of peak No. 2, S 4 The peak area of peak No. 4.
Further, when the sample to be detected is decoction pieces or medicinal materials, the detection is carried out by adopting a formula II, when beta 2 is more than 1.8, the sample to be detected is areca, and when beta 2 is less than or equal to 1.8, the sample to be detected is scorched areca;
Figure BDA0002651539740000041
wherein S is 2 Is the peak area of peak No. 2, S 4 The peak area of peak No. 4.
The technical scheme of the invention has the following advantages:
1. according to the method for constructing the characteristic spectrum of the betel nut or the burnt betel nut, the repeatability and the accuracy of the characteristic spectrum obtained by the method are good, the repeatability and the stability of the characteristic spectrum can be ensured by controlling an elution gradient program, and the obtained characteristic peak has good peak shape and high separation degree.
The characteristic spectrum obtained by the method has high accuracy, can be used for evaluating the quality of the betel nuts and the burnt betel nuts, and can be used as an evaluation reference for controlling each process link in the production of the betel nuts or the burnt betel nuts; and the characteristic map can accurately express the consistency of the drug effect substances of the betel nut and the scorched betel nut under different properties.
The characteristic spectrum obtained by the construction method provided by the invention can be used for identifying the betel nut and the burnt betel nut.
2. The method for detecting the betel nuts or the burnt betel nuts, provided by the invention, is simple, convenient and quick, and can accurately identify the betel nuts and the burnt betel nuts by detecting the peak area ratio of the No. 1 peak to the No. 2 peak and the peak area ratio of the No. 4 peak to the No. 2 peak in the characteristic spectrum, particularly identifying and distinguishing betel nut formula particles and burnt betel nut formula particles.
The method for detecting the betel nuts and the burnt betel nuts can be used for identifying medicinal materials, decoction pieces, standard decoction freeze-dried powder or formula granules of the betel nuts and the burnt betel nuts, and realizes quality control of the betel nuts and the burnt betel nuts.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
Fig. 1 is a characteristic spectrum of 18 batches of betel nut pieces in example 1 of the present invention;
fig. 2 is a characteristic spectrum of 18 batches of burnt areca nut pieces in example 1 of the present invention;
FIG. 3 is a characteristic spectrum of 18 batches of betel nut standard decoction lyophilized powder in example 1 of the present invention;
fig. 4 is a characteristic spectrum of 18 standard decoction lyophilized powders of semen arecae scorch in embodiment 1 of the present invention;
FIG. 5 is a characteristic map of a reference solution in example 1 of the present invention;
FIG. 6 is a characteristic spectrum of the betel nut formula granule in example 1 of the present invention;
FIG. 7 is a characteristic spectrum of the burnt betel nut formula granule in example 1 of the present invention;
FIG. 8 is a characteristic map obtained in Experimental group 1 in Experimental example 1 of the present invention;
FIG. 9 is a characteristic map obtained for control group 1 in Experimental example 1 of the present invention;
FIG. 10 is a characteristic map obtained by the control group 2 in Experimental example 1 of the present invention;
FIG. 11 is a characteristic map obtained in comparative group 1 in Experimental example 2 of the present invention;
FIG. 12 is a characteristic map obtained by the control group in Experimental example 3 of the present invention.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
Reagent and instrument
Reagent testing:
a hydrobromic acid arecoline reference substance (batch number: 111684-201602, china institute for testing food and drug);
catechin control (batch No. 110877-201203, china institute for food and drug testing);
areca control (batch No. 120915-201312, china institute for food and drug testing);
the preparation method of the betel nut standard decoction freeze-dried powder comprises the following steps: soaking Arecae semen decoction pieces in casserole for 30min, adding water 6 times the amount of the decoction pieces, boiling with strong fire (500 w), decocting with slow fire for 30min, filtering, and rapidly cooling; adding water 5 times the amount of decoction pieces, boiling with strong fire, decocting with slow fire (200 w) for 20min, filtering, and rapidly cooling; mixing filtrates, concentrating (below 50 deg.C) to obtain a concentrate with a material-liquid ratio of 1:1 (relative density of 1.05-1.10 (50 deg.C)), and freeze drying. 18 batches of areca standard decoction freeze-dried powder are prepared according to the method, and the batch numbers are respectively as follows: 190617-537000-01, 190617-570100-02, 190617-570100-03, 190617-571500-04, 190617-571600-05, 190617-571200-06, 190617-571400-07, 190617-571300-08, 190617-571500-09, 190617-571500-10, 190617-571600-11, 190617-571600-12, 190617-571600-13, 190617-570100-14, 190617-571200-15, 190617-571400-16, 190617-571400-17, and 190617-571300-18, which are respectively marked as B1-B18. The lot numbers of the areca nut decoction pieces corresponding to the areca nut standard decoction freeze-dried powder are respectively Y1-Y18.
The preparation method of the lyophilized powder of standard decoction of semen Arecae Preparatum comprises: placing semen Arecae Preparata decoction pieces in casserole, soaking for 30min, adding water 6 times the decoction pieces, boiling with strong fire (500 w), decocting with slow fire for 30min, filtering, and rapidly cooling; adding water 5 times the amount of decoction pieces, boiling with strong fire, decocting with slow fire (200 w) for 20min, filtering, and rapidly cooling; mixing filtrates, concentrating (below 50 deg.C) to obtain a concentrate with a material-liquid ratio of 1:1 (relative density of 1.05-1.10 (50 deg.C)), and freeze drying. 18 batches of the standard decoction freeze-dried powder of the areca catechu are prepared according to the method and are respectively marked as J1-J18, and the batch numbers are respectively as follows: j190617-537000-01, J190617-570100-02, J190617-570100-03, J190617-571500-04, J190617-571600-05, J190617-571200-06, J190617-571400-07, J190617-571300-08, J190617-571500-09, J190617-571500-10, J190617-571600-11, J190571600-12, J190617-571600-13, J190617-570100-14, J190617-5715, J190617-571400-16, J190617-571400-17, J617-571300-18, respectively correspond to J JY1-JY18.
The preparation method of the betel nut formula particle comprises the following steps: adding areca-nut decoction pieces, decocting twice, adding 10 times of water for boiling extraction for 2h in the first decoction, adding 7 times of water for boiling extraction for 1.5h in the second decoction, filtering with 150-mesh filter cloth while hot, combining filtrates, concentrating under reduced pressure (less than or equal to 60 ℃), spray drying, adding an appropriate amount of auxiliary materials at an air inlet temperature of 170 +/-5 ℃, mixing uniformly, and granulating. 3 batches of areca nut formula granules are prepared according to the method, wherein the batches comprise: KL190617-537000-01, KL 190617-570100-02, KL 190617-570100-03), denoted as K1-K3;
the preparation method of the burnt betel nut formula particle comprises the following steps: feeding semen Arecae Preparata decoction pieces, decocting twice, adding 10 times of water into the first decoction, boiling for 2 hr, adding 7 times of water into the second decoction, boiling for 1.5 hr, filtering while hot, mixing filtrates, concentrating under reduced pressure (less than or equal to 60 deg.C), spray drying, adding appropriate amount of adjuvant at air inlet temperature of 170 + -5 deg.C, mixing, and granulating. The burnt areca formula granules with 3 batches are prepared according to the method, and the batches are respectively as follows: KLJ190617-537000-01, KLJ190617-570100-02, KLJ190617-570100-03, and designated as KLJ1-KLJ3.
The producing areas of 18 batches of different decoction pieces provided by the invention are respectively,
y190617-537000-01 origin: guangxi Zhuang autonomous area Yulin City Yuzhou district;
y190617-570100-02 origin: the Haikou city, hainan province, longhua district;
y190617-570100-03 origin: the Haikou city, hainan province, longhua district;
y190617-571500-04 origin: wanning city, hainan province;
y190617-571600-05 origin: tunchang county, hainan province;
y190617-571200-06 origin: ding an county, hainan province;
y190617-571400-07 origin: johnson city, hainan province;
y190617-571300-08 origin: wenchang city, hainan province;
y190617-571500-09 origin: wanning city, hainan province;
y190617-571500-10 origin: wanning city, hainan province;
y190617-571600-11 origin: tunchang county, hainan province;
y190617-571600-12 origin: tunchang county, hainan province;
y190617-571600-13 origin: tunchang county, hainan province;
y190617-570100-14 origin: the Haikou city, hainan province, longhua district;
y190617-571200-15 origin: ding an county, hainan province;
y190617-571400-16 origin: johnhai city, hainan province;
origin of Y190617-571400-17: johnson city, hainan province;
y190617-571300-18 origin: wenchang city, hainan province;
the batch numbers of the areca-nut decoction pieces are respectively abbreviated as Y1-Y18;
the charred Arecae semen decoction pieces are prepared by heating a roller medicine parching machine to 240 deg.C, adding Arecae semen decoction pieces at 20r/min, parching for 30min to obtain charred Arecae semen decoction pieces, wherein the obtained charred Arecae semen decoction pieces have batch numbers corresponding to Y1-Y18, and are marked as JY1-JY18.
Acetonitrile is chromatographically pure, fisher;
the ethanol is analytically pure, and is prepared by chemical reagents of national drug group;
phosphoric acid is analytically pure, fisher;
the water is purified Chen's water.
The instrument comprises the following steps:
waters e2695 HPLC high performance liquid chromatograph, 2489UV/Vis Detector ultraviolet Detector, empower 3 chromatographic workstation;
ME104E electronic balance (mettler toledo), JY20002 electronic balance (mettler toledo);
KQ-300DB ultrasonic cleaner (Kunshan ultrasonic instruments Co., ltd.);
an electronic constant temperature water bath DZKW-4 (Beijing Zhongxing Weiwei instruments Co., ltd.).
Example 1
The embodiment provides a method for constructing a characteristic map of betel nut or burnt betel nut, which comprises the following steps,
preparation of reference solutions: accurately weighing arecoline hydrobromide reference substance, adding 50% ethanol, and making into reference substance solution of arecoline hydrobromide reference substance of 60 μ g/ml;
accurately weighing catechin reference substance, adding 50% ethanol, and making into reference substance solution of catechin reference substance of 120 μ g/ml.
Preparation of reference solution of reference drug: taking 0.5g of standard areca-nut medicinal material, placing in a conical flask, adding 25ml of ethanol with volume fraction of 50%, sealing, performing ultrasonic treatment for 30min at ultrasonic power of 300W and frequency of 40kHz, taking out, cooling, shaking up, filtering, and taking subsequent filtrate to obtain reference areca-nut medicinal material solution with concentration of 20mg/ml.
Preparing a particle or freeze-dried powder test solution: grinding a sample to be tested, taking 0.1g of the ground sample, precisely weighing, placing the ground sample in a conical flask, precisely adding 25ml of ethanol with the volume fraction of 50%, weighing, carrying out ultrasonic treatment for 30min, wherein the ultrasonic power is 300W, the frequency is 40kHz, cooling, weighing again, complementing the weight loss by using the ethanol with the volume fraction of 50%, shaking up, filtering, and taking the subsequent filtrate to obtain a sample solution with the concentration of 4mg/ml;
preparing a test solution of decoction pieces: grinding a sample (decoction piece) to be tested, precisely weighing 0.5g, placing the sample (decoction piece) in a conical flask, precisely adding 25ml of ethanol with the volume fraction of 50%, weighing the sample, carrying out ultrasonic treatment for 30min, wherein the ultrasonic power is 300W and the frequency is 40kHz, cooling, weighing again, complementing the weight loss by using the ethanol with the volume fraction of 50%, shaking up, filtering, and taking a subsequent filtrate to obtain the sample solution with the concentration of 20mg/ml.
Chromatographic conditions are as follows: measuring by high performance liquid chromatography, wherein the chromatographic column adopts Agilent ZORBAX Eclipse XDB-C18 (column length 250mm, inner diameter 4.6mm, particle diameter 5 μm), and octadecylsilane chemically bonded silica is used as filler; taking acetonitrile as a mobile phase A, taking phosphoric acid with the volume fraction of 0.1% as a mobile phase B, and performing gradient elution; the detection wavelength is 215nm; the flow rate is 1.0ml/min; the column temperature is 30 ℃; the theoretical plate number calculated by arecoline peak should be not less than 3000.
Wherein the gradient elution procedure is 0-5min, and the mobile phase A: the volume ratio of the mobile phase B is 3%:97 percent; 5-6min, mobile phase A: the volume ratio of the mobile phase B is 3-8%:97-92%;6-45min, mobile phase A: the volume ratio of the mobile phase B is 8%:92 percent.
And (3) determination: respectively sucking 10 μ L of reference substance solution of reference substance, reference solution of reference medicinal material and sample solution, injecting into high performance liquid chromatograph, and measuring.
Identification of characteristic peaks: taking 18 batches of Arecae semen decoction pieces and 18 batches of parched Arecae semen decoction pieces, making into sample solution according to the above method, and determining to obtain characteristic chromatogram according to "chromatogram condition" and "determination" item, wherein the characteristic chromatograms of Arecae semen decoction pieces and parched Arecae semen decoction pieces are shown in figures 1 and 2. Taking 18 batches of areca standard decoction lyophilized powder and 18 batches of burnt areca standard decoction lyophilized powder, preparing into test solution according to the above method, and measuring according to the 'chromatographic condition' and 'measurement' term to obtain characteristic maps, wherein the characteristic maps of the areca standard decoction lyophilized powder and the burnt areca standard decoction lyophilized powder are shown in fig. 3 and 4.4 peaks with better repeatability are selected as common characteristic peaks of the betel nut or the burnt betel nut, namely a No. 1 common characteristic peak, a No. 2 common characteristic peak, a No. 3 common characteristic peak and a No. 4 common characteristic peak respectively, the relative retention time of the characteristic peaks on the characteristic spectra of the betel nut and the burnt betel nut is consistent, and the difference is only that the peak areas of the characteristic peaks are different. The characteristic spectrum of Arecae semen or burnt Arecae semen should exhibit 4 characteristic peaks, and correspond to 4 characteristic peaks in reference substance characteristic spectrum of reference medicinal material, wherein peak 2 and peak 4 correspond to arecoline hydrobromide and catechin, respectively. Taking the No. 2 peak as a reference peak, wherein the relative retention time of other characteristic peaks is +/-10% of a specified value, and the relative retention time of the No. 1 peak is 0.83,2 peak is 1.00; taking the No. 4 peak as a reference peak, wherein the relative retention time of other characteristic peaks is +/-10% of a specified value, and the relative retention time of the No. 3 peak is 0.80; peak No. 4 had a relative retention time of 1.00. The characteristic spectrum of the arecoline hydrobromide reference substance solution and the characteristic spectrum of the catechin reference substance solution are shown in fig. 5, and the characteristic spectrum of the areca or the burnt areca is compared with the characteristic spectrum of the reference substance solution, so that the peak No. 2 is the arecoline hydrobromide peak, and the peak No. 4 is the catechin peak.
Taking 3 batches of betel nut formula particles and 3 batches of burnt betel nut formula particles, preparing a test solution according to the method, measuring according to a chromatographic condition and a measurement item to obtain a characteristic spectrum, and calculating retention time and relative retention time of the betel nut formula particles, the burnt betel nut formula particles, the betel nut standard decoction freeze-dried powder and the burnt betel nut standard decoction freeze-dried powder, the betel nut decoction pieces and the burnt betel nut decoction pieces at the same time as shown in figures 6 and 7, and the retention time and the relative retention time are shown in tables 1 and 2. As can be seen from the results in tables 1 and 2, the betel nut decoction pieces, the scorched betel nut decoction pieces, the betel nut standard decoction lyophilized powder, the scorched betel nut standard decoction lyophilized powder, the betel nut formula particles and the scorched betel nut formula particles meet the characteristic spectrum obtained by the construction method of the present invention, and the method can realize quality control of the betel nuts and the scorched betel nuts.
TABLE 1 relative retention time of characteristic peaks of Areca catechu decoction pieces, standard decoction, and formula granule
Figure BDA0002651539740000091
Figure BDA0002651539740000101
Figure BDA0002651539740000111
TABLE 2 relative retention time of characteristic peaks of semen Arecae decoction pieces, standard decoction and formula granule
Figure BDA0002651539740000112
Figure BDA0002651539740000121
Example 2
The embodiment provides a method for detecting areca formula particles and burnt areca formula particles, which comprises the steps of constructing a characteristic map of a sample to be detected by adopting the method in embodiment 1 to obtain the characteristic map of the sample to be detected; then, detecting the peak area ratio of the No. 1 peak to the No. 2 peak and the peak area ratio of the No. 4 peak to the No. 2 peak in the characteristic spectrum of the sample to be detected; the method comprises the following specific steps:
the preparation method of the sample solution to be detected comprises the following steps: grinding a sample to be tested of the formula particles, precisely weighing 0.1g of the sample, placing the sample in an erlenmeyer flask, precisely adding 25ml of ethanol with the volume fraction of 50%, weighing the sample, carrying out ultrasonic treatment for 30min, wherein the ultrasonic power is 300W and the frequency is 40kHz, cooling, weighing again, complementing the lost weight with the ethanol with the volume fraction of 50%, shaking up, filtering, and taking a subsequent filtrate to obtain the sample solution with the concentration of 4mg/ml.
The amount of sample was 10. Mu.L as determined by the "chromatographic conditions" in example 1, and a characteristic spectrum was obtained.
Calculating the peak area ratio of the No. 1 peak to the No. 2 peak and the peak area ratio of the No. 4 peak to the No. 2 peak in the characteristic spectrum of the sample to be detected of the formula particles according to the formula I and the formula II, wherein when beta 1 is more than 2.2 and beta 2 is more than 3.6, the sample to be detected of the formula particles is the betel nut formula particles; when beta 1 is less than or equal to 2.2 and beta 2 is less than or equal to 3.6, the sample to be tested of the formula particle is the burnt areca seed formula particle;
Figure BDA0002651539740000122
wherein S is 1 Is the peak area of peak No. 1, S 2 Is the peak area of peak No. 2, S 4 The peak area of peak No. 4.
Example 3
The embodiment provides a method for detecting areca-nut decoction pieces and scorched areca-nut decoction pieces, which comprises the steps of constructing a characteristic map of a sample to be detected by adopting the method in embodiment 1 to obtain the characteristic map of the sample to be detected; then, detecting by using the peak area ratio of the No. 1 peak to the No. 2 peak and the peak area ratio of the No. 4 peak to the No. 2 peak in the characteristic spectrum of the sample to be detected; the method comprises the following specific steps:
the preparation method of the sample solution to be tested comprises the following steps: taking 0.5g of a sample to be tested, precisely weighing, placing in a conical flask, precisely adding 25ml of ethanol with the volume fraction of 50%, weighing, ultrasonically treating for 30min at the ultrasonic power of 300W and the frequency of 40kHz, standing for cooling, weighing again, complementing the weight loss by using the ethanol with the volume fraction of 50%, shaking up, filtering, and taking the subsequent filtrate to obtain the sample solution with the concentration of 20mg/ml.
The sample size was 10. Mu.L as determined by the "chromatographic conditions" in example 1, and a characteristic spectrum of the decoction pieces was obtained.
According to the formula II, calculating the peak area ratio of the No. 4 peak to the No. 2 peak in the characteristic spectrum of the sample to be measured in the decoction piece, wherein when beta 2 is more than 1.8, the sample to be measured in the decoction piece is areca, and when beta 2 is less than or equal to 1.8, the sample to be measured in the decoction piece is scorched areca;
Figure BDA0002651539740000131
wherein S is 2 Is the peak area of peak No. 2, S 4 The peak area of peak No. 4.
Example 4
The embodiment provides a method for detecting areca standard decoction freeze-dried powder and burnt areca standard decoction freeze-dried powder, which comprises the steps of constructing a characteristic map of a sample to be detected by adopting the method in embodiment 1 to obtain the characteristic map of the sample to be detected; then, detecting the peak area ratio of the No. 1 peak to the No. 2 peak and the peak area ratio of the No. 4 peak to the No. 2 peak in the characteristic spectrum of the sample to be detected; the method comprises the following specific steps:
the preparation method of the sample solution to be detected comprises the following steps: grinding a sample to be detected of the standard decoction freeze-dried powder, taking 0.1g, precisely weighing, placing in a conical flask, precisely adding 25ml of ethanol with the volume fraction of 50%, weighing, carrying out ultrasonic treatment for 30min, wherein the ultrasonic power is 300W and the frequency is 40kHz, cooling, weighing again, complementing the loss weight by using the ethanol with the volume fraction of 50%, shaking up, filtering, and taking a subsequent filtrate to obtain the sample solution with the concentration of 4mg/ml.
According to the determination of the chromatographic condition in the embodiment 1, the sampling amount is 10 mu L, and the characteristic spectrum of the standard decoction freeze-dried powder is obtained.
Calculating the peak area ratio of the No. 1 peak to the No. 2 peak and the peak area ratio of the No. 4 peak to the No. 2 peak in the characteristic spectrum of the standard decoction freeze-dried powder sample to be detected according to the formula I and the formula II, wherein when beta 1 is more than 2.2 and beta 2 is more than 3.6, the standard decoction freeze-dried powder sample to be detected is areca; on the contrary, the product is semen Arecae;
Figure BDA0002651539740000132
wherein S is 1 Is the peak area of peak No. 1, S 2 Peak area of peak No. 2, S 4 Peak area of peak No. 4.
Experimental example 1 examination of elution gradient
Taking the areca standard decoction freeze-dried powder (B1) of the same batch, and preparing 2 parts of a test solution according to the method under the item of the preparation of the test solution in example 1; experimental group 1A characteristic map was obtained as shown in FIG. 8, according to the "chromatographic conditions" and "measurement" items in example 1.
The control group 1 differs from the experimental group 1 only in the elution gradient, and the elution gradient program of the control group is: 0-5min, mobile phase A: the volume ratio of the mobile phase B is 3%:97 percent; 5-45min, mobile phase A: the volume ratio of the mobile phase B is 3-20%:97-80% to obtain a characteristic spectrum shown in figure 9.
Control 2 the experimental group 1 differed only in the elution gradient, which was programmed as follows: 0-5min, mobile phase A: the volume ratio of the mobile phase B is 4%:96 percent; 5-6min, mobile phase A: the volume ratio of the mobile phase B is 4-15%:96 to 85 percent; 6-30min, mobile phase A: the volume ratio of the mobile phase B is 15%:85, obtaining a characteristic map, which is shown in figure 10.
In the experimental group 1, the baseline was relatively flat and the resolution of the chromatographic peak was relatively good in comparison with the control groups 1 and 2, and therefore, the elution gradient of the experimental group 1 was selected.
Experimental example 2 examination of Mobile phase
Taking the areca standard decoction freeze-dried powder (B1) of the same batch, and preparing 2 parts of a test solution according to the method under the item of the preparation of the test solution in example 1; experimental group 1 the characteristic maps were obtained by performing the "chromatographic conditions" and "assay" procedures in example 1, as shown in FIG. 8.
The difference between the control group 1 and the experimental group 1 is that the mobile phase and the elution gradient are different, the mobile phase of the control group is acetonitrile (a) -water (B), the elution gradient is 0-12min, and the mobile phase a: the volume ratio of the mobile phase B is 8%:92 percent; 12-40min, mobile phase A: the volume ratio of the mobile phase B is 8-25%:92-75%;40-55min, mobile phase A: the volume ratio of the mobile phase B is 25-70%:75-30% to obtain a characteristic map, as shown in FIG. 11.
Compared with the control group 1, the experimental group 1 has poor peak shape and separation degree of arecoline hydrobromide chromatographic peak in the control group 1.
Experimental example 3 examination of sample solution extraction solvent
Taking the areca standard decoction freeze-dried powder (B1) of the same batch, taking 2 parts in total, preparing the test solution according to the method under the item of preparation of the test solution in the example 1 in the experimental group 1, and measuring according to the item of chromatographic conditions and measuring in the example 1 to obtain a characteristic spectrum, which is as shown in the figure 8.
The control group 1, which differs from the experimental group 1 only in the extraction solvent, replaced 50% ethanol with water.
The control group 2, which differs from the experimental group 1 only in the extraction solvent, was 70% ethanol instead of 50% ethanol.
The control group 3, which differs from the experimental group 1 only in the extraction solvent, was 30% ethanol instead of 50% ethanol.
The control group 4, which differs from the experimental group 1 only in the extraction solvent, replaced 50% ethanol with 70% methanol.
The control group 5, which differs from the experimental group 1 only in the extraction solvent, replaced 50% ethanol with 50% methanol.
The control group 6, which differs from the experimental group 1 only in the extraction solvent, replaced 50% ethanol with 30% methanol.
The characteristic maps obtained from the control groups 1 to 6 are shown in fig. 12, and it can be seen from fig. 12 that when water and methanol of each concentration are used as extraction solvents, no chromatographic peak is present at the position corresponding to arecoline hydrobromide, and the substance information presented by the characteristic maps obtained by using 70% ethanol, 50% ethanol and 30% ethanol as solvents is basically the same.
Experimental example 4 methodological verification
(1) Repeatability survey
Taking the same batch of areca standard decoction freeze-dried powder (B1), preparing 6 parts of test solution according to the method under the item of preparation of test solution in example 1, respectively numbering 1-6, determining according to the chromatographic conditions and determination in example 1 to obtain a characteristic spectrum, and calculating the relative retention time, wherein the results are shown in Table 3.
Taking the same batch of standard decoction lyophilized powder of semen Arecae Premna (J1), preparing into sample solution according to the method of "preparation of sample solution" in example 1, total 6 parts, respectively numbering 7-12, determining according to "chromatographic conditions" and "determination" in example 1 to obtain characteristic spectrum, calculating relative retention time, and see Table 4.
TABLE 3 relative Retention time of Standard decoction of Areca catechu
Figure BDA0002651539740000151
TABLE 4 relative Retention time of standard decoction of semen Arecae
Figure BDA0002651539740000161
The results in tables 3 and 4 show that RSD of the betel nut standard decoction lyophilized powder and the burnt betel nut standard decoction lyophilized powder are in the range of 0.0-0.1%, indicating that the repeatability of the characteristic spectrum is better.
(2) Intermediate precision investigation
Taking the areca standard decoction freeze-dried powder (B1) of the same batch, preparing 6 parts of the sample solution according to the method under the item of preparation of the sample solution in the embodiment 1, respectively numbering 1-6, then measuring by adopting different high performance liquid chromatographs according to the item of chromatographic conditions and measuring methods in the embodiment 1 to obtain a characteristic spectrum, and then calculating the relative retention time, which is shown in Table 5.
Taking the same batch of lyophilized powder of standard decoction of semen Arecae Preareta (J1), preparing into test solution according to the method of "preparation of test solution" in example 1, 6 parts in total, numbering 7-12 respectively, measuring with different high performance liquid chromatographs according to the "chromatographic conditions" and "measurement" items in example 1 to obtain characteristic spectrum, and calculating relative retention time, as shown in Table 6.
TABLE 5 relative Retention time of Areca catechu standard decoction
Figure BDA0002651539740000162
TABLE 6 relative retention time of standard decoction of semen Arecae
Figure BDA0002651539740000171
As can be seen from the results in tables 5 and 6, the RSD of the standard betel nut decoction lyophilized powder is in the range of 0.0-0.1%, and the RSD of the standard betel nut decoction lyophilized powder is in the range of 0.0-1.8%. The relative retention time RSD of the standard decoction freeze-dried powder of the semen arecae preparatum is in the range of 0.1% -0.2%, and the relative retention time RSD range among different instruments is 0.0-1.9%, which indicates that the detection results among different instruments meet the analysis requirements.
(3) Stability survey
Taking the same batch of areca standard decoction freeze-dried powder (B1), preparing 7 parts of test solution according to the method under the item of preparation of test solution in example 1, then respectively placing at room temperature for 0, 2, 4, 8, 10, 12 and 24h, and then determining according to the items of chromatography conditions and determination in example 1 to obtain a characteristic spectrum, and calculating the relative retention time, which is shown in Table 7.
Taking the same batch of standard decoction lyophilized powder of semen Arecae Preareta (J1), preparing into test solution 7 parts by the method of "preparation of test solution" in example 1, standing at room temperature for 0, 2, 4, 8, 10, 12, and 24h, respectively, and determining according to the "chromatographic conditions" and "determination" in example 1 to obtain characteristic spectrum, and calculating relative retention time, see Table 8.
TABLE 7 relative Retention time of standard decoction of semen Arecae
Figure BDA0002651539740000172
TABLE 8 relative retention time of standard decoction of semen Arecae
Figure BDA0002651539740000181
As can be seen from the results in tables 7 and 8, the relative retention time RSD of each characteristic peak of betel nut is in the range of 0.0% to 0.1%, and the relative retention time RSD of each characteristic peak of betel nut is in the range of 0.0% to 0.1%, indicating that the chemical components in the test solution have better stability within 24 hours.
(4) Durability examination
A. Investigation of flow Rate
Taking the same batch of areca standard decoction freeze-dried powder (B1), preparing 3 parts of test solution according to the method under the item of preparation of test solution in example 1, determining the experimental group 1 according to the item of chromatographic conditions and determination in example 1 to obtain a characteristic spectrum, and then calculating the relative retention time, wherein the result is shown in Table 9.
The control group 1 is different from the experimental group 1 only in the flow rate, and the flow rate of the control group 1 is 0.8ml/min;
the control group 2 was different from the experimental group 1 only in the flow rate, and the flow rate of the control group 2 was 1.2ml/min.
Taking the same batch of standard decoction lyophilized powder of semen Arecae Preparatum (J1), preparing into test solution according to the method of "preparation of test solution" in example 1, 3 parts in total, determining experimental group 2 according to the "chromatographic conditions" and "determination" in example 1 to obtain characteristic spectrum, calculating relative retention time, and finding out the result shown in Table 10.
The control group 3 is different from the experimental group 2 only in the flow rate, and the flow rate of the control group 3 is 0.8ml/min;
the control group 4 differed from the experimental group 2 only in the flow rate, and the flow rate of the control group 4 was 1.2ml/min.
TABLE 9 relative Retention time of Areca catechu standard decoction
Figure BDA0002651539740000182
TABLE 10 relative retention time of standard decoction of semen Arecae
Figure BDA0002651539740000191
As can be seen from the results in tables 9 and 10, the relative retention time RSD of the standard betel nut decoction lyophilized powder is in the range of 0.0% to 1.5%, and the relative retention time RSD of the standard betel nut decoction lyophilized powder is in the range of 0.3% to 1.5%, which indicates that different flow rates have no influence on the construction of the characteristic spectrum.
B. Investigation of column temperature
Taking the same batch of areca standard decoction freeze-dried powder (B1), preparing the same batch of areca standard decoction into a test solution according to the method under the item of preparation of the test solution in example 1, wherein the total amount of the test solution is 3 parts, determining the experiment group 1 according to the chromatographic conditions and the determination in example 1 to obtain a characteristic spectrum, and then calculating the relative retention time, wherein the result is shown in a table 11.
The control group 1 is different from the experimental group 1 only in column temperature, and the column temperature of the control group 1 is 28 ℃;
the control group 2 was different from the experimental group 1 only in the column temperature, and the column temperature of the control group 2 was 32 ℃.
Taking the same batch of standard decoction lyophilized powder of semen Arecae Preparatum (J1), preparing into test solution according to the preparation of test solution in example 1, 3 parts in total, determining experimental group 2 according to the chromatographic conditions and determination items in example 1 to obtain characteristic spectrum, and calculating relative retention time, the result is shown in Table 12.
The difference between the control group 3 and the experimental group 2 is only that the column temperature is different, and the column temperature of the control group 3 is 28 ℃;
the control group 4 was different from the experimental group 2 only in the column temperature, and the column temperature of the control group 4 was 32 ℃.
TABLE 11 relative Retention time of Standard decoction of Areca catechu
Figure BDA0002651539740000192
TABLE 12 relative retention time of standard decoction of semen Arecae
Figure BDA0002651539740000193
Figure BDA0002651539740000201
As can be seen from the results in tables 11 and 12, the RSD of the betel nut standard decoction lyophilized powder is in the range of 0.0% to 0.6%, and the RSD of the burnt betel nut standard decoction lyophilized powder is in the range of 0.0% to 0.6%, indicating that the column temperature has no influence on the construction of the characteristic spectrum.
C. Examination of acidity
Taking the same batch of areca standard decoction freeze-dried powder (B1), preparing 3 parts of test solution according to the method under the item of preparation of test solution in example 1, determining the experimental group 1 according to the item of chromatographic conditions and determination in example 1 to obtain a characteristic spectrum, and then calculating the relative retention time, wherein the result is shown in Table 13.
The control group 1 is different from the experimental group 1 only in acidity, and the mobile phase B in the control group 1 is phosphoric acid with volume fraction of 0.05%;
the control group 2 is different from the experimental group 1 only in acidity, and the mobile phase B in the control group 2 is phosphoric acid with a volume fraction of 0.15%.
Taking the same batch of standard decoction lyophilized powder of semen Arecae Preparatum (J1), preparing into test solution according to the method of "preparation of test solution" in example 1, 3 parts in total, determining experimental group 2 according to the "chromatographic conditions" and "determination" in example 1 to obtain characteristic spectrum, calculating relative retention time, and finding the result in Table 14.
The control group 3 is different from the experimental group 2 only in acidity, and the mobile phase B in the control group 3 is phosphoric acid with volume fraction of 0.05%;
the control group 4 is different from the experimental group 2 only in acidity, and the mobile phase B in the control group 4 is 0.15% phosphoric acid by volume fraction.
From the results in tables 13 and 14, the acidity had no effect on the construction of the characteristic map.
TABLE 13 relative retention time of standard decoction of Arecae semen
Figure BDA0002651539740000202
TABLE 14 relative retention time of standard decoction of semen Arecae
Figure BDA0002651539740000203
Figure BDA0002651539740000211
D. Inspection of chromatographic columns
Taking the same batch of areca standard decoction freeze-dried powder (B1), preparing 3 parts of test solution according to the method under the item of preparation of test solution in example 1, determining the experimental group 1 according to the item of chromatographic conditions and determination in example 1 to obtain a characteristic spectrum, and then calculating the relative retention time, wherein the result is shown in Table 13.
Control 1 differed from experimental 1 only in the column, which was Welch Xtimate C18 (4.6 x 250mm,5 μm) in control 1;
control 2 differed from experimental 1 only in the column, which was Waters XTerra MS C18 (4.6X 250mm,5 μm) in control 2.
Taking the same batch of standard decoction lyophilized powder of semen Arecae Preparatum (J1), preparing into test solution according to the method of "preparation of test solution" in example 1, 3 parts in total, determining experimental group 2 according to the "chromatographic conditions" and "determination" in example 1 to obtain characteristic spectrum, calculating relative retention time, and finding the result in Table 14.
Control 3 differed from experimental 2 only in the column, which was Welch Xtimate C18 (4.6 x 250mm,5 μm) in control 3;
control 4 differed from experimental 2 only in the column, which was Waters XTerra MS C18 (4.6X 250mm,5 μm) in control 4.
TABLE 15 relative retention time of standard Areca catechu decoction
Figure BDA0002651539740000212
TABLE 16 relative retention time of standard decoction of semen Arecae
Figure BDA0002651539740000213
(5) Investigation of beta value
A. Beta 1, beta 2 investigation of standard decoction lyophilized powder
Beta 1 values and beta 2 values of 18 batches of areca catechu standard decoction freeze-dried powder and 18 batches of burnt areca catechu standard decoction freeze-dried powder are calculated, and the results are shown in a table 17.
TABLE 17 beta 1 and beta 2 values of standard decoction lyophilized powder of Arecae semen and burnt Arecae semen
Figure BDA0002651539740000221
The range of beta 1 of the areca standard decoction is 2.46-4.48, and the range of beta 2 is 3.98-5.07; the range of beta 1 of the standard decoction of the semen arecae preparatum is 1.24-2.14, and the range of beta 2 is 1.71-3.37. Beta 1 and beta 2 are used as identification points for detecting the areca and burnt areca standard decoction freeze-dried powder.
B. Beta 1, beta 2 investigation of formulation granules
Beta 1 and beta 2 values were calculated for 3 batches of areca nut formula and 3 batches of burnt areca nut formula, as shown in table 18.
TABLE 18 beta 1 and beta 2 values for Areca catechu and Areca catechu formulations
Figure BDA0002651539740000222
The beta 1 range of the betel nut formula particle is 5.88 to 6.39, and the beta 2 range is 6.27 to 7.12; the range of beta 1 of the burnt betel nut formula particle is 1.95-2.15, and the range of beta 2 is 3.22-3.47; beta 1 and beta 2 are used as identification points for detecting the betel nut and the burnt betel nut formula particles.
C. Beta 2 investigation of decoction pieces
Taking 18 batches of areca-nut decoction pieces and 18 batches of burnt areca-nut decoction pieces, preparing a test solution according to the method in the embodiment 1, measuring according to the chromatographic condition in the embodiment 1, obtaining a characteristic spectrum by using the sample volume of 10 mu L, and calculating the beta 2 value, wherein the table is 19.
TABLE 19 beta 2 value of Arecae semen and parched Arecae semen decoction pieces
Figure BDA0002651539740000231
In table 19, the range of β 2 of betel nut decoction pieces is 1.88-2.30; the range of the beta 2 of the burnt areca formula particles is 1.02-1.72; beta 2 is used as the identification point for detecting the betel nut and the burnt betel nut formula particles.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the scope of the invention.

Claims (9)

1. A method for constructing a characteristic spectrum of areca nuts or charred areca nuts is characterized by comprising the steps of obtaining the characteristic spectrum of the areca nuts or the charred areca nuts, wherein the characteristic peaks of the characteristic spectrum comprise 4 common characteristic peaks which are respectively a No. 1 peak, a No. 2 peak, a No. 3 peak and a No. 4 peak, wherein the No. 2 peak is an arecoline hydrobromide peak, and the No. 4 peak is a catechin peak;
taking peak No. 2 as a reference peak, and the relative retention time of peak No. 1 is 0.83; taking the No. 4 peak as a reference peak, and the relative retention time of the No. 3 peak is 0.80;
wherein the relative retention time of the common characteristic peak is +/-10% of a specified value;
preparation of reference solutions: preparing arecoline hydrobromide reference substance solution and catechin reference substance solution from arecoline hydrobromide and catechin reference substance;
preparing reference solution of semen Arecae reference medicinal material;
preparation of a test solution: taking a sample to be detected to prepare a test solution;
chromatographic conditions are as follows: measuring by high performance liquid chromatography, and using octadecylsilane chemically bonded silica as filler; taking acetonitrile as a mobile phase A and phosphoric acid as a mobile phase B, and performing gradient elution; the detection wavelength is 200-250nm; the flow rate is 0.5-1.5ml/min;
and (3) determination: respectively sucking reference substance solution and sample solution, injecting into high performance liquid chromatograph, and measuring;
wherein the procedure of gradient elution comprises: 0-5min, mobile phase A: the volume ratio of the mobile phase B is 3%:97 percent; 5-6min, mobile phase A: the volume ratio of the mobile phase B is 3-8%:97-92%;6-45min, mobile phase A: the volume ratio of the mobile phase B is 8%:92 percent.
2. The construction method according to claim 1, wherein the chromatographic conditions are: measuring by high performance liquid chromatography, and using octadecylsilane chemically bonded silica as filler; taking acetonitrile as a mobile phase A and taking phosphoric acid with the volume fraction of 0.1% as a mobile phase B, and carrying out gradient elution; the detection wavelength is 215nm; the flow rate was 1.0ml/min.
3. The construction method according to claim 1 or 2, wherein the preparation step of the reference substance solution comprises: adding arecoline hydrobromide and catechin control into 40-60% alcohol solution;
the preparation method of the reference solution of the reference medicinal material comprises the following steps: adding 40-60% alcoholic solution into Arecae semen control, extracting for 20-30min, filtering, and collecting filtrate;
the preparation method of the test solution comprises precisely weighing sample to be tested, adding alcohol solution, extracting for 20-40min, adding solvent, filtering, and collecting filtrate.
4. The method according to claim 1 or 2, wherein the concentration of the arecoline hydrobromide reference solution is 20-70 μ g/ml; the concentration of the catechin reference substance solution is 50-135 mu g/ml;
the concentration of the reference solution of the reference medicinal material is 15-30mg/ml;
the concentration of the test solution is 3-30mg/ml;
the sample to be detected is a standard decoction freeze-dried powder sample to be detected, a formula particle sample to be detected, a medicinal material sample to be detected or a decoction piece sample to be detected.
5. A method for detecting betel nuts and burnt betel nuts is characterized in that a characteristic map of a sample to be detected is obtained according to the construction method of any one of claims 1 to 4 and the same operation method, and the detection is carried out according to the peak area ratio of the No. 1 peak to the No. 2 peak and the peak area ratio of the No. 4 peak to the No. 2 peak in the characteristic map of the sample to be detected.
6. The method according to claim 5, wherein when the sample to be tested is a standard decoction lyophilized powder sample to be tested or a formula granule sample to be tested, the peak height of the No. 1 peak is higher than the No. 2 peak, the sample to be tested is betel nut, and when the peak height of the No. 2 peak is higher than the No. 1 peak, the sample to be tested is taro betel nut.
7. The method of claim 6, wherein when the sample to be tested is standard decoction lyophilized powder, the test is performed by using formula I and formula II, when β 1 is greater than 2.2 and β 2 is greater than 3.6, the sample to be tested is betel nut standard decoction lyophilized powder;
when beta 1 is less than or equal to 2.2 and beta 2 is less than or equal to 3.6, the sample to be detected is the standard decoction freeze-dried powder of the burnt areca nut;
Figure QLYQS_1
the compound has a structure shown in a formula I,
Figure QLYQS_2
formula II;
wherein S is 1 Is the peak area of peak No. 1, S 2 Peak area of peak No. 2, S 4 Peak area of peak No. 4.
8. The method of claim 6, wherein when the sample to be tested is a formula particle sample to be tested, the test is performed by using formula I and formula II, when β 1 is greater than 2.2 and β 2 is greater than 3.6, the sample to be tested is a betel nut formula particle, and vice versa is a burnt betel nut formula particle;
Figure QLYQS_3
the compound has a structure shown in a formula I,
Figure QLYQS_4
formula II;
wherein S is 1 Is the peak area of peak No. 1, S 2 Is the peak area of peak No. 2, S 4 The peak area of peak No. 4.
9. The method of claim 5, wherein the detection is performed by using formula ii when the sample to be detected is decoction pieces or medicinal materials, the sample to be detected is betel nut when β 2 is greater than 1.8, and the sample to be detected is scorched betel nut when β 2 is less than or equal to 1.8;
Figure QLYQS_5
formula II;
wherein S is 2 Is the peak area of peak No. 2, S 4 The peak area of peak No. 4.
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