A method for constructing a characteristic chromatogram of a laminaria (kelp) formula granular preparation, a quality detection method, and a counterfeit identification method

Abstract

The present application relates to the field of traditional Chinese medicine detection and quality detection, and particularly relates to a method for constructing a characteristic chromatogram of kelp (laminaria japonica) formula granules, a method for quality detection, and a method for identifying adulterants. The present application provides a method for constructing a characteristic chromatogram of kelp (laminaria japonica) formula granules, which comprises using ultra-high performance liquid chromatography, and the chromatographic conditions include: using octadecylsilane-bonded silica gel as the filler, using acetonitrile as the mobile phase A, and using 0.03-0.07% phosphoric acid aqueous solution as the mobile phase B for gradient elution. The above method for constructing a characteristic chromatogram can comprehensively control the quality of kelp (laminaria japonica) formula granules, the characteristic peaks and identified components in the constructed characteristic chromatogram are more, which can comprehensively reflect the characteristic peak information of the sample, and the peak type is good, the purity is high, the separation degree is good, the precision is high, and the reproducibility is good, and the method can be used for identifying kelp (laminaria japonica) formula granules and four common adulterants thereof.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine testing and quality inspection, specifically to a method for constructing a characteristic spectrum of kelp (seaweed) formula granule preparations, a quality inspection method, and a method for identifying adulterated and counterfeit products. Background Technology

[0002] Kelp is the dried thallus of *Laminaria japonica* Aresch. (family Laminariaceae) or *Ecklonia kurome* Okam. (family Eckloniaceae), both perennial algae. Kelp has a long history of medicinal use in my country and is a commonly used medicinal material. It is cold in nature and salty in taste, and enters the liver, stomach, and kidney meridians. It belongs to the category of traditional Chinese medicine for softening and dispersing nodules, and its functions include resolving phlegm, softening and dispersing nodules, and promoting diuresis and reducing swelling. It is used for goiter, scrofula, testicular swelling and pain, and edema due to phlegm retention.

[0003] Kelp has a wide range of clinical applications. For example, when combined with fritillaria, green tangerine peel, and pinellia, it can be used to treat early-stage goiter, which may be hard or swollen. When combined with green tangerine peel, aloe vera, and seaweed powder, it can be used to clear liver stagnation, relieve depression, and resolve phlegm. When combined with ginseng, angelica, and prepared rehmannia, it can be used to treat long-standing goiter with qi and blood deficiency. When combined with notopterygium, saposhnikovia, seaweed, and forsythia, it can be used to treat chills and fever, and early-stage scrofula. When combined with ginseng, angelica, and cyperus, it can be used to treat scrofula caused by liver qi stagnation and qi and blood deficiency. When combined with bupleurum, seaweed, gentian, and sparganium, it can be used to treat scrofula that is widespread on the lower jaw, hard and not ulcerated, with excessive heat and toxicity. When combined with seaweed, tangerine seed, cinnamon bark, and corydalis, it can be used to treat testicular swelling and pain caused by cold and dampness in the lower jiao, qi stagnation, and blood stasis. When combined with tangerine peel, pinellia, and poria, it can be used to treat globus hystericus. Combined with other herbs such as Areca peel, Stephania tetrandra, and Plantago seed, it can be used to treat beriberi edema. Modern clinical applications include the treatment of retinal concussion, thyroid diseases, hypertension, chronic pelvic inflammatory disease, cancer, breast hyperplasia, phlebitis, viral anicteric hepatitis, constipation, vitreous opacity, and senile cataracts. Modern pharmacodynamic studies indicate that this product possesses a variety of pharmacological effects, including influencing the cardiovascular system, lowering blood lipids, lowering blood sugar, anticoagulation, anti-radiation, antitumor, antitussive, antiasthmatic, antimutagenic, antioxidant, antiviral, antibacterial, and immune-influencing properties.

[0004] Common adulterants of kelp include the dried thallus of *Ulva actuca* L. and *Ulva pertusakjellm* (family Ulvaaceae), the dried thallus of *Undaria pinnatifida* (Harv.) Sur (family Asparagaceae), the dried thallus of *Brasenia schreberi* JFGmel. (family Arundinaceae), and the dried thallus of *Sargassum pallidum* (Turn.) C.Ag (commonly known as "large-leaf seaweed") or *Sargassum fusiforme* (Harv.) Setch. (commonly known as "small-leaf seaweed") (family Sargassumaceae).

[0005] Currently, the existing chromatographic detection methods for kelp (kelp) formulation granules have limited specified characteristic peaks and identified few components. Furthermore, the identified components are mostly nucleoside compounds, which are common to most medicinal materials. This is detrimental to the quality control of kelp (kelp) formulation granules and other preparations, and it also fails to distinguish between adulterated and counterfeit products. Compared to raw kelp (kelp), kelp (kelp) formulation granules undergo processes such as decoction and extraction, resulting in significantly different active ingredients. Therefore, the chromatographic detection methods for raw kelp (kelp) are not applicable to kelp (kelp) formulation granules, making it difficult to separate the important characteristic peaks and indicative components. Summary of the Invention

[0006] Therefore, the technical problem to be solved by the present invention is to overcome the fact that the characteristic spectrum of kelp (seaweed) preparations in the prior art has few characteristic peaks and few identified components, making it difficult to comprehensively control its quality and preventing adulteration. To this end, the present invention provides a method for constructing a characteristic spectrum of kelp (seaweed) preparations, a quality detection method, and a method for identifying adulterants. The constructed characteristic spectrum has many characteristic peaks and identified components, with high peak type and purity, and can be distinguished from four kinds of adulterants.

[0007] Therefore, the present invention provides the following technical solution:

[0008] This invention provides a method for constructing characteristic chromatograms of kelp (kelp) preparations, including detection using ultra-high performance liquid chromatography (UHPLC). The chromatographic conditions include: using octadecylsilane-bonded silica gel as the stationary phase, acetonitrile as mobile phase A, and 0.03–0.07% phosphoric acid aqueous solution as mobile phase B, performing gradient elution. The elution program is as follows: 0–10 minutes, mobile phase A 15 v / v% → 27 v / v%, mobile phase B 85 v / v% → 73 v / v%; 10–25 minutes, mobile phase A 27 v / v% → 87 v / v%. v%, mobile phase B 73 v / v% → 13 v / v%; 25–28 minutes, mobile phase A 87 v / v%, mobile phase B 13 v / v%; 28–29 minutes, mobile phase A 87 v / v% → 100 v / v%, mobile phase B 13 v / v% → 0 v / v%; 29–40 minutes, mobile phase A 100 v / v%, mobile phase B 0 v / v%; 40–45 minutes, mobile phase A 100 v / v% → 15 v / v%, mobile phase B 0 v / v% → 85 v / v.

[0009] In some embodiments, the chromatographic conditions further include at least one of the following conditions:

[0010] 1) The flow rate is 0.9–1.1 ml per minute; optionally, the flow rate is 1.0 ml per minute.

[0011] 2) Column temperature is 29–31℃; optionally, the column temperature is 30℃.

[0012] 3) The detection wavelength is 210nm to 330nm; optionally, the detection wavelength is 210nm for 0 to 13 min and 28 to 45 min, and 330nm for 13 to 28 min.

[0013] 4) The injection volume is 15 μl; optional, the injection volume is 15 μl.

[0014] 5) The column specifications are: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm; optional, the column specifications are: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

[0015] 6) Mobile phase B is a 0.05% aqueous solution of phosphoric acid.

[0016] In some embodiments, the preparation of the test solution is also included, comprising: taking the test sample, adding an extraction solvent, extracting, separating solids and liquids, drying, and dissolving in a redissolving solvent;

[0017] And / or, the test sample includes an aqueous extract of kelp (kelp), a standard decoction, or a formulation granule.

[0018] In some embodiments, the preparation of the test solution includes at least one of the following conditions:

[0019] 1) The extraction solvent is water, 30-100 v / v% methanol or its aqueous solution, or 30-100 v / v% ethanol or its aqueous solution; optionally, methanol is selected.

[0020] 2) The extraction method used in the extraction step is ultrasonic extraction or reflux extraction;

[0021] 3) The extraction time in the extraction step is 30-50 minutes;

[0022] 4) The mass-to-volume ratio of the test sample to the extraction solvent is 0.5–2 g: 20 ml;

[0023] 5) The resolvent is 50-100 v / v methanol or its aqueous solution; optionally, a 70 v / v methanol aqueous solution is selected.

[0024] 6) The mass-to-volume ratio of the test sample to the reconstitution solvent is 0.5–2 g: 2 ml.

[0025] In some embodiments, the preparation of a reference solution is also included, including:

[0026] Preparation of reference herb solution: Take the reference herb, add extraction solvent, extract, separate solid and liquid, dry, and dissolve in redissolving solvent;

[0027] And / or, preparation of reference solution: Take linoleic acid reference standard and add solvent to prepare a solution containing 0.05 to 0.15 mg per ml; optionally, a solution containing 0.1 mg per ml; optionally, the solvent is 50 to 100 v / v% methanol or its aqueous solution; optionally, a 70 v / v% methanol aqueous solution is selected.

[0028] In some embodiments, the preparation of the control herbal solution includes at least one of the following conditions:

[0029] 1) The extraction solvent is water, 30-100 v / v% methanol or its aqueous solution, or 30-100 v / v% ethanol or its aqueous solution;

[0030] 2) The extraction method used in the extraction step is ultrasonic extraction or reflux extraction;

[0031] 3) The extraction process is repeated 1 to 2 times;

[0032] 3) The extraction time in the extraction step is 30-50 minutes;

[0033] 4) The mass-to-volume ratio of the reference medicinal material to the extraction solvent is 0.5–2 g: 20–50 ml;

[0034] 5) The resolvent is 50-100 v / v methanol or its aqueous solution; optionally, a 70 v / v methanol aqueous solution is selected.

[0035] 6) The mass-to-volume ratio of the reference medicinal material to the reconstitution solvent is 0.5–2 g: 2 ml.

[0036] This invention provides a method for detecting the content of kelp (kelp) preparations, wherein the test sample solution and the reference solution are taken and detected by ultra-high performance liquid chromatography in the method for constructing the characteristic spectrum of the kelp (kelp) preparations;

[0037] The reference standard includes linoleic acid.

[0038] This invention provides a quality detection method for kelp (seaweed) preparations, including the step of comparing the characteristic spectrum of the sample to be tested with the control characteristic spectrum of the kelp (seaweed) preparation;

[0039] The feature spectrum of the sample to be tested was constructed according to the construction method described above.

[0040] The comparative characteristic chromatogram of the kelp (seaweed) preparation is selected from any one of the following (1)-(2):

[0041] (1) It has 10 common characteristic peaks. Peak 10, which corresponds to the peak of the linoleic acid reference standard, is the S peak. Calculate the relative retention times of peaks 1-9 and peak S. The relative retention times should be within ±10% of the specified values. The specified values ​​of peaks 1-9 are as follows: 0.27, 0.29, 0.34, 0.67, 0.88, 0.92, 0.94, 0.95, 0.98.

[0042] (2) Using at least one batch of kelp (seaweed) preparation standard, the characteristic spectrum obtained according to the construction method described above is used to prepare a control characteristic spectrum by the average value or median method.

[0043] In some embodiments, in (1), peak 2 corresponds to isodesoprolol; peak 3 corresponds to desoprolol; peak 6 corresponds to arachidonic acid; peak 7 corresponds to α-linolenic acid; peak 8 corresponds to γ-linolenic acid; peak 9 corresponds to arachidonic acid; and peak 10(S) corresponds to linoleic acid.

[0044] This invention provides a method for identifying kelp (seaweed) preparations and their adulterants, including testing according to the quality testing method for kelp (seaweed) preparations described above;

[0045] Compared with the characteristic chromatogram of the control kelp preparation or the characteristic chromatogram of the kelp preparation standard as the test sample, when the adulterant is wakame or its derivative, its characteristic chromatogram contains at least one chromatographic peak at 35-40 minutes and lacks chromatographic peak 1.

[0046] Compared with the characteristic chromatogram of the control kelp preparation or the characteristic chromatogram of the kelp preparation standard as the test sample, when the adulterant is small-leaved seaweed or its derivative, its characteristic chromatogram lacks chromatographic peak 8 and chromatographic peak 1.

[0047] Compared with the characteristic chromatogram of the control chromatogram of kelp preparation or the characteristic chromatogram of the kelp preparation standard as the test sample, when the adulterant is seaweed or its derivative, the peak areas of peak 2 and peak 3 in its characteristic chromatogram are greater than the peak areas of chromatographic peaks 4 to 10, and the peak area of ​​peak 4 is greater than the peak area of ​​peak 6, and chromatographic peak 1 is missing.

[0048] Compared to the characteristic chromatograms of kelp (kelp) preparations or kelp (kelp) preparation standards used as test samples, when the adulterant is water shield or its derivatives, the peak area of ​​peak 7 in its characteristic chromatogram is greater than that of peak 6, and chromatographic peak 1 is missing.

[0049] In this invention, the kelp (seaweed) preparation includes water extracts of kelp (seaweed), standard decoctions, or formulated granules.

[0050] The technical solution of this invention has the following advantages:

[0051] 1. This invention provides a method for constructing a characteristic chromatogram of a kelp (kelp) preparation, comprising detection using ultra-high performance liquid chromatography (UHPLC). The chromatographic conditions include: using octadecylsilane-bonded silica gel as the stationary phase, acetonitrile as mobile phase A, and 0.03–0.07 v / v% phosphoric acid aqueous solution as mobile phase B, performing gradient elution. The elution program is as follows: 0–10 minutes, mobile phase A 15 v / v% → 27 v / v%, mobile phase B 85 v / v% → 73 v / v%; 10–25 minutes, mobile phase A 27 v / v% → 87 v / v%, mobile phase B 73 v / v% → 13 v / v%; 25–28 minutes, mobile phase A 87 v / v%, mobile phase B ...% → mobile phase B 87 v / v% → mobile phase B 87 v / v% → mobile phase B 87 v / v% → mobile phase B 87 v / v% → mobile phase B 87 v / v% Mobile phase B was 13 v / v%. At 28–29 minutes, mobile phase A changed from 87 v / v% to 100 v / v%, and mobile phase B changed from 13 v / v% to 0 v / v%. At 29–40 minutes, mobile phase A changed from 100 v / v%, and mobile phase B changed from 0 v / v%. At 40–45 minutes, mobile phase A changed from 100 v / v% to 15 v / v%, and mobile phase B changed from 0 v / v% to 85 v / v%. The above-mentioned method for constructing characteristic spectra can comprehensively control the quality of kelp (seaweed) preparations. The constructed characteristic spectra contain numerous characteristic peaks and identifying components, comprehensively reflecting the characteristic peak information of the sample. Furthermore, the peaks exhibit good shape, high purity, good separation, high precision, and good reproducibility.

[0052] Furthermore, the above method can be used to identify kelp (seaweed) preparations and their four common adulterants, which can enhance the specific identification of kelp (seaweed) preparations.

[0053] Furthermore, the above method can also be used to determine the content of kelp preparations, including the determination of indicative components such as isodicyclopentaenoic acid, isodicyclopentaenoic acid, arachidonic acid, α-linolenic acid, γ-linolenic acid, arachidonic acid, and linoleic acid. Attached Figure Description

[0054] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0055] Figure 1 These are the characteristic chromatograms of the test sample in Example 1 of the present invention; (1) is the characteristic chromatogram of the test sample; (2) is the characteristic chromatogram of the reference sample; (3) is the characteristic chromatogram of the reference medicinal material;

[0056] Figure 2 This is a comparative characteristic spectrum of kelp (seaweed) formulation particles in Example 2 of the present invention;

[0057] Figure 3 This refers to the results of the delay test in Embodiment 3 of the present invention;

[0058] Figure 4 These are characteristic spectra under different extraction solvent conditions in Example 4 of the present invention;

[0059] Figure 5 These are feature maps under different extraction methods in Embodiment 4 of the present invention;

[0060] Figure 6 These are feature maps at different extraction times in Embodiment 4 of the present invention;

[0061] Figure 7 These are the feature maps under different extraction amounts in Example 4 of the present invention;

[0062] Figure 8 These are the characteristic spectra under different reconstitution solvents in Example 4 of the present invention;

[0063] Figure 9 This is a characteristic spectrum of the test solution in Example 5 of the present invention;

[0064] Figure 10 This is a characteristic chromatogram of the linoleic acid reference solution in Example 5 of the present invention;

[0065] Figure 11 This is a characteristic spectrum of the anion exchange solution of the excipient in Example 5 of the present invention;

[0066] Figure 12 This is the characteristic spectrum of the blank solvent in Example 5 of the present invention;

[0067] Figure 13 These are the characteristic spectra of each adulterated product in Embodiment 6 of the present invention;

[0068] Figure 14 yes Figure 13 A magnified view of a portion of the image;

[0069] Figure 15 This is a feature map of the comparative example of the present invention. Detailed Implementation

[0070] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0071] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0072] Instruments, reagents and reagents

[0073] Instruments: Shimadzu LC-2010A HT high performance liquid chromatograph; ME104E electronic balance (Mettler Toledo Scientific Instruments (Shanghai) Co., Ltd.), JY20002 electronic balance (Shanghai Sunny Hengping Instrument Co., Ltd.), BSA124S electronic balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.), XP26 electronic balance (Mettler Toledo Scientific Instruments (Shanghai) Co., Ltd.), KQ-500DB ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

[0074] Chromatographic column:

[0075] Agilent ZORBAX Eclipse XDB-C18 (4.6mm*250mm, 5μm);

[0076] Agilent ZORBAX Extend-C18 (4.6mm*250mm, 5μm);

[0077] Agilent ZORBAX Eclipse Plus C18 (4.6mm*250mm, 5μm).

[0078] Drug trials:

[0079] Kelp (kelp) formula granules (batch numbers: K402CP01, K402CP02, K402CP03, self-made by Beijing Kangrentang Pharmaceutical Co., Ltd.); The preparation method of kelp (kelp) formula granules is as follows: Take kelp (kelp), heat and reflux extract twice. For the first extraction, add 14 times the weight of water to kelp (kelp), heat and reflux extract for 1 hour, filter. For the second extraction, add 12 times the weight of water to kelp (kelp), heat and reflux extract for 1 hour, filter, combine the filtrates, concentrate the filtrate to a relative density of 1.0 g / mL (measured at 60℃), spray dry, add maltodextrin (the total weight of the dried powder after adding maltodextrin accounts for 45.4% of the kelp (kelp) feed amount) to the spray-dried powder, mix evenly, and then dry granulate to make granules.

[0080] Preparation method of standard decoction of kelp (batch number: K402BJ01~15, self-made by Beijing Kangrentang Pharmaceutical Co., Ltd.): Take 100-150g of kelp (kelp) slices and place them in a decoction pot. For the first decoction, add 16 times the amount of water as the slices and soak in cold water for 30 minutes. After boiling over high heat (500W), simmer over low heat (200W) for 30 minutes. Filter while hot through a 150-mesh filter cloth and cool quickly for later use. For the second decoction, add 14 times the amount of water as the slices and boil over high heat (500W) and simmer over low heat (200W) for 20 minutes. Filter while hot through a 150-mesh filter cloth and cool quickly for later use. Combine the filtrates and concentrate under reduced pressure (temperature set at 65℃, vacuum degree -0.1MPa) until the material-to-liquid ratio is about 1:1 (relative density of 1.05-1.10 (65℃)). Freeze dry to obtain a fine powder.

[0081] Kelp (kelp) reference medicinal material (batch number: AY300005-202305, Foshan Aoyu Biotechnology Co., Ltd.).

[0082] Linoleic acid reference standard (batch number: 111622-202105, purity 99.6%, China National Institutes for Food and Drug Control);

[0083] Reagents: Acetonitrile (Merk) and phosphoric acid (Fisher Scientific) were chromatographically pure; water was distilled water (Watson's); methanol and ethyl acetate were analytically pure.

[0084] Example 1: Method for constructing characteristic spectra of kelp (seaweed) preparations

[0085] This embodiment provides a method for constructing characteristic chromatograms of kelp (kelp) preparations, including detection using ultra-high performance liquid chromatography:

[0086] The chromatographic conditions were as follows: Octadecylsilane-bonded silica gel was used as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm; column: ZORBAX Extend-C18); acetonitrile was used as mobile phase A, and 0.05 v / v% phosphoric acid was used as mobile phase B, with gradient elution as specified in the table below; the flow rate was 1.0 mL / min; the column temperature was 30 °C; and the detection wavelengths were 210 nm (0–13 min and 28–45 min) and 330 nm (13–28 min). The theoretical plate number, calculated based on the linoleic acid peak, should be no less than 5000.

[0087] Table 1. Gradient elution program

[0088] Time (minutes) Mobile phase A (v / v%) Mobile phase B (v / v%) 0～10 15→27 85→73 10～25 27→87 73→13 25～28 87 13 28～29 87→100 13→0 29～40 100 0 40～45 100→15 0→85

[0089] Preparation of reference solution: Take about 2.0g of kelp reference material, add 50ml of water, reflux for 30 minutes, cool, filter, concentrate the filtrate to dryness under reduced pressure, add 20ml of methanol to the residue, sonicate (250W power, 40kHz frequency) for 40 minutes, remove, cool, filter, evaporate the filtrate to dryness, and dissolve in 2ml of 70% methanol solution to prepare the reference material solution. Separately, take an appropriate amount of linoleic acid reference standard, accurately weigh it, and add 70% methanol to prepare a solution containing 0.1mg per ml to prepare the linoleic acid reference standard solution.

[0090] Preparation of the test solution: Take an appropriate amount of kelp (kelp) formula granules (batch numbers K402CP01, K402CP02, K402CP03), grind them into a fine powder, weigh about 1.0g, place them in a stoppered conical flask, add 20ml of methanol, sonicate (power 250W, frequency 40kHz) for 40 minutes, cool, filter, evaporate the filtrate to dryness, and dissolve it in 2ml of 70% methanol solution to obtain the test solution.

[0091] Determination method: Accurately pipette 15 μl of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0092] The measured characteristic spectra are shown in the table below.

[0093] Table 2. Relative retention times of three batches of kelp (seaweed) formulation granules

[0094]

[0095] Characteristic chromatograms of the test sample, reference standard, and reference medicinal material, as shown below. Figure 1 As shown in the figure and the table above, the characteristic spectrum of the test sample presents 10 characteristic peaks, which correspond to the retention times of the 10 characteristic peaks in the control characteristic spectrum. Among them, peak 10 should correspond to the retention time of the linoleic acid reference peak. The peak corresponding to the linoleic acid reference peak is the S peak. The relative retention times of the other characteristic peaks and the S peak are calculated. The relative retention times should be within ±10% of the specified values, which are: 0.27 (peak 1), 0.29 (peak 2), 0.34 (peak 3), 0.67 (peak 4), 0.88 (peak 5), 0.92 (peak 6), 0.94 (peak 7), 0.95 (peak 8), and 0.98 (peak 9). Among them, peak 2 corresponds to isogepiperidine; peak 3 corresponds to digepiperidine; peak 6 corresponds to arachidonic acid; peak 7 corresponds to α-linolenic acid; peak 8 corresponds to γ-linolenic acid; peak 9 corresponds to arachidonic acid; and peak 10 (S) corresponds to linoleic acid. Using the above method, the quality of the test sample can be tested. The test sample shows 10 characteristic peaks, which is relatively numerous. There are 7 identified components, which can be used for comprehensive quality control. Moreover, the characteristic spectrum has good separation, stable baseline, good peak shape, and high purity.

[0096] Example 2: Establishment of a control characteristic chromatogram for kelp (seaweed) preparations

[0097] Fifteen batches of standard kelp (kelp) decoction and three batches of kelp (kelp) granules were taken respectively, and their characteristic spectra were obtained according to the method in Example 1. The characteristic spectra of the 15 batches of standard kelp (kelp) decoction are shown in the table below, and the characteristic spectra of the three batches of kelp (kelp) granules are shown in Table 2 in Example 1. Using the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (version 2021.1), one of the characteristic spectra was used as the reference spectra, and the median was used to calculate the reference spectra of the kelp (kelp) preparation as follows. Figure 2 As shown, common peaks are identified, with 10 common peaks identified.

[0098] Table 3. Retention Time of 15 Batches of Kelp (Seaweed) Standard Decoction

[0099]

[0100]

[0101] Table 4. Peak area and relative peak area of ​​15 batches of kelp (seaweed) standard decoction

[0102]

[0103]

[0104] Table 5. Relative retention times of the characteristic chromatograms of kelp (seaweed) formulation granules

[0105]

[0106] Table 6. Relative peak areas of the characteristic chromatograms of kelp (seaweed) formulation particles.

[0107]

[0108]

[0109] The characteristic peaks in the established chromatogram of kelp (seaweed) formula granules were identified. Using reference standards, peak 2 was identified as isopropenolone; peak 3 as isopropenolone; peak 6 as arachidonic acid; peak 7 as α-linolenic acid; peak 8 as γ-linolenic acid; peak 9 as arachidonic acid; and peak 10 as linoleic acid. Peak 10 (linoleic acid) was determined as the S peak.

[0110] Finally, it was determined that the chromatogram of the test sample should show 10 characteristic peaks, among which peak 10 should correspond to the retention time of the linoleic acid reference peak. The peak corresponding to the linoleic acid reference peak is designated as peak S. The relative retention times of peaks 1, 2, 3, 4, 5, 6, 7, 8, and 9 with peak S were calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.27 (peak 1), 0.29 (peak 2), 0.34 (peak 3), 0.67 (peak 4), 0.88 (peak 5), 0.92 (peak 6), 0.94 (peak 7), 0.95 (peak 8), and 0.98 (peak 9). Among them, peak 2 corresponds to isoglucosinolate; peak 3 corresponds to digoxin; peak 6 corresponds to arachidonic acid; peak 7 corresponds to α-linolenic acid; peak 8 corresponds to γ-linolenic acid; peak 9 corresponds to arachidonic acid; and peak 10 (S) corresponds to linoleic acid.

[0111] Example 3 Delay Test

[0112] Kelp (kelp) granules (batch number K402CP01) were prepared according to the method for preparing the test solution in Example 1. Then, the chromatographic conditions in Example 1 were followed for detection, and the elution time of twice the mobile phase was recorded. The results are as follows: Figure 3 As shown.

[0113] The results showed that no obvious hysteresis peak appeared after 40 minutes, indicating that the chromatographic method met the analytical requirements.

[0114] Example 4 System Suitability Test

[0115] Kelp (kelp) granules (batch number K402CP01) were prepared according to the method for preparing the test solution in Example 1. Then, a system suitability test was conducted under the chromatographic conditions in Example 1, using the linoleic acid peak as a reference. The results are shown in the table below, indicating that the theoretical plate number, tailing factor, and retention time of the linoleic acid peak all meet the analytical requirements.

[0116] Table 7. Results of System Suitability Test

[0117]

[0118]

[0119] Example 5: Investigation of the preparation of the test solution

[0120] 1. Investigation of extraction solvents

[0121] Nine portions of kelp (kelp) granules (batch number: K402CP01), each 1.0 g, were weighed, ground finely, and accurately weighed. Each portion was placed in a stoppered conical flask, and water, 30% methanol, 50% methanol, 70% methanol, methanol, 30% ethanol, 50% ethanol, 70% ethanol, and 20 ml of ethanol were accurately added respectively. The mixture was weighed, and the mixture was sonicated (250 W, 40 kHz) for 40 minutes. After cooling, the corresponding solvents were added to make up the weight. The mixture was filtered, evaporated to dryness, and dissolved in 2 ml of the corresponding solvent. 15 μl of the filtrate was accurately injected into a high-performance liquid chromatograph (HPLC) and analyzed under the chromatographic conditions described in Example 1. The peak areas were measured, and the results are shown in the table below. Figure 4 Chromatographic and data analysis showed that when ethanol was used as the extraction solvent, it was difficult to extract the substances with the intended characteristic peaks. The extraction efficiency of 70% methanol was basically the same as that of methanol. However, during sample preparation, 70% methanol would extract more sugars, making reconstitution difficult. Considering all factors, in order to ensure sufficient extraction and facilitate the preservation of organic solvents, methanol was selected as the optimal extraction solvent for preparing the kelp (kelp) formulation granule characteristic chromatogram test sample.

[0122] Table 8. Analysis of Extraction Solvents

[0123] Extraction solvent Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 water 88377 379303 415718 31264 267992 185131 74741 28525 109218 38338 30% methanol 130971 347168 384818 56910 231334 126115 51219 39483 57588 21887 50% methanol 172329 377798 410489 71138 604503 268308 101759 88886 69650 29280 70% methanol 185201 430447 470335 85173 1402586 1092852 408035 357485 508198 211611 methanol 369853 354603 369824 61984 1481711 1147561 434799 376496 550329 229555 30% ethanol 41836 374057 403183 67989 409698 237505 93131 77205 112516 41336 50% ethanol 65950 303631 347516 63562 1003248 785694 294176 256590 373303 154939 70% ethanol 48046 344587 361547 59633 1000630 814864 300038 267222 391182 158225 ethanol 8234 60623 53584 34194 712237 628242 234182 193395 301232 124825

[0124] 2. Examination of extraction methods

[0125] Two portions of 1.0g each of kelp (kelp) granules (batch number: K402CP01) were weighed, ground finely, and accurately weighed. Each portion was placed in a stoppered conical flask, and 20ml of methanol was accurately added. The flasks were then subjected to ultrasonic treatment (250W, 40kHz) for 40 minutes and reflux for 40 minutes, respectively. After cooling, the weight was adjusted with methanol, filtered, and evaporated to dryness. The residue was dissolved in 2ml of methanol to obtain the final product. 15μl of the filtrate was accurately injected into a high-performance liquid chromatograph (HPLC) and analyzed under the chromatographic conditions described in Example 1. The peak areas were measured, and the results are shown in the table below. Figure 5 Chromatographic and data analysis showed no significant difference between reflux extraction and ultrasonic extraction, although ultrasonic extraction had a higher peak area. Considering all factors, ultrasonic extraction was selected as the optimal extraction method for preparing the kelp (seaweed) formulation granule sample.

[0126] Table 9. Examination of Extraction Methods

[0127] Extraction method Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 ultrasound 369853 354603 369824 61984 1481711 1147561 434799 376496 550329 229555 reflux 392721 296866 310853 59575 1162598 927555 357750 311379 476600 225988

[0128] 3. Examination of extraction time

[0129] Weigh out three portions of kelp (kelp) granules (batch number: K402CP01), approximately 1.0g each, grind them finely, accurately weigh them, and place them in stoppered conical flasks. Accurately add 20ml of methanol to each flask, weigh them, and sonicate them (250W power, 40kHz frequency) for 30, 40, and 50 minutes respectively. Cool them, make up the weight with methanol, filter, evaporate to dryness, and dissolve in 2ml of methanol to obtain the final product. Accurately pipette 15μl of the filtrate and inject it into a high-performance liquid chromatograph. Determine the peak area according to the chromatographic conditions in Example 1. The results are shown in the table below. Figure 6 The results showed that the peak areas of each characteristic peak were basically the same in the characteristic chromatograms obtained from different extraction times. In order to ensure that the peak areas of each chromatographic peak were of appropriate size, the optimal extraction time was determined to be 40 minutes.

[0130] Table 10. Extraction Time Assessment Table

[0131] Extraction time Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 30min 247897 214197 218573 40860 1016914 793508 298610 258001 398547 156355 40min 369853 354603 369824 61984 1481711 1147561 434799 376496 550329 229555 50min 298122 254104 263015 46260 1081251 865212 318527 273269 421612 168243

[0132] 4. Examination of extraction dosage

[0133] Three portions of kelp (kelp) granules (batch number: K402CP01), approximately 0.5, 1.0, and 2.0 g respectively, were weighed, ground finely, and accurately weighed. Each portion was placed in a stoppered conical flask, and 20 ml of methanol was accurately added. The flasks were then sonicated (250 W, 40 kHz) for 40 minutes, cooled, and the weight was adjusted with methanol. The flasks were filtered, evaporated to dryness, and dissolved in 2 ml of methanol. 15 μl of the filtrate was accurately injected into a high-performance liquid chromatograph (HPLC) and analyzed under the chromatographic conditions described in Example 1. The peak areas were measured, and the results are shown in the table below. Figure 7 The results showed that the total peak area of ​​the test samples with sampling amounts of 0.5g, 1g, and 2g was not significantly different after conversion of the sampling amount. This indicates that the extraction efficiency of the proposed characteristic peaks in the test samples with sampling amounts of 0.5g, 1g, and 2g was basically the same. This shows that the extraction parameters determined above can stably extract the characteristic peak substances of 1g. Taking all factors into consideration, 1g was determined to be the optimal sampling amount for the characteristic spectrum of kelp (seaweed) formula granules.

[0134] Table 11. Extraction Dosage Assessment Table

[0135]

[0136]

[0137] 5. Investigation of the reconstitution solvent

[0138] Weigh out three portions of kelp (kelp) granules (batch number: K402CP01), each approximately 1.0g, grind them finely, accurately weigh them, place them in stoppered conical flasks, accurately add 20ml of methanol, weigh them, sonicate (power 250W, frequency 40kHz) for 40 minutes, cool, make up the weight with methanol, filter and evaporate to dryness, dissolve in 2ml of methanol, 70% methanol, and 50% methanol respectively to obtain the final product. Accurately pipette 15μl of the filtrate and inject it into a high-performance liquid chromatograph. Measure the peak area according to the chromatographic conditions in Example 1. The results are shown in the table below. Figure 8 During the experiment, it was found that crystallization occurred in the methanol-redissolved samples. Redissolving with 50% methanol affected the peak area of ​​the characteristic peaks. Taking all factors into consideration, 70% methanol was determined to be the optimal redissolving solvent for the characteristic spectrum of kelp (seaweed) formulation particles.

[0139] Table 12. Evaluation of Reconstitution Solvents

[0140] Reconstituted solvent Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Peak 8 Peak 9 Peak 10 methanol 369853 354603 369824 61984 1481711 1147561 434799 376496 550329 229555 70% methanol 380065 374555 410694 91333 1400743 1068036 413598 357904 478894 214283 50% methanol 405308 409026 428405 84290 687955 341327 131709 114635 110369 44810

[0141] Example 5 Methodological Validation

[0142] 1. Exclusivity

[0143] Linoleic acid reference standard (prepared according to the linoleic acid reference standard solution in Example 1), test solution (kelp (seaweed) formulation granules (batch number: K402CP01, prepared according to the test solution in Example 1), blank solvent (70 v / v % methanol), and excipient negative sample were taken and determined according to the chromatographic conditions in Example 1. The results are as follows: Figures 9-12 As shown, this illustrates that the method of the present invention has strong specificity.

[0144] Preparation of excipient anion solution: Take an appropriate amount of maltodextrin, grind it into a fine powder, weigh about 1.0g, place it in a stoppered conical flask, add 20ml of methanol, sonicate (power 250W, frequency 40kHz) for 40 minutes, cool, filter, evaporate the filtrate to dryness, and dissolve it in 2ml of 70% methanol solution to obtain the solution.

[0145] 2. Precision

[0146] Six portions of kelp (kelp) granules (batch number: K402CP01) were weighed and finely ground. The characteristic chromatograms were obtained under the chromatographic conditions described in Example 1. Peak 10 was used as the reference peak, and the relative peak area and relative retention time were calculated. The RSD was also calculated. The results are shown in the table below. The RSD of the relative peak area for each characteristic peak ranged from 0.77% to 3.91%, with slightly large differences in relative peak area. Considering the large error in the reconstitution process during sample preparation, which may lead to significant differences in the area of ​​each characteristic peak, no specific requirement was placed on the relative peak area. The RSD of the relative retention time for each characteristic peak ranged from 0% to 0.11%, with small differences in relative retention time, indicating good repeatability of the characteristic chromatogram.

[0147] Table 13. Retention Time and Relative Retention Time for Repeatability Study of Kelp (Seaweed) Formulation Granules

[0148]

[0149] Table 14. Peak area and relative peak area of ​​kelp (seaweed) in repeatability studies

[0150]

[0151] 2. Intermediate precision

[0152] Six portions of kelp (kelp) formulation granules (batch number: K402CP01) were analyzed using a high-performance liquid chromatography (HPLC) instrument and a PDA detector, according to the chromatographic conditions described in Example 1. Characteristic chromatograms were obtained, with peak 10 as the reference peak. The relative peak area and relative retention time were calculated, and the RSD was also calculated. The results are shown in the table below. In the characteristic chromatograms obtained using HPLC (PDA detector), the RSD of the relative retention time for each characteristic peak was 0.02–0.05%, and the RSD of the relative peak area was in the range of 1.20–2.03%. The RSD of the relative retention time between different instruments (two HPLC instruments (PDA detectors)) was 0–0.20%, while the relative peak area varied slightly between instruments, with an RSD of 0.95–18.20%, which may be related to different detectors. Therefore, no specific requirements were set for the relative peak area. The experimental results show that the relative retention time of this characteristic chromatogram method meets the analytical requirements between different instruments.

[0153] Table 15. Retention Time and Relative Retention Time for Intermediate Precision Study of Kelp (Seaweed)

[0154]

[0155] Table 16. Peak area and relative peak area of ​​kelp (seaweed) intermediate precision test

[0156]

[0157]

[0158] 3. Stability

[0159] Kelp (kelp) formulation granules (batch number: K402CP01) were finely ground and used to prepare the test solution according to Example 1. The solution was analyzed at 0, 2, 4, 8, 12, and 24 hours under the chromatographic conditions of Example 1 to obtain characteristic chromatograms. Peak 10 was used as the reference peak, and the relative peak area and relative retention time were calculated. The RSD was also calculated. The results are shown in the table below. The stability test results show that the RSD of the relative peak area is in the range of 0.34%–1.25%, and the RSD of the relative retention time of each characteristic peak is in the range of 0%–0.32%, indicating that the relative retention time of this characteristic chromatographic method meets the analytical requirements during stability testing.

[0160] Table 17. Retention Time and Relative Retention Schedule for Stability Study of Kelp (Seaweed)

[0161]

[0162] Table 18. Peak area and relative peak area of ​​kelp (seaweed) stability test

[0163]

[0164]

[0165] 4. Durability

[0166] 4.1 Investigation at different column temperatures:

[0167] A test solution was prepared using kelp (kelp) granules (batch number: K402CP01) according to Example 1. The solution was then analyzed at different column temperatures (29℃, 30℃, and 31℃) under the chromatographic conditions of Example 1 to examine the robustness of the experimental method to column temperature. The results are shown in the table below. The relative retention times (RSDs) of the characteristic peaks obtained at different column temperatures ranged from 0.03% to 0.66%, and the RSDs of the relative peak areas ranged from 0.56% to 2.88%. The results indicate that column temperature has a relatively small impact on the relative peak area and relative retention time. This invention does not specify a relative peak area; therefore, this method exhibits good column temperature robustness.

[0168] Table 19. Retention times and relative retention times of kelp (seaweed) at different column temperatures.

[0169]

[0170] Table 20. Peak area and relative peak area of ​​kelp (seaweed) at different column temperatures

[0171]

[0172] 4.2 Investigation of different flow velocities:

[0173] Kelp (kelp) formulation granules (batch number: K402CP01) were used to prepare the test solution according to Example 1. Chromatographic measurements were performed at different flow rates (0.9 ml / min, 1.0 ml / min, and 1.1 ml / min) under the chromatographic conditions described in Example 1 to investigate the robustness of the experimental method to different flow rates. The results are shown in the table below. The results show that the relative retention times (RSDs) of the characteristic peaks in the chromatograms obtained at different flow rates were in the range of 0.10–5.38% (meeting the specified value ±10%), and the RSDs of the relative peak areas were in the range of 0.04–1.76%. This method exhibits good robustness to column temperature.

[0174] Table 21. Retention Time and Relative Retention Time of Kelp (Seaweed) under Different Flow Velocities

[0175]

[0176] Table 22. Peak area and relative peak area of ​​kelp (kelp) under different flow velocities.

[0177]

[0178] 4.3 Investigation of different chromatographic columns:

[0179] Kelp (kelp) formulation granules (batch number: K401CP01) were used to prepare test solutions according to Example 1. Different types of chromatographic columns (Column 1: Agilent ZORBAX Eclipse XDB-C18 (4.6mm*250mm, 5μm), Column 2: Agilent ZORBAX Extend-C18 (4.6mm*250mm, 5μm), Column 3: Agilent ZORBAX Eclipse PlusC18 (4.6mm*250mm, 5μm)) were used to determine the chromatographic properties under the conditions of Example 1, examining the robustness of the experimental method to different types of chromatographic columns. The results are shown in the table below. The results show that the relative retention times (RSDs) of the characteristic peaks in the chromatograms obtained under different columns ranged from 0.18% to 7.88%, while the relative peak areas had relatively large RSDs, ranging from 0.27% to 3.64%. This indicates that the method has good robustness to different chromatographic columns.

[0180] Table 23. Retention times and relative retention times of kelp (kelp) on different chromatographic columns.

[0181]

[0182] Table 24. Peak areas and relative peak areas of kelp (seaweed) on different chromatographic columns.

[0183]

[0184] Example 6: Identification Method of Kelp (Seaweed) Preparations and Adulterants

[0185] This embodiment provides a method for identifying kelp (seaweed) preparations from adulterants. The sample to be tested is:

[0186] Large-leaved seaweed (dried thallus of Sargassum pallidum (Turn.) C.Ag, a plant of the Sargassum family): Take an appropriate amount of large-leaved seaweed, grind it into a fine powder, weigh about 1.0 g, place it in a stoppered conical flask, add 20 ml of methanol, sonicate (power 250 W, frequency 40 kHz) for 40 minutes, cool, filter, evaporate the filtrate to dryness, and dissolve it in 2 ml of 70 v / v% methanol solution to obtain the sample.

[0187] Small-leaved seaweed (dried thallus of Sargassum fusiforme (Harv.) Setch.): Take an appropriate amount of small-leaved seaweed, grind it into a fine powder, weigh about 1.0 g, place it in a stoppered conical flask, add 20 ml of methanol, sonicate (power 250 W, frequency 40 kHz) for 40 minutes, cool, filter, evaporate the filtrate to dryness, and dissolve it in 2 ml of 70 v / v% methanol solution to obtain the sample.

[0188] Sample of wakame (dried thallus of Undaria pinnatifida (Harv.) Sur, a plant of the family Algaceae): Take an appropriate amount of wakame, grind it into a fine powder, weigh about 1.0 g, place it in a stoppered conical flask, add 20 ml of methanol, sonicate (power 250 W, frequency 40 kHz) for 40 minutes, cool, filter, evaporate the filtrate to dryness, and dissolve it in 2 ml of 70 v / v% methanol solution to obtain the sample.

[0189] Dried thallus of Brasenia schreberi JFGmel. (family Brasenaceae): Take an appropriate amount of Brasenia schreberi, grind it into a fine powder, weigh about 1.0 g, place it in a stoppered conical flask, add 20 ml of methanol, sonicate (power 250 W, frequency 40 kHz) for 40 minutes, cool, filter, evaporate the filtrate to dryness, and dissolve it in 2 ml of 70 v / v% methanol solution to obtain the sample.

[0190] The above-mentioned samples (each sample consists of 3 batches) were used to construct feature maps according to the method in Example 1. The feature map of one batch of the exemplary samples is shown below. Figure 13 The comparison results of the characteristic spectra with those of kelp (seaweed) formula granules are as follows: Figure 14 As shown. We can see:

[0191] Compared with the control characteristic chromatogram of kelp (kelp) preparation, when the adulterant is wakame or its derivative, its characteristic chromatogram contains at least one chromatographic peak at 35-40 minutes and lacks chromatographic peak 1; the comparison results of the remaining batches of samples are consistent with the above.

[0192] Compared with the control characteristic chromatogram of kelp (seaweed) preparations, when the adulterant is small-leaved seaweed or its derivative, its characteristic chromatogram lacks chromatographic peak 8 and chromatographic peak 1; the comparison results of the remaining batches of samples are consistent with the above.

[0193] Compared with the control characteristic chromatogram of kelp (seaweed) preparations, when the adulterant is seaweed or its derivative, the peak areas of peak 2 and peak 3 in its characteristic chromatogram are greater than the peak areas of chromatographic peaks 4 to 10, and the peak area of ​​peak 4 is greater than the peak area of ​​peak 6, and chromatographic peak 1 is missing; the comparison results of the remaining batches of samples are consistent with the above.

[0194] Compared to the control chromatogram of kelp (kelp) preparations, when the adulterant is water shield or its derivative, the peak area of ​​peak 7 in its characteristic chromatogram is greater than that of peak 6, and chromatographic peak 1 is missing. The comparison results of the remaining batches of samples are consistent with the above.

[0195] Therefore, the present invention can distinguish kelp (seaweed) preparations from adulterated products.

[0196] Comparative Example

[0197] The kelp (kelp) formula granules (batch number: K401CP01) were tested according to the test sample preparation method and chromatographic conditions in "Study on UPLC Fingerprint and Chemical Pattern Recognition of Kelp" (Huang Huahua, Zhang Yiping, Lü Shishi, Journal of Chinese Medicinal Materials, Vol. 43, No. 7, July 2020). The results are as follows. Figure 15 As shown, the characteristic peaks are basically lost.

[0198] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing a characteristic map of a laminaria preparation, characterized by, The detection includes high performance liquid chromatography, and the chromatographic conditions include: The chromatographic column is filled with octadecylsilane-bonded silica gel, has a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm, acetonitrile is used as a mobile phase A, 0.03-0.07 v / v % of a phosphoric acid aqueous solution is used as a mobile phase B, and gradient elution is performed at a detection wavelength of 210 nm at 0-13 min and 28-45 min, 330 nm at 13-28 min, and an elution procedure is as follows: The preparation of the test sample solution further includes: taking the test sample, adding an extraction solvent, extracting, solid-liquid separation, drying, and dissolving by adding a redissolving solvent; the extraction solvent is 70-100 v / v % of methanol; The test sample is a standard Laminaria japonica decoction or a Laminaria japonica formula granule; The characteristic spectrum has 10 common characteristic peaks, peak 10 corresponding to the peak of the linoleic acid reference substance is an S peak, the relative retention times of peaks 1-9 and the S peak should be within ±10 % of the specified values, and the specified values of peaks 1-9 are 0.27, 0.29, 0.34, 0.67, 0.88, 0.92, 0.94, 0.95, and 0.98, respectively; Peak 2 corresponds to isogeijipinolide, peak 3 corresponds to geijipinolide, peak 6 corresponds to arachidonate, peak 7 corresponds to α-linolenic acid, peak 8 corresponds to γ-linolenic acid, peak 9 corresponds to arachidonic acid, and peak 10 corresponds to linoleic acid.

2. The method of constructing a characteristic pattern of a kelp preparation according to claim 1, characterized by, The chromatographic conditions further include at least one of the following conditions: 1) the flow rate is 0.9-1.1 ml / min; 2) the column temperature is 29-31 ℃; 3) the injection volume is 15 μl; 4) the mobile phase B is 0.05 % of a phosphoric acid aqueous solution.

3. The method of claim 2, wherein the method is characterized by, The flow rate is 1.0 ml / min; Or The column temperature is 30 ℃.

4. The method of constructing a characteristic pattern of a kelp preparation according to claim 3, characterized by, The preparation of the test sample solution includes at least one of the following conditions: 1) the extraction method used in the extraction step is ultrasonic extraction or reflux extraction; 2) the extraction time in the extraction step is 30-50 min; 3) the mass-volume ratio of the test sample to the extraction solvent is 0.5-2 g:20 ml; 4) the redissolving solvent is 50-100 v / v % of methanol; 5) the mass-volume ratio of the test sample to the redissolving solvent is 0.5-2 g:2 ml.

5. The characteristic spectrum construction method of the Laminaria japonica preparation according to claim 4, characterized in that: The redissolving solvent is 70 v / v % of methanol.

6. The method of constructing a characteristic pattern of a kelp preparation according to any one of claims 1 to 5, characterized in that, The preparation of the reference solution further includes: The preparation of the control medicinal material solution: taking the control medicinal material, adding an extraction solvent, extracting, solid-liquid separation, drying, and dissolving by adding a redissolving solvent; The preparation of the reference substance solution: taking the linoleic acid reference substance, adding a solvent to prepare a 1-ml solution containing 0.05-0.15 mg.

7. The characteristic spectrum construction method of the Laminaria japonica preparation according to claim 5, characterized in that: The reference substance solution is a 1-ml solution containing 0.1 mg of linoleic acid; Or In the reference substance solution, the solvent is 50-100 v / v % of methanol.

8. The characteristic spectrum construction method of the Laminaria japonica preparation according to claim 7, characterized in that: In the reference substance solution, the solvent is 70 v / v % of a methanol aqueous solution.

9. The method of claim 6, wherein the method is characterized by, The preparation of the control medicinal material solution comprises at least one of the following conditions: 1) the extraction solvent is 70-100 v / v% methanol; 2) the extraction method used in the extraction step is ultrasonic extraction or reflux extraction; 3) the number of extraction times in the extraction step is 1-2 times; 4) the extraction time in the extraction step is 30-50 min; 5) the mass-volume ratio of the control medicinal material and the extraction solvent is 0.5-2 g:20-50 ml; 6) the redissolving solvent is 50-100 v / v% methanol; 7) the mass-volume ratio of the control medicinal material and the redissolving solvent is 0.5-2 g:2 ml.

10. The method for constructing a characteristic map of a laminaria preparation according to claim 9, characterized in that, the redissolving solvent is 70 v / v% methanol.

11. A method for quality control of a laminaria preparation, characterized by, comparing the characteristic map of the sample to be tested with the control characteristic map of the laminaria preparation; the characteristic map of the sample to be tested is constructed according to the method for constructing a characteristic map of a laminaria preparation according to any one of claims 1-10; the laminaria preparation is a laminaria standard decoction or a laminaria formula granule; the control characteristic map of the laminaria preparation is obtained by averaging or mediating the characteristic maps of at least one batch of laminaria preparation standard products obtained according to the method for constructing a characteristic map of a laminaria preparation according to any one of claims 1-10.

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