A method for constructing a characteristic map of mistletoe formula granules and application thereof

Abstract

The application provides a construction method and application of a mistletoe preparation characteristic spectrum, wherein the construction method of the mistletoe preparation characteristic spectrum adopts a high performance liquid chromatography method to obtain a characteristic spectrum of a to-be-detected substance; and specific chromatographic conditions of the high performance liquid chromatography method are provided, wherein an octadecylsilane bonded silica gel is used as a filler of a chromatographic column; a detection wavelength is 248-252 nm; methanol is used as a mobile phase A, and a 0.09-0.11% phosphoric acid aqueous solution is used as a mobile phase B, and elution is performed according to a specific gradient program. The characteristic spectrum obtained by the construction method is comprehensive in material information, and fully represents key characteristic chemical components of the mistletoe preparation and common adulterated water extraction preparations of the mistletoe; differences in chemical components of the mistletoe and the preparation thereof and different adulterants characterized by the method can realize overall quality control of the mistletoe and identification of adulterated mistletoe.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of traditional Chinese medicine identification, and particularly relates to a construction method and application of a characteristic spectrum of mistletoe dispensing granules. BACKGROUND

[0002] Mistletoe is the dry leafy branch of Viscum coloratum (Komar.) Nakai of the family of Loranthaceae, and has a bitter and flat taste, and belongs to the liver and kidney channels.

[0003] Mistletoe has a complex base and is a semi-parasitic plant. Although the commercially available medicinal materials are mistletoe, they may actually be medicinal materials of different species in the same family, and may have problems such as adulteration, which may affect the use effect and even the safety of mistletoe to some extent. Commonly confused products include Viscum liquidambaricolum Hayata (Yunnan Viscum liquidambaricolum, Sichuan Viscum liquidambaricolum), Taxillus chinensis (DC.) Danser and Taxillus sutchuensis (Hemsl.) Danser. Viscum liquidambaricolum Hayata is the branch and leaf of Viscum liquidambaricolum Hayata of the family of Loranthaceae; Taxillus chinensis (DC.) Danser is the dry leafy branch of Taxillus chinensis (DC.) Danser of the family of Loranthaceae; and Taxillus sutchuensis (Hemsl.) Danser is the most common confused product of mistletoe on the market. These species are in the same family as mistletoe and have similar names and similar properties, and are often adulterated.

[0004] The commonly used identification methods are plant ecology identification and medicinal material appearance identification, which require the identifier to have rich experience in medicinal material identification. However, after being prepared into a preparation, especially a dispensing granule, the medicinal material loses the original appearance characteristics, and it is difficult for the prior art to determine whether the preparation is from mistletoe or whether it is adulterated with other counterfeit products, and the quality control of mistletoe preparations lacks monitoring, which affects the quality stability and effectiveness of mistletoe preparations. SUMMARY

[0005] The prior art discloses a characteristic spectrum construction method and application, which cannot achieve the identification of mistletoe and its adulterants. Therefore, the technical problem to be solved by the present application is to provide a mistletoe preparation characteristic spectrum construction method which can be used to identify mistletoe and its adulterants, and the present application also provides the application of the characteristic spectrum construction method in the identification of mistletoe and its adulterants, and the application of the characteristic spectrum construction method in the quality control of mistletoe.

[0006] A mistletoe preparation characteristic spectrum construction method, which uses high performance liquid chromatography to obtain the characteristic spectrum of the test object;

[0007] The chromatographic conditions of the high performance liquid chromatography are as follows:

[0008] A chromatographic column with octadecylsilane-bonded silica gel as the filler; a detection wavelength of 248-252 nm; elution with methanol as the mobile phase A and 0.09-0.11% phosphoric acid aqueous solution as the mobile phase B according to the following gradient elution program:

[0009]

[0010] In the chromatographic conditions of the high-performance liquid chromatography, the column length of the chromatographic column is 100 mm, the inner diameter of the column is 2.1 mm, and the particle size is 1.6-1.8 μm;

[0011] And / or, the chromatographic column is an ACQUITY UPLC HSS T3, a CORTECS UPLC T3 or a Syncronis aQ;

[0012] And / or, the column temperature is 27-33 ℃;

[0013] And / or, the flow rate is 0.12-0.18 ml / min;

[0014] And / or, the theoretical plate number should be not less than 10,000 according to the peak of syringin.

[0015] When the to-be-tested substance includes a preparation of I. orientale, the preparation process of the to-be-tested substance test sample is as follows: the to-be-tested substance is precisely weighed, a solvent is added, the weight is weighed, ultrasonic treatment is performed, the sample is taken out, cooled, and weighed again, the lost weight is made up with the corresponding solvent, shaken, filtered, and the filtrate is taken, to obtain the test sample solution;

[0016] When the to-be-tested substance is a control medicinal material, the preparation process of the to-be-tested substance test sample is as follows: the to-be-tested substance control medicinal material is taken, water is added, hot reflux treatment is performed, cooled, filtered, and the filtrate is taken, to obtain the control medicinal material reference solution;

[0017] When the to-be-tested substance is a control substance, the control substance is taken, precisely weighed, and water is added to prepare a control substance reference solution containing 20 μg of the control substance per 1 ml; the control substance includes uridine, adenosine, guanosine, thymidine, ferulic acid, caffeic acid and syringin.

[0018] The I. orientale preparation includes an I. orientale water-extracted extract, an I. orientale water-extracted dried powder, an I. orientale formula granule or an I. orientale water-extracted preparation;

[0019] And / or, the solvent is water or a methanol aqueous solution.

[0020] The characteristic peaks in the characteristic spectrum include peaks 1-9;

[0021] The relative retention times of the peaks 1, 3 and 4 are 0.91±10%, 1.14±10% and 1.45±10% respectively, according to the characteristic peak of adenosine as the peak 2;

[0022] With the characteristic peak corresponding to syringin as peak 8, the relative retention times of peaks 5-7 and peak 9 are 0.53±10%, 0.91±10%, 0.94±10%, and 1.12±10%, respectively, based on peak 8.

[0023] A construction method of a mistletoe preparation characteristic spectrum and application of the mistletoe preparation characteristic spectrum in quality control of mistletoe preparations.

[0024] Application of a mistletoe preparation characteristic spectrum in determination of mistletoe adulterants, wherein the characteristic spectrum is obtained by the construction method described above;

[0025] In the characteristic spectrum, the characteristic peak corresponding to the retention time and thymidine is peak a, the characteristic peak corresponding to the retention time and adenosine is peak 2, the characteristic peak corresponding to the retention time and caffeic acid is peak 7, the peak at a position with a relative retention time of 0.87±5% to peak 7 is peak b, the peak at a position with a relative retention time of 0.53±10% to peak 8 is peak 5, and the peak at a position with a relative retention time of 1.12±10% to peak 8 is peak 9.

[0026] The ratio of the peak area of peak b to peak 7 is K b7 , the ratio of the peak area of peak a to peak 2 is K a2 , and the ratio of the peak area of peak 5 to peak 9 is K 59 .

[0027] K b7 ≤0.20, K a2 ≤0.45, and K 59 ≥0.60 is mistletoe, and the rest is mistletoe adulterants.

[0028] K b7 >0.20 is flat branch mistletoe adulterants.

[0029] K b7 ≤0.20 and K a2 >0.45 is Ruscus aculeatus adulterants.

[0030] K b7 ≤0.20, K a2 ≤0.45, and K 59 <0.60 is black mistletoe adulterants.

[0031] The technical scheme of the present application has the following advantages:

[0032] 1. The application provides a construction method of a mistletoe preparation characteristic spectrum, the characteristic spectrum obtained by the construction method is comprehensive in material information, and fully represents key characteristic chemical components of mistletoe preparations and common adulterants of water extraction preparations of the mistletoe, the mistletoe and the preparation thereof represented by the method and different adulterants are different in chemical components, so that the overall quality control of the mistletoe and identification of adulterants of the mistletoe can be realized.

[0033] 2. In the application, the characteristic spectrum obtained by the construction method of the application is comprehensive in material information, and includes different chemical components of the mistletoe and different adulterants, so that the overall quality control of the mistletoe can be effectively applied.

[0034] 3. In the application, in the characteristic spectrum obtained by the construction method of the application, K b7 , K a2 and K 59 can effectively realize analysis and identification of the mistletoe and adulterants thereof, and effectively realize the differentiation between the mistletoe, adulterants of Phoradendron juniperinum, adulterants of Taxilli Herba, adulterants of Taxilli Herba and adulterants of Taxilli Herba. BRIEF DESCRIPTION OF DRAWINGS

[0035] In order to more clearly illustrate the specific embodiments of the application or the technical solutions in the prior art, the drawings needed in the specific embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0036] Figure 1 is the characteristic spectrum of the mistletoe in Example 1 of the application;

[0037] Figure 2 is the positioning control diagram of peak a and peak b in the characteristic spectrum of the application.

[0038] Figure 3 is the characteristic spectrum control diagram of the mistletoe and adulterants thereof in the application.

[0039] Figure 4 is the characteristic spectrum of the specificity study in Example 3 of the application.

[0040] Figure 5 is the characteristic spectrum of the delay test in Example 3 of the application.

[0041] Figure 6 is the characteristic spectrum of Comparative Example 1 of the application.

[0042] Figure 7 is the characteristic spectrum of Comparative Example 2 of the application. DETAILED DESCRIPTION

[0043] Unless otherwise indicated, the experimental procedures or conditions in the examples were carried out according to the conventional experimental procedures described in the literature or under the conditions described in the examples. Unless otherwise indicated, the reagents or instruments used were conventional reagent products that can be obtained commercially.

[0044] Instruments: ME104E electronic balance (Mettler Toledo), JY2002 electronic balance (Mettler Toledo) DZKW-4 electronic constant temperature water bath (Beijing Zhongxingweiye Instrument Co., Ltd.); KQ-300DB type numerical control ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

[0045] Reagents: Control drug material of Taxillus delavayi (batch number: 121038-201906, China Institute for Drug Control), Taxillus delavayi medicinal materials (15 batches, batch numbers: HJS01-HJS15); Taxillus sutchuensis medicinal materials (3 batches, batch numbers: BZHJS01-BZHJS03); Taxillus vishnui medicinal materials (3 batches, batch numbers: SJS01-SJS03); Taxillus nigris medicinal materials (3 batches, batch numbers: black JS01-black JS03), the batch numbers and sources are shown in Table 1 below;

[0046] Table 1

[0047]

[0048] Uridine, adenosine, guanosine, thymidine, ferulic acid, caffeic acid, and syringin control samples were purchased from the China Institute for Drug Control.

[0049] Reagents: methanol (Merck), phosphoric acid (Fisher Chemical) were chromatographically pure, methanol, ethanol were analytically pure, and water was distilled water from Aesop.

[0050] Example 1

[0051] A method for constructing a characteristic chromatogram of a Taxillus delavayi preparation, the specific process being as follows:

[0052] 1. Preparation of test sample:

[0053] Pre-treatment of medicinal materials (processing): According to the method under Taxillus delavayi decoction pieces (processing) in the 2020 edition of the People's Republic of China Pharmacopoeia, take Taxillus delavayi medicinal materials, Taxillus vishnui medicinal materials, Taxillus sutchuensis medicinal materials and Taxillus nigris medicinal materials, remove impurities, wash slightly, soak thoroughly, cut into thick slices, dry to obtain Taxillus delavayi decoction pieces, Taxillus vishnui decoction pieces, Taxillus sutchuensis decoction pieces and Taxillus nigris decoction pieces.

[0054] Standard decoction water extraction dry powder (lyophilized powder) preparation method: take 100g of mistletoe decoction pieces, put them in a sand pot, soak for 30 minutes, add 10 times the amount of water, boil with a strong fire, then simmer for 30 minutes with a weak fire, filter while hot, and reserve; add 8 times the amount of water to the second decoction, boil with a strong fire, then simmer for 20 minutes with a weak fire, filter while hot; combine the filtrates, concentrate (concentration temperature ≤ 65°C), concentrate to a ratio of about 1:1, transfer the concentrated solution to a lyophilization tray, and place it in a freeze dryer to obtain mistletoe water extraction dry powder (mistletoe lyophilized powder); take the remaining decoction pieces of the confused products to prepare the corresponding flat branch mistletoe water extraction dry powder (flat branch mistletoe lyophilized powder), mulberry mistletoe water extraction dry powder (mulberry mistletoe lyophilized powder), and black mistletoe water extraction dry powder (black mistletoe lyophilized powder) by the same method.

[0055] Formulation granule preparation method: take 4000g of mistletoe decoction pieces, extract twice, add 14 times and 12 times the amount of water respectively, each time boil for 0.5 hours, filter, combine the filtrates, reduce pressure to concentrate to a density of 1.05-1.10, spray dry, add an appropriate amount of excipients, mix well, and dry granulation to obtain mistletoe formulation granules; take the remaining decoction pieces of the confused products to prepare the corresponding flat branch mistletoe formulation granules, mulberry mistletoe formulation granules, and black mistletoe formulation granules by the same method.

[0056] 2. Test sample characteristic spectrum

[0057] 2.1, Solution preparation

[0058] Test sample solution preparation: take about 0.2g of sample (mistletoe lyophilized powder, flat branch mistletoe lyophilized powder, mulberry mistletoe lyophilized powder, black mistletoe lyophilized powder, mistletoe formulation granules, flat branch mistletoe formulation granules, mulberry mistletoe formulation granules, or black mistletoe formulation granules), accurately weigh, place in a conical flask with a plug, accurately add 10% methanol 15ml, weigh, ultrasonic treat for 30 minutes, take out, cool, re-weigh, make up the weight loss with 10% methanol, shake well, filter, and take the filtrate to obtain the test sample solution.

[0059] Reference solution preparation of control medicinal materials: take 0.5g of mistletoe control medicinal materials, place in a conical flask with a plug, add water 15ml, hot reflux for 30 minutes, cool, filter, and the filtrate is used as the reference solution of the control medicinal materials.

[0060] Reference solution of control: take an appropriate amount of uridine, adenosine, guanosine, thymidine, ferulic acid, caffeic acid, and syringin, accurately weigh, add water to prepare a solution containing 20μg of control per 1ml, which is used as the reference solution of the control.

[0061] 2.2, Chromatographic method

[0062] The following chromatographic conditions are used for detection:

[0063] Octadecylsilane bonded silica gel was used as the packing agent (column length 100 mm, column inner diameter 2.1 mm, particle size 1.8 μm, ACQUITY UPLC HSS T3); methanol (chromatographically pure) was used as the mobile phase A, and 0.10% phosphoric acid aqueous solution was used as the mobile phase B, gradient elution was carried out according to the provisions in Table 2 below; the detection wavelength was 250 nm; the column temperature was 30°C; and the flow rate was 0.15 ml / min. The theoretical plate number should not be less than 10000 according to the peak of syringin.

[0064] Table 2

[0065]

[0066] 1 ul of the test sample solution, the reference material solution of the control drug material, and the reference material solution of the control sample were respectively injected into the liquid chromatograph for determination.

[0067] The test sample solution was prepared by using the 15 batches of processed Phoradendron chinese drug materials shown in Table 1 according to the above standard decoction water extraction and drying powder (lyophilized powder) preparation method, and 3 batches of Phoradendron chinese formula granules were prepared according to the above formula granule preparation method, and the determination results are shown in Table 3 below.

[0068] Table 3

[0069]

[0070]

[0071] The characteristic chromatograms of the 15 batches of Phoradendron chinese lyophilized powder and 3 batches of Phoradendron chinese formula granules obtained in this example showed that the relative retention time difference of each characteristic peak was small, and the RSD range was 0.26% to 3.19%. At the same time, the similarity of the characteristic chromatograms of the 15 batches of Phoradendron chinese lyophilized powder and 3 batches of Phoradendron chinese formula granules was calculated, and the similarity range was 0.898 to 0.998, and the correlation was high; which met the quality control requirements.

[0072] Using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012.1 version), taking the S1 liquid chromatogram as the reference chromatogram, calculating the median, matching the full spectrum peaks, and fitting to generate the characteristic chromatogram as shown in Figure 1 .

[0073] Figure 1In the characteristic map, 9 characteristic peaks should be presented, wherein the retention time of peak 1, peak 2, peak 4, peak 6, peak 7 and peak 8 should correspond to the retention time of the reference peak of uridine, adenosine, guanosine, ferulic acid and syringa against the reference peak of caffeic acid, respectively. The peak (peak 2) corresponding to the retention time of the reference peak of adenosine is counted as S1 peak, and the relative retention time of peaks 1-4 to S1 peak is calculated, which should be within ±10% of the specified value, and the specified value is 0.91 (peak 1), 1.14 (peak 3) and 1.45 (peak 4); the peak (peak 8) corresponding to the retention time of the reference peak of syringa is counted as S2 peak, and the relative retention time of peaks 5-9 to S2 peak is calculated, which should be within ±10% of the specified value, and the specified value is 0.53 (peak 5), 0.91 (peak 6), 0.94 (peak 7) and 1.12 (peak 9), with an allowable error of ±10%.

[0074] Example 2

[0075] A method for constructing a characteristic map of an ivy preparation is used to determine the adulteration of ivy. The same method as in Example 1 is used to obtain the characteristic map of the test sample, and the peak areas of peaks 2, 5, 7 and 9 in the characteristic map are obtained, and the positions of peaks a and b and the peak areas of peaks a and b are labeled.

[0076] The positions of peaks a and b are as follows: the retention time of peak a is consistent with the retention time of the reference peak of thymidine, and the average relative retention time of peak b and peak 7 (caffeic acid) is 0.87, as shown in Figure 2 Table 3. Three batches of ivy freeze-dried powder and three batches of ivy formula granules are used to verify the position of peak b, and the verification results are shown in Table 4.

[0077] Table 4

[0078]

[0079] As shown in Table 4 above, the average relative retention time of peak b and peak 7 (caffeic acid) is 0.87, and the RSD value is 0.23%, so the position of peak b is determined as the relative retention time of peak 7 is 0.87, with an allowable error of ±5%.

[0080] Characteristic maps of ivy freeze-dried powder and formula granules, Phryta freeze-dried powder and formula granules, Morinda freeze-dried powder and formula granules, and black freeze-dried powder and formula granules are detected respectively, and the difference peaks in the spectra are analyzed and the relative peak area ratios of K a2 , K b7 and K 59 are calculated, and the results are shown in Figure 3 and Tables 5-8 below.

[0081] Table 5 Ka2 K b7 and K 59 result

[0082]

[0083] Results analysis: Mistletoe freeze-dried powder and formulation granules K a2 The range was 0.045–0.326, with a mean +3SD of 0.421, determining the K values ​​for the mistletoe freeze-dried powder and formulation granules. a2 ≤0.45; Mistletoe freeze-dried powder and formulation granules K b7 The range was 0.0–0.192, with a mean of +30% being 0.121. This determined the K values ​​for the lyophilized mistletoe powder and the formulated granules. b7 ≤0.20; Mistletoe freeze-dried powder and formulation granules K 59 The range was 0.618–2.917, with a mean -3SD of 0.701, determining the K values ​​for the mistletoe freeze-dried powder and formulation granules. 59 ≥0.60. Therefore, the above-mentioned mistletoe freeze-dried powder and formulation granules in this invention both satisfy K. b7 ≤0.20, K a2 ≤0.45 and K 59 The requirement of ≥0.60 confirms that the above 15 batches of mistletoe freeze-dried powder and 3 batches of mistletoe formulation granules are consistent with the actual results and are all mistletoe.

[0084] The difference peaks between *Viscum rotundifolia* and *Viscum spp.* are peak a, peak 9, and peak b, with peak 9 missing. The results of the relevant peak areas are shown in Table 6 below:

[0085] Table 6 Freeze-dried powder and formulation granules of *Mistletoe spp.* a2 and K b7 result

[0086]

[0087]

[0088] Results analysis: Freeze-dried powder of *Mistletoe spp.* and formulation granules K b7 The range was 0.364–0.704, with a mean of 0.484 and a mean -1.5 SD value of 0.227. This determined the K values ​​for the freeze-dried powder and formulation granules of *Viscum album*. b7 >0.25; Freeze-dried powder and formulation granules of *Mistletoe spp.* a2 The range is 3.38–5.40. As can be seen from the above, the K values ​​of the aforementioned freeze-dried powder and formulated granules of *Viscum album* are... b7 Satisfying K b7 The requirement of >0.2 proves that it is consistent with the actual result and is a counterfeit of *Viscum album*.

[0089] The difference peaks of black parasite and mistletoe are peak 5, peak 7, peak 8 and peak 9, and peak b is not detected, and the related peak area results are shown in the following table 7:

[0090] Table 7 black parasite freeze-dried powder and formula granule K a2 and K 59 Results

[0091]

[0092] Result analysis: black parasite freeze-dried powder and formula granule K 59 The actual range is 0.209-0.320, the average is 0.273, and the average+30% value is 0.355, so the black parasite freeze-dried powder and formula granule K 59 ≤0.35; the black parasite freeze-dried powder and formula granule K a2 range is 0.093-0.162, so the black parasite freeze-dried powder and formula granule K a2 ≤0.2; since peak b is not detected, K b7 is about 0. From the above, the K a2 , K 59 and K b7 of the above black parasite freeze-dried powder and formula granule meet the requirements of K b7 ≤0.20, K a2 ≤0.45 and K 59 <0.60, which proves that they are consistent with the actual results and are black parasite counterfeit products.

[0093] The difference peaks of taxillus and mistletoe are peak a, peak 5, peak 7, peak 8, peak b and peak 9 are missing, and the related peak area results are as follows:

[0094] Table 8 taxillus freeze-dried powder and formula granule K a2 Results

[0095]

[0096]

[0097] Result analysis: taxillus freeze-dried powder and formula granule K a2 The actual range is 10.522-19.273, the average is 14.88, and the average-30% value is 10.419, so the taxillus freeze-dried powder and formula granule K a2 ≥10.0, and peak b and peak 5 are missing, so K 59 , K b7 is about 0. From the above, the K a2 and K b7 of the above taxillus freeze-dried powder and formula granule meet the requirements of K b7 ≤0.20 and K a2The requirement of >0.45 proves that it is consistent with the actual result and all are Morindae Radicis adulterants.

[0098] Example 3

[0099] This example is used to verify the methodology of the chromatographic conditions in Example 1.

[0100] 1. Specificity study

[0101] Take Morindae Radicis Dispensing Granules with auxiliary material dextrin and Morindae Radicis Dispensing Granules 0.2 g respectively, accurately add 10% methanol 15 ml, and prepare according to the test sample solution preparation method in the text; take the test sample solution of the dispensing granules and the negative control solution to obtain their chromatograms according to the text method, and the results are shown in Figure 4 .

[0102] Summary: The chromatogram shows that the negative solution has no interference with the characteristic peak, and the method has good specificity.

[0103] 2. Delay test

[0104] Take the test sample solution of Morindae Radicis Dispensing Granules, inject according to the chromatographic conditions in the text method, record the 2-fold mobile phase retention time chromatogram, and the results are shown in Figure 5 .

[0105] Summary: The results of the above figure show that there is no obvious lag peak after 50 minutes, and this chromatographic method has good delay.

[0106] Example 4

[0107] A method for constructing a characteristic chromatogram of Morindae Radicis Dispensing Granules, which is different from Example 1 in the preparation of the test sample solution.

[0108] The preparation method of the test sample solution is as follows: take Morindae Radicis Dispensing Granules about 0.2 g, accurately weigh, put it in a conical flask with a plug, accurately add water and 10% methanol 15 ml respectively, tightly plug, weigh, ultrasonic treatment (power 250 W, frequency 40 kHz) for 30 minutes, take out, cool down, weigh again, make up the weight loss with the corresponding solvent, shake well, filter, take the filtrate, and get it. Inject and detect according to the method in the text, and compare the peak areas of the characteristic peaks, see Table 9.

[0109] Table 9 shows the results of the extraction solvent research

[0110]

[0111] Summary: Under the conditions of 10% methanol and water as the extraction solvent, the peak areas of the characteristic peaks have little difference. Considering that the 10% methanol solution has better stability, the extraction solvent for Morindae Radicis Dispensing Granules characteristic chromatogram is determined as 10% methanol.

[0112] Example 5

[0113] A mistletoe preparation characteristic map construction method, which is different from example 1 in that the chromatographic conditions are different, and the specific conditions are as follows:

[0114] (1) Investigation of different flow rates

[0115] Take mistletoe freeze-dried powder, appropriate amount of sample, grind finely, take 0.2g, prepare the test sample according to the preparation method of the test sample solution in example 1, and determine at flow rates of 0.12ml / min, 0.15ml / min and 0.18ml / min, respectively. The results are shown in Table 10.

[0116] Table 10 Determination results of relative retention time and relative peak area of characteristic map at different flow rates

[0117]

[0118]

[0119] Summary: The data shows that under the above different flow rate conditions, the relative retention time RSD of each characteristic peak in the characteristic map is in the range of 0.13% to 4.24%; the relative peak area RSD of each characteristic peak in the characteristic map is in the range of 0.34% to 6.93%, both of which are less than 10%; the method has good robustness to flow rate at 0.12-0.18ml / min.

[0120] (2) Investigation of different column temperatures

[0121] Take mistletoe freeze-dried powder, appropriate amount of sample, grind finely, take 0.2g, prepare the test sample according to the preparation method of the test sample solution in example 1, and determine at column temperatures of 27℃, 30℃ and 33℃, respectively. The results are shown in Table 11.

[0122] Table 11 Determination results of relative retention time and relative peak area of characteristic map at different column temperatures

[0123]

[0124] Summary: The data shows that under different column temperature conditions, the relative retention time RSD of each characteristic peak in the characteristic map is in the range of 0.13-1.03; the relative peak area RSD of each characteristic peak is in the range of 0.32% to 6.97%, both of which are less than 10%, and the method has good robustness to column temperature between 27-33℃.

[0125] (3) Investigation of different chromatographic columns and different detection wavelengths

[0126] Take the freeze-dried powder of mistletoe, take an appropriate amount of sample, grind it finely, take 0.2 g, prepare the test sample according to the test sample solution preparation method in Example 1, and determine it at detection wavelengths of 248 nm, 250 nm and 252 nm using chromatographic column 1: CORTECS UPLC T3 (2.1 x 100 mm, 1.6 μm), chromatographic column 2: ACQUITY UPLC HSS T3 (2.1 x 100 mm, 1.8 um), and chromatographic column 3: Syncronis aQ (2.1 x 100 mm, 1.7 um). The results are shown in Table 12.

[0127] Table 12 Determination results of relative retention time and relative peak area of characteristic spectrum under different chromatographic columns and detection wavelengths

[0128]

[0129] Summary: The data show that under different chromatographic columns and different detection wavelengths, the RSD of the relative retention time of each characteristic peak in the characteristic spectrum is in the range of 1.11% to 5.78%; the RSD of the relative peak area of each characteristic peak in the characteristic spectrum is in the range of 3.41% to 7.98%, both of which are less than 10%; and the method has good characteristic peak durability under the conditions of the three chromatographic columns CORTECS UPLC T3 (2.1 x 100 mm, 1.6 μm), ACQUITY UPLC HSS T3 (2.1 x 100 mm, 1.8 um) and Syncronis aQ (2.1 x 100 mm, 1.7 um) and detection wavelengths of 248 nm-252 nm.

[0130] (4) Investigation of different phosphoric acid concentrations

[0131] Take the freeze-dried powder of mistletoe, take an appropriate amount of sample, grind it finely, take 0.2 g, prepare the test sample according to the test sample solution preparation method in Example 1, and determine it at detection wavelengths of 248 nm, 250 nm and 252 nm using chromatographic column 1: CORTECS UPLC T3 (2.1 x 100 mm, 1.6 μm), chromatographic column 2: ACQUITY UPLC HSS T3 (2.1 x 100 mm, 1.8 um), and chromatographic column 3: Syncronis aQ (2.1 x 100 mm, 1.7 um). The results are shown in Table 12.

[0132] Table 13 Determination results of relative retention time and relative peak area of characteristic spectrum under different phosphoric acid concentrations

[0133]

[0134] Summary: The data show that under different chromatographic columns and different detection wavelengths, the RSD of the relative retention time of each characteristic peak in the characteristic spectrum is in the range of 1.11% to 5.78%; the RSD of the relative peak area of each characteristic peak in the characteristic spectrum is in the range of 3.41% to 7.98%, both of which are less than 10%; and the method has good characteristic peak durability under the conditions of the three chromatographic columns CORTECS UPLC T3 (2.1 x 100 mm, 1.6 μm), ACQUITY UPLC HSS T3 (2.1 x 100 mm, 1.8 um) and Syncronis aQ (2.1 x 100 mm, 1.7 um) and detection wavelengths of 248 nm-252 nm.

[0135] Comparative Example 1

[0136] A mistletoe preparation characteristic spectrum construction method, which is different from example 1 in high performance liquid chromatography, is as follows:

[0137] Waters ACQUITY UPLC○RBEH Shield RP18 chromatographic column (2.1*100mm, 1.7um) chromatographic column, with acetonitrile-0.1% phosphoric acid solution as the mobile phase; gradient elution is carried out according to the following table 14, the detection wavelength is 210nm, the column temperature is 30℃, and the flow rate is 0.3ml / min.

[0138] Table 14

[0139]

[0140] The spectrum obtained under this condition is shown in Figure 6 , except that there is one peak larger at 210nm, other chromatographic peaks are smaller, and the peak shape and separation degree are not good; and the chromatographic peak of syringin is smaller and the separation is not good.

[0141] Comparative example 2

[0142] A mistletoe preparation characteristic spectrum construction method, which is different from example 1 in high performance liquid chromatography, is as follows:

[0143] Waters ACQUITY HSS T3 (2.1*100mm, 1.8um) chromatographic column, with acetonitrile-0.1% formic acid solution as the mobile phase; gradient elution is carried out according to the following table 15. The detection wavelength is 250nm, the column temperature is 30℃, and the flow rate is 0.3ml / min.

[0144] Table 15

[0145]

[0146] The spectrum obtained under this condition is shown in Figure 7 , the peak of syringin is smaller, and the separation is basically obtained, but it is not stable and the repeatability is not good.

[0147] Obviously, the above examples are only examples for the purpose of clear illustration, and are not limited to the embodiments. For ordinary skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, it is not necessary and impossible to exhaust all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A method for constructing a characteristic map of a mistletoe preparation, characterized by, The characteristic spectrum of the test substance is obtained by using ultra-high performance liquid chromatography; The chromatographic conditions of the ultra-high performance liquid chromatography are as follows: The chromatographic column is CORTECS UPLC T3 with a size of 2.1*100 mm and a particle size of 1.6 μm, ACQUITY UPLC HSS T3 with a size of 2.1*100 mm and a particle size of 1.8 μm, or Syncronis aQ with a size of 2.1*100 mm and a particle size of 1.7 μm; the detection wavelength is 248-252 nm; methanol is used as the mobile phase A, and 0.09-0.11% phosphoric acid aqueous solution is used as the mobile phase B, and elution is carried out according to the following gradient program: The preparation process of the test substance sample solution is as follows: the test substance is accurately weighed, solvent is added, the weight is determined, ultrasonic treatment is carried out, the solution is taken out, cooled, and the weight is determined again, the lost weight is made up with the corresponding solvent, shaken uniformly, filtered, and the filtrate is taken as the test substance sample solution; The preparation process of the reference solution of the control substance is as follows: the control substance is accurately weighed, water is added to prepare the reference solution of the control substance, and the control substance includes uridine, adenosine, guanosine, thymidine, ferulic acid, caffeic acid and syringin; The mistletoe preparation is mistletoe water extraction, mistletoe water extraction and drying powder or mistletoe formula granules; The solvent is water or methanol aqueous solution.

2. The construction method of claim 1, wherein, In the chromatographic conditions of the ultra-high performance liquid chromatography, the column temperature is 27-33 ℃; And / or, the flow rate is 0.12-0.18 ml / min; And / or, the theoretical plate number calculated according to the peak of syringin should be not less than 10,000.

3. The construction method according to claim 2, characterized in that, The preparation process of the reference solution of the control medicinal material is as follows: the control medicinal material is added with water, hot reflux treatment is carried out, cooled, filtered, and the filtrate is taken as the reference solution of the control medicinal material; The preparation process of the reference solution of the control substance is as follows: the control substance is accurately weighed, water is added to prepare the reference solution of the control substance containing 20 μg of the control substance per 1 ml.

4. The construction method according to any one of claims 1 to 3, characterized in that, The characteristic peaks in the characteristic spectrum include peaks 1-9; Taking the characteristic peak corresponding to adenosine as peak 2, the relative retention times of peaks 1, 3 and 4 are 0.91±10%, 1.14±10% and 1.45±10% respectively according to peak 2; Taking the characteristic peak corresponding to syringin as peak 8, the relative retention times of peaks 5-7 and 9 are 0.53±10%, 0.91±10%, 0.94±10% and 1.12±10% respectively according to peak 8.

5. The application of the characteristic spectrum constructed by the construction method of any one of claims 1-4 in the quality control of mistletoe preparations.

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