Thin layer identification, content determination and characteristic spectrum determination method of coix seed

Abstract

The invention discloses a thin layer identification, content determination and characteristic spectrum determination method of coix seed. The present invention uses ethyl acetate to replace ether, which can effectively solve the harm to the liver, kidney and nervous system of the test personnel in the existing identification process of coix seed; at the same time, the spots of the thin layer chromatogram obtained by using 10±1vol% sulfuric acid ethanol solution as a color developer can be intuitive and clear under ultraviolet light, ensuring the accuracy of analysis and discrimination. The present invention can simultaneously detect 1,2-linoleic acid-3-oleic acid glyceride and triolein, and 7 main characteristic peaks are presented in the test sample spectrum, the separation between the peaks is high, the peak shape is good, and the overall appearance of the multi-components of coix seed can be reflected, and the result is reliable.

Description

Technical Field

[0001] The invention relates to the technical field of coix seed detection, and in particular to a method for thin layer identification, content determination and characteristic spectrum determination of coix seed. Background Art

[0002] Coix seeds are the dried mature kernels of Coix lacryma-jobi L. var. mayuen (Roman.) Stapf, a plant of the Gramineae family. The plants are harvested in autumn when the fruits are ripe, dried in the sun, the fruits are beaten, dried again, the shells, yellow-brown seed coats and impurities are removed, and the kernels are collected. Coix seeds are one of the commonly used Chinese medicines with high medicinal value. They have the effects of promoting diuresis and dispelling dampness, strengthening the spleen and stopping diarrhea, removing numbness, discharging pus, and detoxifying and dispersing knots. They are mainly used for edema, beriberi, dysuria, spleen deficiency diarrhea, dampness numbness and cramps, lung abscess, intestinal abscess, warts, and cancer.

[0003] Chinese Pharmacopoeia Committee. Pharmacopoeia of the People's Republic of China: 2020 Edition Volume 1 [S]. Beijing: China Medical Science and Technology Press, 2020. It contains coix seed and makes relevant provisions for the thin layer chromatography identification, liquid phase identification, content determination and other detection methods of this variety. In the thin layer chromatography identification method of coix seed in this standard, the developing solvent system used is petroleum ether (60-90℃)-ether-glacial acetic acid, the color developer is 5% vanillin sulfuric acid solution, and the inspection method is white light. Ether is a controlled and easily made drug reagent, which is not easy to obtain, and has hepatotoxicity, nephrotoxicity, and neurotoxicity, which is harmful to the liver, kidneys, and nervous system of the inspectors; the thin layer chromatogram obtained with 5% vanillin sulfuric acid solution as the color developer has some spots that are not intuitive and clear enough under white light, affecting analysis and discrimination. In the content determination method of coix seed in this standard, the mobile phase used is acetonitrile-dichloromethane (65:35). Before using dichloromethane, the water phase in the HPLC pump and pipeline needs to be emptied, which increases the complexity of the experiment; the use of dichloromethane will increase the wear of HPLC pump components, and the halogen elements in dichloromethane will corrode the metal tube wall and chromatographic column of the HPLC; this standard has few detection indicators, only introduces triolein as a detection indicator, and does not introduce other active ingredients as detection indicators, which fails to more fully reflect the true quality of the test product. The coix seed liquid phase identification method in this standard is roughly the same as the content determination method, and no relative retention time is specified for each characteristic peak, which is not conducive to accurate analysis by test personnel.

[0004] Therefore, the prior art has defects and needs to be improved. Summary of the invention

[0005] The technical problem to be solved by the present invention is: to provide a thin layer identification, content determination and characteristic spectrum determination method of coix seed, reduce the damage to the operator's body during the detection process, can be clearly displayed on the chromatogram, and ensure the accuracy of the judgment; it is convenient to effectively and reliably detect the true quality of coix seed, and facilitate accurate analysis by detection personnel.

[0006] The technical scheme of the present invention is as follows: A thin layer identification method for coix seed is provided, comprising the following steps. S1: Prepare a first test solution; take an appropriate amount of test sample powder, add an appropriate amount of petroleum ether, ultrasonically treat, filter, and take the filtrate, which is the test solution. The ultrasonic treatment time is 10-60 minutes. Prepare a control solution; take an appropriate amount of coix seed medicinal material powder, add an appropriate amount of petroleum ether, ultrasonically treat, filter, and take the filtrate, which is the control medicinal material solution; take coix seed oil control extract, add petroleum ether, and prepare a solution of coix seed oil control extract as the first control extract solution. The ultrasonic treatment time is 10-60 minutes. The boiling point range of the petroleum ether is 60 to 90°C. S2: Take the same volume of the first test solution, the control medicinal material solution, and the first control extract solution, and spot them on the same silica gel G thin layer plate, use petroleum ether-ethyl acetate-glacial acetic acid as the developing agent, develop, take out, dry, spray with 10±1vol% sulfuric acid ethanol solution, heat at 105±2℃ until the spots are clearly colored, and check under ultraviolet light; the volume ratio of petroleum ether-ethyl acetate-glacial acetic acid is: 8.5±0.85:0.8±0.08:0.1±0.01. S3: Result judgment: whether the chromatogram of the first test sample shows spots of the same color at the corresponding positions of the chromatograms of coix seed medicinal material and coix seed oil control extract; if spots of the same color are shown, the test sample is coix seed, if spots of the same color are not shown, the test sample is not coix seed. Preferably, the first control extract solution is a solution containing 2±0.2 mg of coix seed oil control extract per 1 ml; the wavelength of the ultraviolet light emitted by the ultraviolet lamp is 365 nm; the volume ratio of petroleum ether-ethyl acetate-glacial acetic acid is: 8.5:0.8:0.1. The mass of the test sample powder is the same as the mass of the coix seed control medicinal material powder, and the volume of petroleum ether added to the two is the same.

[0007] The present invention also provides a method for determining the content of coix seed, and the content of the aforementioned test sample powder is detected, comprising the following steps. M1: Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel is used as a filler, acetonitrile is used as a mobile phase A, and isopropanol is used as a mobile phase B, and gradient elution is performed; flow rate: 1.0 ml / min; detector: ELSD; column temperature: 30°C; the theoretical plate number should be not less than 5000 calculated according to the triolein peak. M2: Preparation of reference solution: Take appropriate amounts of triolein reference substance and 1,2-linoleic acid-3-olein reference substance respectively, weigh them, place them in a volumetric flask, add a mixed solvent with a volume ratio of acetonitrile: isopropanol = 55-65:45-35, and prepare a first reference solution containing 0.3±0.03 mg of triolein and 0.4±0.04 mg of 1,2-linoleic acid-3-olein per 1 ml. M3: Preparation of the second test solution: Take the test powder, weigh it, put it in a stoppered conical flask, add a mixed solvent with a volume ratio of acetonitrile: isopropanol = 55-65:45-35, plug it, weigh it, soak it for 30min-6h, ultrasonically treat it for 10-60min, let it cool, make up the lost weight, shake it well, filter it, take the filtrate, and get the second test solution. The test powder is passed through a No. 3 sieve; the power of the ultrasonic treatment is 250W and the frequency is 50kHz. M4: Absorb the reference solution, absorb the second test solution, inject it into the liquid chromatograph, measure it, and calculate it with the external standard two-point method logarithmic equation to obtain the content of triolein and 1,2-linoleic acid-3-oleic acid glyceride components in the test powder. Preferably, the volume ratio of acetonitrile: isopropanol = 60:40. In step M4, the two groups of reference solution are 5 μl and 10 μl respectively, and the second test solution is 10-15 μl.

[0008] The present invention also provides a characteristic spectrum determination method of coix seed, which is used to determine the characteristic spectrum of the aforementioned test sample powder, and includes the following steps: A1: Chromatographic conditions and system suitability test: using octadecylsilane bonded silica gel as a filler, acetonitrile as a mobile phase A, and isopropanol as a mobile phase B, and performing gradient elution; flow rate: 1.0 ml / min; detector: ELSD; column temperature: 30°C; the theoretical plate number calculated based on the triolein peak should be no less than 5000. A2: Preparation of reference solution: Take appropriate amount of triolein reference substance and 1,2-linoleic acid-3-olein reference substance, weigh them, put them in a volumetric flask, add a mixed solvent of acetonitrile:isopropanol in a volume ratio of 55-65:45-35 to prepare a mixed solution containing 0.3±0.03 mg of triolein and 0.4±0.04 mg of 1,2-linoleic acid-3-olein per 1 ml, thus obtaining a reference solution; take coix seed oil reference extract, add a mixed solvent of acetonitrile:isopropanol in a volume ratio of 55-65:45-35 to prepare a second reference extract solution containing 1±0.1 mg per 1 ml. A3: Preparation of the third test solution: Take the test powder, weigh it, put it in a stoppered conical flask, add a mixed solvent with a volume ratio of acetonitrile: isopropanol = 55-65:45-35, plug it, weigh it, soak it for 30min-6h, ultrasonically treat it for 10-60min, let it cool, make up the lost weight, shake it well, filter it, and take the filtrate to get the third test solution. The test powder is passed through a No. 3 sieve; the power of the ultrasonic treatment is 250W and the frequency is 50kHz. A4: Take the second control extract solution, take the reference solution, take the third test solution, inject them into the liquid chromatograph respectively, measure them, and obtain the characteristic spectrum of the test product. Preferably, the volume ratio of acetonitrile: isopropanol = 60:40. In step A4, the second control extract solution is 5μl, the reference solution is 10μl, and the third test solution is 10-15μl.

[0009] By adopting the above scheme, the present invention provides a thin layer identification, content determination and characteristic spectrum determination method of coix seed, and uses ethyl acetate to replace ether, which can effectively solve the damage to the liver, kidney and nervous system of the test personnel in the existing coix seed identification process; at the same time, the spots of the thin layer chromatogram obtained by using 10±1vol% sulfuric acid ethanol solution as a color developer can be intuitive and clear under ultraviolet light, ensuring the accuracy of analysis and discrimination. The present invention can simultaneously detect 1,2-linoleic acid-3-oleic acid glyceride and triolein, and 7 main characteristic peaks are presented in the test sample spectrum, the separation between the peaks is high, the peak shape is good, and the overall appearance of the multi-components of coix seed can be reflected, and the result is reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1It is the thin layer chromatogram of coix seed (1,2-coix seed, 3,4-coix seed oil reference extract, 5,6-coix seed reference medicinal material);

[0011] Figure 2 It is the content determination spectrum of coix seed (1-1,2-linoleic acid-3-oleylglycerol, 2-triolein);

[0012] Figure 3 The reference characteristic spectrum of coix seed (2-1,2-linoleic acid-3-oleylglycerol, 6-triolein);

[0013] Figure 4 Chromatograms were investigated for different color developers and inspection methods (the color developer was 5% vanillin sulfuric acid and the inspection method was white light, 1,2-Coix seed, 3,4-Coix seed oil as control extract, and 5,6-Coix seed as control herbal medicine);

[0014] Figure 5 Chromatograms were investigated for different color developers and inspection methods (the color developer was 10% ethanolic sulfuric acid and the inspection method was white light, 1,2-Coix seed, 3,4-Coix seed oil as control extract, and 5,6-Coix seed as control herbal medicine);

[0015] Figure 6 The chromatograms were investigated for different color developers and viewing methods (the color developer was 5% vanillin sulfuric acid and the viewing method was 365 nm, 1,2-Coix seed, 3,4-Coix seed oil control extract, 5,6-Coix seed control herb);

[0016] Figure 7 Chromatograms were investigated for different color developers and viewing methods (the color developer was 10% ethanolic sulfuric acid and the viewing method was 365 nm, 1,2-Coix seed, 3,4-Coix seed oil as control extract, and 5,6-Coix seed as control medicinal material);

[0017] Figure 8 Chromatograms were investigated for different developing solvent ratios (developing solvent was petroleum ether (60-90°C)-ethyl acetate-glacial acetic acid (8.5:0.9:0.1), 1-Coix seed, 2,3-Coix seed oil control extract, 4,5-Coix seed control medicinal material, 6-negative control);

[0018] Fig. 9 Chromatograms were investigated for different developing solvent ratios (developing solvent was petroleum ether (60-90°C)-ethyl acetate-glacial acetic acid (8.5:1.0:0.1), 1,2-Coix seed, 3,4-Coix seed oil as control extract, and 5,6-Coix seed as control medicinal material);

[0019] Fig.10The chromatograms were investigated for different developing solvent ratios (the developing solvent was petroleum ether (60-90°C)-ethyl acetate-glacial acetic acid (8.5:0.8:0.15), 1,2-Coix seed, 3,4-Coix seed oil as control extract, and 5,6-Coix seed as control medicinal material);

[0020] Fig.11 The chromatograms were investigated for different developing solvent ratios (the developing solvent was petroleum ether (60-90°C)-ethyl acetate-glacial acetic acid (8.5:0.8:0.1), 1,2-Coix seed, 3,4-Coix seed oil as control extract, and 5,6-Coix seed as control medicinal material);

[0021] Fig.12 Chromatograms for specificity investigation (1,2-Coix seed, 3-Coix seed oil control extract, 4,5-Coix seed control medicinal material, 6-negative control);

[0022] Fig.13 To investigate the chromatograms at different temperatures (temperature 10°C, 1,2-Coix seed, 3,4-Coix seed oil control extract, 5,6-Coix seed control medicinal material);

[0023] Fig.14 To investigate the chromatograms at different temperatures (temperature 25°C, 1,2-Coix seed, 3,4-Coix seed oil control extract, 5,6-Coix seed control medicinal material);

[0024] Fig.15 Chromatograms were investigated at different humidity levels (humidity 32%, 1,2-Coix seed, 3,4-Coix seed oil control extract, 5,6-Coix seed control medicinal material);

[0025] Fig.16 Chromatograms were investigated at different humidity levels (humidity 58%, 1,2-Coix seed, 3,4-Coix seed oil control extract, 5,6-Coix seed control medicinal material);

[0026] Fig.17 Chromatograms were investigated at different humidity levels (humidity 72%, 1,2-Coix seed, 3,4-Coix seed oil control extract, 5,6-Coix seed control medicinal material);

[0027] Fig.18 Chromatograms were investigated for thin-layer plates from different manufacturers (Silica G thin-layer plates from Qingdao Ocean Chemical Plant, 1,2-Coix seed, 3,4-Coix seed oil reference extracts, and 5,6-Coix seed reference medicinal materials);

[0028] Fig.19 Chromatograms were examined for thin layer plates from different manufacturers (silica gel G thin layer plates from Yantai Jiangyou Silica Gel Development Co., Ltd., 1,2-Coix seed, 3,4-Coix seed oil reference extracts, and 5,6-Coix seed reference medicinal materials);

[0029] Fig. 20 Chromatograms were investigated for thin layer plates from different manufacturers (silica gel G thin layer plates from Yantai Huayang New Materials Technology Co., Ltd., 1,2-Coix seed, 3,4-Coix seed oil reference extracts, 5,6-Coix seed reference medicinal materials);

[0030] Fig.21 Examining chromatograms for different detectors;

[0031] Fig. 22 Investigate the chromatogram for the column type (9% carbon loading reversed phase C18 column);

[0032] Fig.23 Investigate the chromatogram for the column type (19% carbon loading reversed phase C18 column);

[0033] Fig.24 Chromatograms for investigation of mobile phase gradient and column length (250 mm reverse C18 column combined with acetonitrile-isopropanol (gradient 1) mobile phase analysis;

[0034] Fig.25 To investigate the chromatogram for mobile phase gradient and column length (150 mm reverse C18 column combined with acetonitrile-isopropanol (gradient 2) mobile phase analysis);

[0035] Fig.26 To investigate the chromatogram at column temperature (25°C);

[0036] Fig. 27 Chromatogram for column temperature investigation (30°C);

[0037] Fig.28 To investigate the chromatogram for the preparation method of the test product;

[0038] Fig.29 For exclusive investigation;

[0039] Fig.30 For durability - column temperature inspection (column temperature 30°C);

[0040] Fig.31 For durability - column temperature inspection (column temperature 35°C);

[0041] Fig.32 For durability-flow rate investigation (flow rate 0.9ml / min);

[0042] Fig.33 For durability-flow rate investigation (flow rate 1.0 ml / min). DETAILED DESCRIPTION

[0043] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0044] The specific experimental process is as follows.

[0045] 1.1 Experimental equipment

[0046] Thin layer imaging system (DD70, produced by Germany's Diseck Company), high performance liquid chromatograph (1260, produced by Agilent Technologies, Inc., USA), one-tenth of a microbalance (ML204, produced by Mettler-Toledo Instruments (Shanghai) Co., Ltd.), one-hundred-thousandth of a microbalance (XS205DU, produced by Mettler-Toledo Instruments (Shanghai) Co., Ltd.), integrated pure water / ultrapure water system (PALL, CascadaⅡ.Ⅰ), and ultrasonic cleaning machine (HNY600, produced by Guangzhou South China Ultrasonic Equipment Co., Ltd.).

[0047] 1.2 Experimental Materials

[0048] Standard substances: coix seed oil (China Food and Drug Inspection Institute, batch number: 111780-201102), coix seed reference medicinal materials (China Food and Drug Inspection Institute, batch number: 121254-202105), triolein (China Food and Drug Inspection Institute, batch number: 111962-202007), 1,2-linoleic acid-3-oleylglycerol (Chengdu Pusi Biotechnology Co., Ltd., batch number: PS220708-03).

[0049] Reagents and drugs: petroleum ether (60-90°C) (analytical grade), ethyl acetate (analytical grade), glacial acetic acid (analytical grade), sulfuric acid (analytical grade), 95% ethanol (analytical grade), vanillin (analytical grade), acetonitrile (chromatographic grade), isopropanol (chromatographic grade), and water is ultrapure water.

[0050] Thin layer plate: Silica gel G thin layer plate (10cm*10cm, Qingdao Ocean Chemical Plant), Silica gel G thin layer plate (10cm*10cm, Yantai Jiangyou Silica Gel Development Co., Ltd.), Silica gel G thin layer plate (10cm*10cm, Yantai Huayang New Material Technology Co., Ltd.).

[0051] Chromatographic column: Symmetry C18 chromatographic column (Waters, 4.6*150*5), Symmetry C18 chromatographic column (Waters, 4.6*250*5), ZORBAX Eclipse Plus C18 chromatographic column (Agilent, 4.6*250*5).

[0052] Test sample: Coix seed medicinal material Y210698.

[0053] Example 1 Thin layer chromatography identification of coix seed

[0054] (1) Preparation of test solution: Take 1 g of the product powder, add 30 ml of petroleum ether (60-90°C), ultrasonically treat for 30 minutes, filter, and take the filtrate as the test solution.

[0055] (2) Preparation of control solution: Take 1g of coix seed control medicinal material, add 30ml of petroleum ether (60-90℃), ultrasonically treat for 30 minutes, filter, and take the filtrate as the control medicinal material solution. Take coix seed oil control extract, add petroleum ether (60-90℃) to make a solution containing 2mg per 1ml, as the control extract solution.

[0056] (3) Determination method: Take 5 μl of each of the above two solutions and spot them on the same silica gel G thin layer plate, use petroleum ether (60-90°C)-ethyl acetate-glacial acetic acid (8.5:0.8:0.1) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clearly colored, and examine under ultraviolet light (365mn).

[0057] (4) Thin layer chromatography analysis: In the chromatogram of the test sample, spots of the same color appear at the corresponding positions in the chromatograms of the control medicinal material and the control extract.

[0058] Example 2 Determination of coix seed content

[0059] (1) Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler, acetonitrile was used as mobile phase A, and isopropanol was used as mobile phase B. Gradient elution was performed according to the requirements in the following table; flow rate: 1.0 ml / min; detector: ELSD; column temperature: 30°C. The theoretical plate number calculated based on the trioleylglycerol peak should be no less than 5000.

[0060]

[0061] (2) Preparation of reference solution: Take appropriate amounts of triolein reference substance and 1,2-linoleic acid-3-olein reference substance, accurately weigh them, place them in a volumetric flask, and add acetonitrile-isopropanol (60:40) to prepare a mixed reference solution containing 0.3 mg of triolein and 0.4 mg of 1,2-linoleic acid-3-olein per 1 ml.

[0062] (3) Preparation of test solution: Take about 0.6 g of the product powder (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 20 ml of acetonitrile-isopropanol (60:40), stopper it tightly, weigh it, soak it for 2 hours, ultrasonically treat it (power 250 W, frequency 50 kHz) for 30 minutes, cool it, make up the lost weight, shake it well, filter it, and take the filtrate to obtain the solution.

[0063] (4) Determination method: Accurately pipette 5 μl and 10 μl of the reference solution and 10-15 μl of the test solution respectively, inject into the liquid chromatograph, determine, and calculate using the external standard two-point method logarithmic equation to obtain the result.

[0064] Example 3 Coix seed characteristic spectrum

[0065] (1) Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler, acetonitrile was used as mobile phase A, and isopropanol was used as mobile phase B. Gradient elution was performed according to the requirements in the following table; flow rate: 1.0 ml / min; detector: ELSD; column temperature: 30°C. The theoretical plate number calculated based on the trioleylglycerol peak should be no less than 5000.

[0066]

[0067] (2) Preparation of reference solution: Take appropriate amount of triolein reference substance and 1,2-linoleic acid-3-oleylglycerol reference substance, accurately weigh them, place them in a volumetric flask, add acetonitrile-isopropanol (60:40) to prepare a mixed reference solution containing 0.3 mg of triolein and 0.4 mg of 1,2-linoleic acid-3-oleylglycerol per 1 ml. Take coix seed oil reference extract, add acetonitrile-isopropanol (60:40) to prepare a reference extract solution containing 1 mg per 1 ml.

[0068] (3) Preparation of test solution: Take about 0.6 g of the product powder (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 20 ml of acetonitrile-isopropanol (60:40), stopper it tightly, weigh it, soak it for 2 hours, ultrasonically treat it (power 250 W, frequency 50 kHz) for 30 minutes, cool it, make up the lost weight, shake it well, filter it, and take the filtrate to obtain the solution.

[0069] (4) Determination method: Accurately pipette 5 μl of the reference solution and 10-15 μl of the test solution respectively, inject into the liquid chromatograph, and determine.

[0070] (5) Characteristic spectrum analysis: The characteristic spectrum of the test sample should show 7 characteristic peaks, and the retention times should correspond to the 7 characteristic peaks in the chromatogram of the coix seed oil control extract. The peak corresponding to 1,2-linoleyl-3-oleylglycerol is the S peak. The relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within ±5% of the specified value. The specified values ​​are: 0.85 (peak 1), 1.00 (peak 2, S peak), 1.05 (peak 3), 1.20 (peak 4), 1.27 (peak 5), 1.47 (peak 6), and 1.56 (peak 7).

[0071] (1) Compared with the prior art, the thin layer chromatography identification method of coix seed of the present invention adopts a developing agent system of petroleum ether (60-90°C)-ethyl acetate-glacial acetic acid, a color developer of 10% sulfuric acid ethanol solution, and an inspection method of ultraviolet light 365min. The developing agent is available and has little harm to the liver, kidney, and nervous system of the test personnel. The obtained thin layer chromatography spot separation is good and the spots are intuitive and clear, which is conducive to identification.

[0072] (2) Please refer to Figure 1 The results of the coix seed thin layer chromatography identification method in this embodiment show that 1 and 2 are test solution, 3 and 4 are control extract solution, 5 and 6 are control medicinal material solution; the control medicinal material solution has 6 spots, the control extract solution has 5 spots, and the test solution has 6 spots. The spot color is light red, the brightness is clear, the separation is good, and the ratio shift value is between 0.1 and 0.8

[0073] (3) Compared with the prior art, the mobile phase system used in the coix seed content determination and characteristic spectrum method of the present invention is acetonitrile-isopropanol, which has moderate polarity and can be miscible with water. There is no need to empty the water phase inside the high-performance liquid chromatograph before using the mobile phase, which increases the convenience of the experiment. Compared with dichloromethane, isopropanol can reduce the wear on the pump components of the high-performance liquid chromatograph and reduce the corrosion to the metal tube wall and chromatographic column of the instrument. The method introduces 1,2-linoleic acid-3-oleylglycerol, another active ingredient of coix seed, as a detection indicator, which can better control the quality of coix seed.

[0074] (4) Please refer to Figure 2-3 The coix seed content determination and characteristic spectrum determination method of the present invention can simultaneously detect 1,2-linoleic acid-3-oleylglycerol and triolein. Seven main characteristic peaks are presented in the test sample spectrum. The separation degree between the peaks is high and the peak shape is good. It can reflect the overall appearance of the multi-components of coix seed, and the result is reliable.

[0075] (5) The present invention is based on the determination method of thin layer chromatography identification, content determination, and characteristic spectrum determination. Methodological investigation suggests that the results of various indicators are good and can be used for quality detection of coix seeds. It provides a new method for quality control of coix seeds and has good application prospects.

[0076] The beneficial effects of the present invention are specifically described below through test examples:

[0077] Experimental Example 1 Establishment of Thin Layer Chromatography Conditions

[0078] (1) Preparation of test solution: Take 1 g of the product powder, add 30 ml of petroleum ether (60-90°C), ultrasonically treat for 30 minutes, filter, and take the filtrate as the test solution.

[0079] (2) Preparation of control solution: Take 1g of coix seed control medicinal material, add 30ml of petroleum ether (60-90℃), ultrasonically treat for 30 minutes, filter, and take the filtrate as the control medicinal material solution. Take coix seed oil control extract, add petroleum ether (60-90℃) to make a solution containing 2mg per 1ml, as the control extract solution.

[0080] (3) Investigation of different color developers and inspection methods: The test solution and control solution prepared by the above method were displayed according to the development method of the 2020 edition of the "Chinese Pharmacopoeia". Under the same conditions, the effects of 5% vanillin sulfuric acid solution and 10% sulfuric acid ethanol solution as color developers were investigated. The results showed that 5% vanillin sulfuric acid solution and 10% sulfuric acid ethanol solution as color developers had similar effects under white light, but the spots were not clear enough. Then, the color development effects of the two color developers were investigated under ultraviolet light at 365nm. The results showed that 10% sulfuric acid ethanol solution as a color developer had the largest number of clearest spots under ultraviolet light at 365nm. Therefore, 10% sulfuric acid ethanol solution was selected as the color developer, and 365nm ultraviolet light was used as the inspection wavelength. See. Figure 4-7 .

[0081] (4) Investigation of different developing agent ratios: The test solution and control solution prepared by the above method were used, with 10% sulfuric acid ethanol solution as the color developer and ultraviolet light (365nm) as the inspection wavelength. The developing agent system was petroleum ether (60-90℃)-ethyl acetate-glacial acetic acid at different ratios. The results showed that petroleum ether (60-90℃)-ethyl acetate-glacial acetic acid (8.5:0.8:0.1) had the best overall development effect. Therefore, petroleum ether (60-90℃)-ethyl acetate-glacial acetic acid (8.5:0.8:0.1) was selected as the developing agent ratio. See Figure 8-11 .

[0082] (5) The TLC conditions were finally determined as follows: ① Developing agent: petroleum ether (60-90°C)-ethyl acetate-glacial acetic acid (8.5:0.8:0.1); ② Color developer: 10% sulfuric acid ethanol solution; ③ Inspection wavelength: UV lamp 365nm.

[0083] Experimental Example 2: Study on Thin Layer Chromatography Methodology

[0084] (1) Specificity: According to the proposed experimental conditions, prepare negative control solution, test solution, control extract solution, and control medicinal material solution. Take 5 μl of each solution and spot it on the same silica gel G thin layer plate. The results show that the negative control solution does not interfere with the identification. Fig.12 .

[0085] (2) Durability - Different temperature test: Take the spotted thin layer plate, develop it at 10℃ (in the refrigerator) and 25℃ (room temperature), and inspect it. The results show that temperature has no obvious effect on the identification. Figure 13-14 .

[0086] (3) Durability - Different humidity test: Take the spotted thin layer plate and develop it in a chromatography cylinder with relative humidity adjusted to 32%, 58%, and 72% with sulfuric acid, develop the color, and inspect it. The results show that the test in the relative humidity range of 32%-72% has no obvious effect on the identification. Figure 15-17 .

[0087] (4) Durability - Investigation of thin layer plates from different manufacturers: Three prefabricated ordinary silica gel G thin layer plates from different manufacturers were tested according to the proposed method. The results showed that there was no significant difference in the separation effect of thin layer plates from different manufacturers. Figure 18-20 .

[0088] Test Example 3: Content determination and characteristic spectrum method establishment

[0089] (1) Investigation of different detectors: The detection effects of ELSD detector and DAD detector on the chemical components of coix seed were investigated. The baseline of ELSD detector was relatively stable, so ELSD detector was selected. Fig.21 .

[0090] (2) Column type investigation: Under the same mobile phase gradient conditions, reverse C18 columns with 9% carbon loading and 19% carbon loading were used for analysis. The results showed that the reverse C18 column with 19% carbon loading had the best separation effect. Therefore, the reverse C18 column with a higher carbon loading (19%) was selected as the analytical column. Figure 22-23 .

[0091] (3) Investigation of mobile phase gradient and column length: Based on the results of the preliminary experiments, the overall chromatographic effect of the chromatogram was compared in the following manner. The results showed that the overall chromatographic effect of the 250 mm reverse C18 column combined with acetonitrile-isopropanol (gradient 1) mobile phase analysis was equivalent to that of the 150 mm reverse C18 column combined with acetonitrile-isopropanol (gradient 2) mobile phase analysis. In order to save analysis time and mobile phase usage, the 150 mm reverse C18 column combined with acetonitrile-isopropanol (gradient 2) mobile phase was selected as the chromatographic condition in this method. Figure 24-25 .

[0092]

[0093] Gradient 1

[0094]

[0095] Gradient 2

[0096]

[0097]

[0098] (4) Column temperature investigation: After determining the ratio of chromatographic column and mobile phase, the chromatographic effects of column temperatures of 25°C and 30°C were investigated under the same conditions. The results showed that the chromatographic peak shape of the column temperature of 30°C was better than that of the column temperature of 25°C, so 30°C was selected as the column temperature in the method. Figure 26-27 .

[0099] (5) Investigation of the test sample preparation method: Referring to the sample weight of the test sample in the Chinese Pharmacopoeia, 0.6 g of the same test sample powder was taken and the test sample solution was prepared in the following manner. The sample was injected and measured according to the established chromatographic conditions, and the content was calculated. The optimal test sample preparation method was comprehensively evaluated. Finally, the optimal test sample preparation method was obtained as the extraction solvent acetonitrile-isopropanol (60:40), the solvent volume was 20 ml, the soaking time was 30 min, and the ultrasonic extraction time was 30 min. Fig.28 .

[0100]

[0101] The final method of content determination and characteristic spectrum was determined as follows: ① Chromatographic conditions: octadecylsilane bonded silica gel as filler (chromatographic column with high carbon load, column length 15 cm, inner diameter 4.6 mm, particle size 5 μm), acetonitrile as mobile phase A, isopropanol as mobile phase B, gradient elution according to gradient 2; flow rate: 1.0 ml / min; detector: ELSD; column temperature: 30 °C. ② Preparation of test solution: the amount of test powder is 0.6 g; the extraction solvent is acetonitrile-isopropanol (60:40) 20 ml; the extraction method is soaking for 2 hours and ultrasonic treatment for 30 minutes.

[0102] Experimental Example 4: Investigation of content determination methodology

[0103] (1) Specificity: Take a conical flask, add 20 ml of the extraction solvent acetonitrile-isopropanol (60:40), follow the established method for preparing the test solution, and perform the analysis under the established chromatographic conditions. The results show that the blank solvent has no interference with the analysis of the test product. Fig.29 .

[0104] (2) Linear relationship: Take 1,2-linoleyl-3-oleylglycerol and trioleylglycerol mixed reference substances to prepare mixed reference substance solutions with concentrations of 0.4082 mg / ml and 0.2748 mg / ml, respectively. Accurately pipette 2.5ul, 5ul, 10ul, 15ul, 20ul, and 30ul, and measure according to the established method. Use the logarithm of the reference substance injection volume as the abscissa (X) and the logarithm of the peak area as the ordinate (Y) for regression processing. The results of the linear relationship test are shown in the table below.

[0105]

[0106] (3) Precision test: Take the same test solution and inject it 6 times continuously according to the established chromatographic conditions. Determine the peak area of ​​1,2-linoleyl-3-oleylglycerol and trioleylglycerol and calculate the RSD. The peak area RSDs are 2.4% and 1.9% respectively, indicating that the experimental system has good precision. The results of the precision test are shown in the table below.

[0107]

[0108]

[0109] (4) Repeatability test: Take 6 portions of the same test sample, prepare the test sample solution according to the established method, and continuously inject and measure according to the established chromatographic conditions. Calculate the content and RSD value of 1,2-linoleyl-3-oleylglycerol and triolein respectively. The RSD values ​​of the content are 3.4% and 2.4%, respectively, indicating that the method has good repeatability. The repeatability test results are shown in the table below.

[0110]

[0111] (5) Stability test: 10 μl of the same test solution was injected at 0 h, 2 h, 5 h, 10 h, 15 h, 20 h, and 24 h. The RSD of the peak areas of 1,2-linoleyl-3-oleylglycerol and triolein were 3.0% and 3.5%, respectively, indicating that the test solution prepared by this method had good stability within 24 h. The results of the stability test are shown in the table below.

[0112]

[0113]

[0114] (6) Accuracy test: Take 6 portions of coix seed powder with known 1,2-linoleic acid-3-oleylglycerol and trioleylglycerol content, 0.3 g per portion, add reference substances equivalent to 0.3 g of the test sample content, prepare sample solutions according to the preparation method of the test sample solution, inject and measure, and calculate the average recovery rate and its RSD value. The average recovery rates of 1,2-linoleic acid-3-oleylglycerol and trioleylglycerol are 100% and 94%, respectively, and the RSD values ​​are 4.0% and 3.1%, respectively, indicating that the recovery rate of this method is good.

[0115] The accuracy test results are shown in the table below.

[0116]

[0117] Experimental Example 5: Characteristic Spectrum Methodology Study

[0118] (1) Specificity: Take a conical flask, add 20 ml of the extraction solvent acetonitrile-isopropanol (60:40), follow the established method for preparing the test solution, and perform the analysis under the established chromatographic conditions. The results show that the blank solvent has no interference with the analysis of the test product. Fig.29 .

[0119] (2) Precision test: Take the same test solution and inject it 6 times continuously according to the established chromatographic conditions. Take Peak 2 (1,2-linoleyl-3-oleylglycerol) as the reference peak s peak, calculate the relative retention time RSD values ​​of the 7 common characteristic peaks and s peak. The relative retention time RSD values ​​of the 7 common characteristic peaks and s peak are 0.1%, 0.0, 0.1%, 0.1%, 0.2%, 0.2%, 0.2%, respectively, indicating that the experimental system has good precision. The results of the precision test are shown in the table below.

[0120]

[0121] (3) Repeatability test: Take 6 portions of the same test sample, prepare the test sample solution according to the established method, and continuously inject and measure according to the established chromatographic conditions. Take Peak 2 (1,2-linoleyl-3-oleylglycerol) as the reference peak s peak, calculate the relative retention time RSD values ​​of the 7 common characteristic peaks and the s peak. The relative retention time RSD values ​​of the 7 common characteristic peaks and the s peak are 0.1%, 0.0, 0.1%, 0.1%, 0.7%, 0.2%, and 0.2%, respectively, indicating that the experimental system has good precision. The repeatability test results are shown in the table below.

[0122]

[0123]

[0124] Durability - Column temperature investigation: Accurately pipette 15ul of the test solution, set the column temperature to 30℃ and 35℃ respectively, and measure according to the established chromatographic conditions. The results show that the column temperature range of 30℃-35℃ has no effect on the method. Figure 30-31 .

[0125] Durability-flow rate investigation: 15ul of the test solution was accurately aspirated and measured at flow rates of 0.9ml / ml and 1.0ml / m respectively according to the established chromatographic conditions. The results showed that the flow rate range of 0.9ml / ml-1.0ml / m had no effect on the method. Figure 32-33 .

[0126] In summary, the present invention provides a thin layer identification, content determination and characteristic spectrum determination method of coix seed, and uses ethyl acetate to replace ether, which can effectively solve the existing damage to the liver, kidney and nervous system of the test personnel in the identification process of coix seed; at the same time, the spots of the thin layer chromatogram obtained by using 10±1vol% sulfuric acid ethanol solution as a color developer can be intuitive and clear under ultraviolet light, ensuring the accuracy of analysis and discrimination. The present invention can simultaneously detect 1,2-linoleic acid-3-oleic acid glyceride and triolein, and 7 main characteristic peaks are presented in the sample spectrum, the separation between the peaks is high, the peak shape is good, and the overall appearance of the multi-components of coix seed can be reflected, and the result is reliable.

[0127] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining the content of coix seed, characterized in that: The following steps are involved: M1: Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel as filler, acetonitrile as mobile phase A, isopropanol as mobile phase B, gradient elution; flow rate: 1.0 ml / min; detector: ELSD; column temperature: 30°C; the theoretical plate number calculated based on the trioleylglycerol peak should not be less than 5000; the gradient elution procedure is as follows: M2: Preparation of reference solution: Take appropriate amount of triolein reference substance and 1,2-linoleic acid-3-olein reference substance, weigh them, put them in a volumetric flask, add a mixed solvent of acetonitrile:isopropanol with a volume ratio of 55-65:45-35, and prepare a reference solution containing 0.3±0.03 mg of triolein and 0.4±0.04 mg of 1,2-linoleic acid-3-olein per 1 ml; M3: Preparation of test solution: Take the test sample powder, weigh it, put it in a stoppered conical flask, add a mixed solvent of acetonitrile: isopropanol in a volume ratio of 55-65:45-35, stopper it tightly, weigh it, soak it for 30min-6h, ultrasonically treat it for 10-60min, cool it, make up the lost weight, shake it well, filter it, and take the filtrate to obtain the test solution; M4: Aspirate the reference solution, aspirate the test solution, inject into the liquid chromatograph, measure, and calculate using the external standard two-point method logarithmic equation to obtain the content of triolein and 1,2-linoleic acid-3-oleylglycerol in the test sample powder.

2. The method for determining the content of coix seed according to claim 1, characterized in that: The sample powder was passed through a No. 3 sieve; the power of ultrasonic treatment was 250 W and the frequency was 50 kHz; the volume ratio of acetonitrile:isopropanol was 60:

40.

3. The method for determining the content of coix seed according to claim 1, characterized in that: In step M4, the two groups of reference solution are 5 μl and 10 μl respectively, and the test solution is 10-15 μl.

4. A method for determining the characteristic spectrum of coix seeds, characterized in that: The following steps are involved: A1: Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel as filler, acetonitrile as mobile phase A, isopropanol as mobile phase B, gradient elution; flow rate: 1.0 ml / min; Detector: ELSD; Column temperature: 30°C; The theoretical plate number calculated based on the trioleylglycerol peak should be no less than 5000; A2: Preparation of reference solution: Take appropriate amount of triolein reference substance and 1,2-linoleic acid-3-olein reference substance, weigh them, put them in a volumetric flask, add a mixed solvent of acetonitrile:isopropanol in a volume ratio of 55-65:45-35 to prepare a mixed solution containing 0.3±0.03 mg of triolein and 0.4±0.04 mg of 1,2-linoleic acid-3-olein per 1 ml, thus obtaining a reference solution; take coix seed oil reference extract, add a mixed solvent of acetonitrile:isopropanol in a volume ratio of 55-65:45-35 to prepare a reference extract solution containing 1±0.1 mg per 1 ml; A3: Preparation of test solution: Take the test sample powder, weigh it, put it in a stoppered conical flask, add a mixed solvent of acetonitrile: isopropanol in a volume ratio of 55-65:45-35, stopper it tightly, weigh it, soak it for 30min-6h, ultrasonically treat it for 10-60min, cool it, make up the lost weight, shake it well, filter it, and take the filtrate to obtain the test solution; A4: Pipette the control extract solution, the reference solution, and the test solution, and inject them into the liquid chromatograph respectively for measurement to obtain the characteristic spectrum of the test sample powder.

5. The method for determining the characteristic spectrum of coix seeds according to claim 4, characterized in that: The sample powder was passed through a No. 3 sieve; the power of ultrasonic treatment was 250 W and the frequency was 50 kHz; the volume ratio of acetonitrile:isopropanol was 60:

40.

6. The method for determining the characteristic spectrum of coix seeds according to claim 4, characterized in that: In step A4, the control extract solution is 5 μl, the reference solution is 10 μl, and the test solution is 10-15 μl.

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