A methargma dipeptide compound, its synthesis method and application

Abstract

The application discloses a methyl guanidine dipeptide compound and a synthesis method and application thereof, and belongs to the technical field of chemical synthesis, and the chemical structural formula of the compound is as follows: the synthesis method of the methyl guanidine dipeptide compound comprises the following steps: L-ornithine hydrochloride and 1,2-dimethyl-2-isothiourea hydroiodide are heated and reacted overnight in an alkaline solution; the pH value of the solution is adjusted to be neutral, purification is carried out, water is added for dissolution and the solution is adjusted to be acidic, ion exchange resin is passed through, and an intermediate 1 is obtained; pure water and isopropyl alcohol are added to the intermediate 1 and adjusted to be alkaline; isovaleryl chloride is added to the solution and adjusted to be alkaline, and the solution is stirred and reacted at room temperature; the solution is kept alkaline, heated to 45-55 DEG C, adjusted to be weakly acidic, cooled to 5-15 DEG C, and the precipitate is collected, so that a crude product is prepared; the crude product is dissolved in a methanol-water mixed solution, gradient elution is carried out through liquid chromatography, and drying is carried out, so that the methyl guanidine dipeptide compound is prepared. The methyl guanidine dipeptide compound synthesized by the method has high purity, the synthesis process is simple, and the operation is convenient.

Description

Technical Field

[0001] This invention belongs to the field of chemical synthesis technology, specifically relating to a methylguanidine dipeptide compound, its synthesis method, and its application. Background Technology

[0002] Pinellia ternata is a plant of the Araceae family. Pinellia ternate The dried tuber of (Thunb.) Breit is a traditional and commonly used Chinese medicinal herb. Pinellia ternata mainly contains chemical components such as alkaloids, organic acids, amino acids, polysaccharides, proteins, volatile oils, sterols, and flavonoids. Current research focuses on organic acids and alkaloids.

[0003] Water pinellia is a plant belonging to the genus Pinellia in the family Araceae. Typhonium flagelliforme The dried tubers of *Pinellia ternata* (Lodd.) Blume, along with *Pinellia ternata* (also known as water pinellia) and *Pinellia ternata* (banxia), both belong to the same family and are traditional Chinese medicines used for drying dampness, resolving phlegm, relieving cough, and calming asthma. Both are widely used in TCM clinical practice for treating cough and excessive phlegm. However, studies have shown that water pinellia is more toxic than *Pinellia ternata*, and its antitussive and antiemetic pharmacological effects are weaker. Therefore, blindly using water pinellia to replace *Pinellia ternata* may pose certain medication safety risks. On the other hand, water pinellia has relatively lower cultivation requirements, is easier to harvest, and has a higher yield. In recent years, the price range of water pinellia has generally been 10-20 yuan, while the price range of *Pinellia ternata* has fluctuated between 70-120 yuan. This nearly five-fold price difference has led some unscrupulous merchants to perform "plastic surgery" on water pinellia to counterfeit genuine *Pinellia ternata*.

[0004] Comparative analysis of the chemical components in Pinellia ternata and Pinellia ternata revealed certain differences. Therefore, the two can be distinguished by detecting the content of differentiating compounds. However, the current purification process for these differentiating compounds is relatively complex, and the amount of purified product obtained is small, which cannot meet the application requirements. Therefore, developing a synthetic method for this compound to prepare purified products in large quantities is particularly important. Summary of the Invention

[0005] To address the aforementioned shortcomings in the prior art, this invention provides a methylguanidine dipeptide compound and its synthesis method. The methylguanidine dipeptide compound synthesized using this application has high purity, and the synthesis process is simple and easy to operate.

[0006] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problem is as follows:

[0007] A methylguanidine dipeptide compound has the following chemical structural formula:

[0008]

[0009] The method for synthesizing the above-mentioned methylguanidine dipeptide compound includes the following steps:

[0010] (1) Dissolve L-ornithine hydrochloride and 1,2-dimethyl-2-isothiourea hydroiodide in an alkaline solution and heat at 70-110℃ overnight;

[0011] (2) Adjust the pH of the solution to neutral, purify, then add water to dissolve, and adjust the pH of the solution to acidic, pass through ion exchange resin, rinse with pure water first, then wash with ammonia water, and freeze dry to obtain intermediate 1;

[0012] (3) Add pure water and isopropanol to intermediate 1, and add alkaline solution to adjust the pH of the solution to be alkaline;

[0013] (4) Add isovaleryl chloride to the solution and add alkaline solution to adjust the pH of the solution to alkaline. Stir the reaction at room temperature for 2-4 hours.

[0014] (5) Keep the pH of the solution alkaline, heat to 45-55℃, then adjust the pH of the solution to weakly acidic, cool to 5-15℃, collect the precipitate, and obtain the crude compound.

[0015] (6) The crude compound was dissolved in a methanol-water mixture, then eluted by gradient liquid chromatography and dried to obtain the product.

[0016] Further, in step (1), the molar ratio of L-ornithine hydrochloride and 1,2-dimethyl-2-isothiourea hydroiodide is 1:3-5; the alkaline solution is a 1M sodium hydroxide solution.

[0017] Further, in step (2), dilute hydrochloric acid is used to adjust the pH of the solution; the purification process is as follows: after evaporating the solvent, methanol is added to dissolve it, the impurities are removed by filtration, and the methanol solvent is recovered; the pH of the solution is adjusted to 3-5; the concentration of ammonia is 0.2M.

[0018] Further, in step (3), the ratio of intermediate 1, pure water and isopropanol is 9-11 mmol: 7-9 ml: 24-26 ml; sodium hydroxide solution is added to adjust the pH of the solution to 10-12.

[0019] Furthermore, in step (4), the molar ratio of isovaleryl chloride to intermediate 1 is 14-16:1.

[0020] Furthermore, in step (5), after heating, concentrated hydrochloric acid is used to adjust the pH of the solution to 5-6.

[0021] Furthermore, in step (6), the volume ratio of methanol to water in the methanol-water mixed solution is 1-2:1-2.

[0022] Furthermore, during elution in step (6), the chromatographic column is a Waters Xbridge 19x250mm 10μm, the mobile phase A is purified water, and the mobile phase B is acetonitrile; the flow rate is 25ml / min.

[0023] Further, the gradient elution conditions in step (6) are as follows: 0 min, 95% of phase A, 5% of phase B; 1.5 min, 95% of phase A, 5% of phase B; 9.5 min, 80% of phase A, 20% of phase B; 9.7 min, 5% of phase A, 95% of phase B; 12.7 min, 5% of phase A, 95% of phase B; 13 min, 95% of phase A, 5% of phase B; 16 min, 95% of phase A, 5% of phase B.

[0024] The above-mentioned methylguanidine dipeptide compound was used as a reference standard in the identification of adulterated Pinellia ternata and Pinellia ternata decoction pieces.

[0025] Furthermore, the specific identification methods are as follows:

[0026] (1) Using methylguanidine dipeptide as a reference standard and a water-water-soluble organic solvent mixture as a solvent, a reference standard solution was prepared;

[0027] (2) Crush the Pinellia ternata medicinal material or decoction pieces, sieve them, add water-water-soluble organic solvent mixture solution, extract by ultrasonication, filter, and obtain Pinellia ternata extract;

[0028] (3) Chromatography-mass spectrometry was performed on the reference solution in step (1) and the Pinellia ternata extract in step (2), respectively; High performance liquid chromatography conditions: Column: Thermo Hypersil GOLD aQ C 18 Chromatographic column; injection volume: 1 μl; flow rate: 0.3 ml / min; column temperature: 35℃; mobile phase A: acetonitrile; mobile phase B: 20 ​​mmol / L ammonium formate-10% methanol solution; elution program: 0 min, mobile phase A 5%, mobile phase B 95%; 2 min, mobile phase A 5%, mobile phase B 95%; 8 min, mobile phase A 22%, mobile phase B 78%.

[0029] Mass spectrometry conditions: Triple quadrupole mass spectrometer, electrospray ionization (ESI) mode, multiple reaction monitoring (MRM); spray voltage: static; positive ion mode: 3500 V; ion transfer tube temperature: 280℃; nebulizer temperature: 280℃; sheath gas: 40 Arb; purge gas: 1 Arb; methylguanidine dipeptide mass spectrometry acquisition parameters:

[0030]

[0031] (4) Compare the chromatograms of the reference solution and the extract of Pinellia ternata or processed medicinal slices. If the chromatogram of Pinellia ternata or processed medicinal slices shows a chromatographic peak with the same retention time as the chromatogram of the reference solution, it is determined that the Pinellia ternata or processed medicinal slices contains adulterated water-processed Pinellia ternata; otherwise, it is determined that the Pinellia ternata is adulterated anhydrous Pinellia ternata.

[0032] Among them, the peak area of ​​methylguanidine dipeptide increased with the increase of the proportion of water-infused Pinellia ternata, and showed a good linear relationship. 2 Not less than 0.99.

[0033] The beneficial effects of this invention are as follows:

[0034] 1. The method in this invention can synthesize high-purity methylguanidine dipeptide compounds for subsequent detection needs; and the synthesis conditions of the synthesis method in this application are mild, the operation is simple, and it is convenient for actual production operation.

[0035] 2. In this invention, the synthesized methylguanidine dipeptide is used as a reference standard to identify Pinellia ternata and Pinellia ternata var. chinensis, which can effectively reduce the difficulty of identification between the two and improve the accuracy of identification. Attached Figure Description

[0036] Figure 1 This is a diagram illustrating the synthesis process of methylguanidine dipeptide;

[0037] Figure 2 It is a methylguanidine dipeptide 1 H NMR spectrum;

[0038] Figure 3 It is a methylguanidine dipeptide 13 C NMR spectrum;

[0039] Figure 4 It is a methylguanidine dipeptide 1 H-1H COSY spectrum;

[0040] Figure 5 It is a methylguanidine dipeptide 1 H- 13 C HSQC spectrum;

[0041] Figure 6 The HMBC spectrum of methylguanidine dipeptide is shown.

[0042] Figure 7 This is a high-resolution mass spectrum of methylguanidine dipeptide;

[0043] Figure 8 The infrared spectrum of methylguanidine dipeptide;

[0044] Figure 9 This is the chromatogram for the detection of methylguanidine dipeptide;

[0045] Figure 10 This is the chromatogram for the detection of methylguanidine dipeptide reference standard;

[0046] Figure 11 Chromatogram for the detection of raw Pinellia ternata;

[0047] Figure 12 The chromatogram for detecting Pinellia ternata is shown below.

[0048] Figure 13 The chromatogram for the detection of Pinellia ternata;

[0049] Figure 14 The chromatogram for the detection of Pinellia ternata;

[0050] Figure 15 Chromatogram of Pinellia ternata in fresh water;

[0051] Figure 16 The chromatogram for detecting Pinellia ternata in ginger water;

[0052] Figure 17 The chromatogram for the detection of Pinellia ternata in water is shown below.

[0053] Figure 18 The chromatogram for the detection of Pinellia ternata in clear water;

[0054] Figure 19 This is a standard curve diagram for the reference standard.

[0055] Figure 20 Linear relationship diagram of different proportions of raw Pinellia ternata mixed with raw water;

[0056] Figure 21 Linear relationship diagram of different proportions of ginger-infused Pinellia ternata;

[0057] Figure 22 Linear relationship diagram of Pinellia ternata added to Pinellia ternata in different proportions;

[0058] Figure 23 Linear relationship diagram of different proportions of Pinellia ternata mixed with Pinellia ternata. Detailed Implementation

[0059] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments.

[0060] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0061] It should be noted that relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes the element.

[0062] The features and performance of the present invention will be further described in detail below with reference to the embodiments and accompanying drawings.

[0063] Example 1

[0064] A methylguanidine dipeptide compound has the following chemical structural formula:

[0065]

[0066] The above-mentioned method for synthesizing methylguanidine dipeptide is as follows:

[0067] (1) L-ornithine hydrochloride (10 g, 60 mmol) and 1,2-dimethyl-2-isothiourea hydroiodide (56 g, 240 mmol) were dissolved in 120 ml of 1 M sodium hydroxide solution and reacted overnight in an oil bath at 110 °C.

[0068] (2) Add 1M dilute hydrochloric acid to adjust the pH of the solution to neutral, concentrate to remove water, add methanol (100 mL), stir for 30 minutes, and filter; add pure water (100 mL) to the filtrate, adjust the pH to 4.0 with 1M dilute hydrochloric acid, pass through ion exchange resin (D101 neutral ion exchange resin), rinse with pure water first, then wash with 0.2M ammonia water, and freeze dry to obtain intermediate 1;

[0069] (3) Add pure water (8 mL) and isopropanol (25 mL) to intermediate 1 (1.9 g, 10 mmol), and add sodium hydroxide solution to adjust the pH of the solution to 11;

[0070] (4) Add isovaleryl chloride (1.8 g, 15 mmol, 1.5 eq) to the solution and stir at room temperature for 3 h;

[0071] (5) Keep the pH of the solution at 11, heat to 50°C, then add concentrated hydrochloric acid to adjust the pH of the solution to 6, cool to 0°C, collect the precipitated white precipitate, and obtain the crude compound.

[0072] (6) The crude compound was dissolved in a methanol-water (1:1) mixture and then eluted by gradient liquid chromatography. The column was a Waters Xbridge 19x250mm 10μm, the mobile phase A was purified water, and the mobile phase B was acetonitrile. The flow rate was 25ml / min. The gradient elution conditions are shown in the table below. The eluent was collected within 9.0-9.5min and dried by rotary evaporation at 60℃ to obtain the product.

[0073]

[0074] Example 2

[0075] A methylguanidine dipeptide compound has the following chemical structural formula:

[0076]

[0077] The above-mentioned method for synthesizing methylguanidine dipeptide is as follows:

[0078] (1) L-ornithine hydrochloride (13.3 g, 80 mmol) and 1,2-dimethyl-2-isothiourea hydroiodide (56 g, 240 mmol) were dissolved in 120 ml of 1 M sodium hydroxide solution and reacted overnight in an oil bath at 100 °C.

[0079] (2) Add 1M dilute hydrochloric acid to adjust the pH of the solution to neutral, concentrate to remove water, add methanol (100 mL), stir for 30 minutes, filter, add pure water (100 mL) to the filtrate, adjust the pH of the solution to 3.0 with 1M dilute hydrochloric acid, pass through ion exchange resin (D101 neutral ion exchange resin), rinse with pure water first, then wash with 0.2M ammonia water, freeze dry to obtain intermediate 1;

[0080] (3) Add pure water (7 mL) and isopropanol (24 mL) to intermediate 1 (1.9 g, 10 mmol), and add sodium hydroxide solution to adjust the pH of the solution to 12;

[0081] (4) Add isovaleryl chloride (1.68 g, 14 mmol, 1.4 eq) to the solution and stir at room temperature for 4 h;

[0082] (5) Keep the pH of the solution at 12, heat to 45°C, then add concentrated hydrochloric acid to adjust the pH of the solution to 5, cool to room temperature, collect the precipitated white precipitate, and obtain the crude compound;

[0083] (6) The crude compound was dissolved in a methanol-water (1:1) mixture and then eluted by gradient liquid chromatography. The column was a Waters Xbridge 19x250mm 10μm, the mobile phase A was purified water, and the mobile phase B was acetonitrile. The flow rate was 25ml / min. The gradient elution conditions are shown in the table below. The eluent was collected within 9.0-9.5min and dried by rotary evaporation at 60℃ to obtain the product.

[0084]

[0085] Example 3

[0086] A methylguanidine dipeptide compound has the following chemical structural formula:

[0087]

[0088] The above-mentioned method for synthesizing methylguanidine dipeptide is as follows:

[0089] (1) Dissolve L-ornithine hydrochloride (10 g, 60 mmol) and 1,2-dimethyl-2-isothiourea hydroiodide (70 g, 300 mmol) in 120 ml of 1 M sodium hydroxide solution and react overnight in an oil bath at 70 °C.

[0090] (2) Adjust the pH of the solution to neutral by adding 1M dilute hydrochloric acid, concentrate to remove water, add methanol (100 mL), stir for 30 minutes, and filter; add pure water (100 mL) to the filtrate, adjust the pH to 5.0 with 1M dilute hydrochloric acid, pass through ion exchange resin (D101 neutral ion exchange resin), rinse with pure water first, then wash with 0.2M ammonia water, and freeze dry to obtain intermediate 1;

[0091] (3) Add pure water (9 mL) and isopropanol (26 mL) to intermediate 1 (1.9 g, 10 mmol), and add sodium hydroxide solution to adjust the pH of the solution to 11;

[0092] (4) Add isovaleryl chloride (1.8 g, 15 mmol, 1.5 eq) to the solution and stir at room temperature for 3 h;

[0093] (5) Keep the pH of the solution at 11, heat to 55°C, then add concentrated hydrochloric acid to adjust the pH of the solution to 6, cool to -20°C, collect the precipitated white precipitate, and obtain the crude compound.

[0094] (6) The crude compound was dissolved in a methanol-water (1:1) mixture and then eluted by gradient liquid chromatography. The column was a Waters Xbridge 19x250mm 10μm, the mobile phase A was purified water, and the mobile phase B was acetonitrile. The flow rate was 25ml / min. The gradient elution conditions are shown in the table below. The eluent was collected within 9.0-9.5min and dried by rotary evaporation at 60℃ to obtain the product.

[0095]

[0096] Example 4

[0097] The above-mentioned methylguanidine dipeptide compound is used as a reference standard in identifying adulterated Pinellia ternata (Pinellia ternata) and its processed forms. The specific detection method is as follows:

[0098] (1) Using methylguanidine dipeptide as a reference standard, a reference standard solution was prepared using 50% ethanol as a solvent;

[0099] (2) Crush the Pinellia ternata medicinal material or decoction pieces, sieve, add 50% ethanol, extract by ultrasonication, filter, and obtain Pinellia ternata extract;

[0100] (3) Chromatography-mass spectrometry was performed on the reference solution in step (1) and the Pinellia ternata extract in step (2) respectively; High performance liquid chromatography conditions: C18 column, injection volume 1 μL, flow rate 0.3 ml / min, column temperature 35℃, mobile phase A is acetonitrile, mobile phase B is 20 mmol / L ammonium formate-10% methanol system solution; Elution program: 0 min, mobile phase A is 5%, mobile phase B is 95%, 2 min, mobile phase A is 5%, mobile phase B is 95%, 8 min, mobile phase A is 22%, mobile phase B is 78%;

[0101] The mass spectrometry detection parameters were as follows: Thermo Scientific Vanquish UHPLC, Thermo Scientific TSQ Altis triple quadrupole mass spectrometer, electrospray ionization positive ion mode (ESI+), multiple reaction monitoring (MRM), Spray Voltage: Static; Positive Ion: 3500 V; Ion Transfer Tube Temp: 280℃; Vaporizer Temp: 280℃; Sheath Gas: 40 Arb; Sweep Gas: 1 Arb; The compound mass spectrometry acquisition parameters are as follows:

[0102]

[0103] (4) Compare the chromatograms of the reference solution and the extract of Pinellia ternata or processed medicinal slices. If the chromatogram of Pinellia ternata or processed medicinal slices shows a chromatographic peak with the same retention time as the chromatogram of the reference solution, it is determined that the Pinellia ternata or processed medicinal slices contains adulterated water-processed Pinellia ternata; otherwise, it is determined that the Pinellia ternata is adulterated anhydrous Pinellia ternata.

[0104] Among them, the peak area of ​​methylguanidine dipeptide increased with the increase of the proportion of water-infused Pinellia ternata, and showed a good linear relationship. 2 Not less than 0.9995.

[0105] Test case

[0106] The synthesis process of the methylguanidine dipeptide in this application is described in [link to application]. Figure 1 .

[0107] I. Taking the methylguanidine dipeptide prepared in Example 1 as an example for detection:

[0108] (1) Nuclear magnetic resonance detection

[0109] The NMR detection data are shown in Table 1 and Figure 2-6 ;

[0110] Table 1: Bruker Avance NEO 600M MRI scanner, DMSO-d6

[0111]

[0112] Figure 2 for 1 H NMR spectrum Figure 3 for 13 C NMR spectrum, Figure 4 for 1 H-1H COSY spectrum, Figure 5 for 1 H- 13 C HSQC spectrum, Figure 6 The above spectrum shows the HMBC spectrum, from which the specific structure of the methylguanidine dipeptide can be determined, with a purity of over 95%.

[0113] (2) High-resolution mass spectrometry, infrared spectroscopy detection and optical rotation measurement;

[0114] For detailed test results, please see [link / details]. Figure 7-8 ; Figure 7 This is a high-resolution mass spectrum. Figure 8 The infrared spectrum is shown, with IR vmax = 3323, 2957, 2874, 1645, 1459, 1399, 1214, 1160, 998, 824, 765, 708, 557, 427. These results further confirm the molecular formula of the methylguanidine dipeptide compound as C0. 12 H 24N4O3. Optical rotation: [α] 20 D +12.632 (c0.095,MeOH).

[0115] (3) Detection by reversed-phase high performance liquid chromatography-mass spectrometry (RP-HPLC-MS);

[0116] Instrument: Agilent 1200 HPLC + 6130MS

[0117] Column: Shimadzu InertSustain C18 4.6x150mm 5μm

[0118] Column temperature: 25℃

[0119] Mobile phase: Mobile phase A: purified water (0.05% TFA)

[0120] Mobile phase B: Acetonitrile (0.05% TFA)

[0121] Flow rate: 1.0 mL / min

[0122] Injection volume: 5 µl

[0123] Detector: λ = 203nm & 230nm

[0124] Mass spectrometry parameters: Drying Gas Flow: 12 L / min; Drying Gas Temp.: 350 °C;

[0125] Capillary Voltage: 2.5kV

[0126] Mode: Positive, Scan 100-1000mW

[0127] Elution method: gradient elution

[0128] Diluent: Methanol-Water (50:50)

[0129] Gradient condition:

[0130]

[0131] For detailed test results, please see [link / details]. Figure 9 , Figure 9 From top to bottom are: chromatogram at 204 nm using a UV detector, total ion chromatogram using a mass spectrometer, and mass spectrum of the corresponding chromatographic peaks, further confirming that the purity of the synthesized methylguanidine dipeptide can reach over 95%.

[0132] II. Exclusivity

[0133] Using the detection method in Example 4, raw Pinellia ternata, ginger-processed Pinellia ternata, processed Pinellia ternata, and purified Pinellia ternata were used to prepare negative drug solutions; raw water Pinellia ternata, ginger-processed water Pinellia ternata, processed water Pinellia ternata, and purified water Pinellia ternata were used to prepare positive drug solutions.

[0134] Preparation of methylguanidine dipeptide reference solution: Accurately weigh 16.99 mg of methylguanidine dipeptide reference standard into a 25 ml volumetric flask, dissolve and dilute to the mark with 50% ethanol, and shake well to obtain a reference solution with a concentration of 645.6 μg / ml ①. Accurately pipette 0.1 ml of reference standard ① into a 25 ml volumetric flask, dilute with 50% ethanol and dilute to the mark, and shake well to obtain a reference solution with a concentration of 2.5824 μg / ml ②. Then accurately measure 0.1 ml of reference solution ② into a 25 ml volumetric flask, dilute with 50% ethanol and dilute to the mark, and shake well to obtain a reference solution with a concentration of 0.01033 μg / ml ③.

[0135] The negative analyte solution, positive analyte solution, and control solution were tested separately. Specific test results are available in the [link to results]. Figure 10-18 .

[0136] Figure 10 This is the chromatogram of the reference standard, methylguanidine dipeptide. Figure 11 The chromatogram of raw Pinellia ternata. Figure 12 The chromatogram of Pinellia ternata is shown below. Figure 13 This is the chromatogram of Pinellia ternata. Figure 14 This is the chromatogram of Pinellia ternata. Figure 15 The chromatogram of raw Pinellia ternata. Figure 16 The chromatogram of ginger-infused Pinellia ternata is shown. Figure 17 The image shows the chromatogram of Pinellia ternata. Figure 18 The chromatogram of Pinellia ternata in clear water;

[0137] Figure 10-18 The results showed that at retention times of 7.8-8 min, no chromatographic peak corresponding to the reference standard methylguanidine dipeptide peak appeared in the chromatograms of raw Pinellia ternata, ginger-processed Pinellia ternata, processed Pinellia ternata, and clear Pinellia ternata. However, chromatographic peaks corresponding to the reference standard methylguanidine dipeptide appeared in the chromatograms of raw water Pinellia ternata, ginger-processed water Pinellia ternata, processed water Pinellia ternata, and clear water Pinellia ternata. This indicates that raw water Pinellia ternata, ginger-processed water Pinellia ternata, processed water Pinellia ternata, and clear water Pinellia ternata are significantly different from raw Pinellia ternata, ginger-processed Pinellia ternata, processed Pinellia ternata, and clear water Pinellia ternata. The Pinellia ternata matrix did not interfere with the determination results, and the specificity was good.

[0138] III. Matrix Effect

[0139] Preparation of solvent reference solutions: Accurately pipette 0.2 ml of reference solution ② (concentration 2.5824 μg / ml) into a 10 ml volumetric flask, dilute with 50% ethanol and bring to volume, and shake well to obtain an intermediate reference solution with a concentration of 51.6480 ng / ml; accurately pipette 4 ml, 0.4 ml, and 0.04 ml of the intermediate reference solution into 10 ml volumetric flasks, dilute with 50% ethanol and bring to volume, and shake well to obtain working solvent reference solutions with concentrations of 20.6592 ng / ml, 2.0659 ng / ml, and 0.2066 ng / ml, respectively.

[0140] Preparation of matrix reference working solutions: Accurately pipette 0.2 ml of reference solution ② (concentration 2.5824 μg / ml) into four 10 ml volumetric flasks. Take negative control solutions of raw Pinellia ternata, ginger-processed Pinellia ternata, prepared Pinellia ternata, and clear Pinellia ternata respectively, dilute and bring to the mark to obtain intermediate solutions for each matrix reference. Then accurately pipette 4 ml, 0.4 ml, and 0.04 ml of the intermediate solutions of matrix references into four 10 ml volumetric flasks respectively. Add the negative control solutions of each negative control to dilute and bring to the mark. Shake well to obtain matrix reference working solutions with concentrations of 0.2066 ng / ml, 2.0659 ng / ml, and 20.6592 ng / ml.

[0141] Take 1 μl of each of the three concentrations of reference standard working solution and matrix reference standard working solution and inject them into the liquid chromatography-mass spectrometer. Perform the determination according to the chromatographic-mass spectrometry conditions proposed in Example 4. The matrix effect of methylguanidine dipeptide in each matrix is ​​evaluated by the ratio (%) of the peak area of ​​the reference standard in the blank matrix solution to the peak area of ​​the reference standard in the pure solvent. The results are shown in Table 2.

[0142] Table 2: Results of matrix effect investigation (peak area ratio %)

[0143]

[0144] The results showed that when the concentrations of the methylguanidine dipeptide reference standard in the negative matrix solutions were 0.2066 ng / mL, 2.0659 ng / mL, and 20.6592 ng / mL, the matrix effect was within the range of 0.80-1.20, indicating that the matrix effect had virtually no impact on the detection results. Therefore, a solvent was selected to prepare the standard curve, and the external standard method was used for quantification.

[0145] IV. Examination of Linear Relationships

[0146] Accurately pipette 0.8 ml, 0.4 ml, and 0.2 ml of reference solution ② (concentration 2.5824 μg / ml) into 10 ml volumetric flasks, respectively. Dilute with 50% ethanol and bring to volume, then mix well to obtain reference solutions with concentrations of 206.5920 ng / ml (ST9), 103.2960 ng / ml (ST8), and 51.6480 ng / ml (ST7). Accurately pipette 1 ml each of reference solutions ST9, ST8, and ST7 into 10 ml volumetric flasks, respectively. Dilute with 50% ethanol and bring to volume, then mix well to obtain reference solutions with concentrations of 20.6592 ng / ml (ST6), 10.3296 ng / ml (ST5), and 5.1648 ng / ml (ST4). Accurately pipette 1 ml each of reference solutions ST6, ST5, and ST4 into 10 ml volumetric flasks, respectively. In a volumetric flask, dilute with 50% ethanol and bring to volume. Mix well to obtain reference solutions with concentrations of 2.0659 ng / ml (ST3), 1.0330 ng / ml (ST2), and 0.5165 ng / ml (ST1). Inject 1 μl of each ST1-ST9 solution into a liquid chromatograph-mass spectrometer and determine the concentrations under the prescribed chromatographic-mass spectrometric conditions. Plot a standard curve with concentration (ng / ml) on the x-axis and peak area on the y-axis. The results are shown in Table 3 and [see attached table]. Figure 19 .

[0147] Table 3: Results of Linear Range Examination

[0148]

[0149] The results showed that the regression equation for the control was y = -27.657 + 2135.573x (R²). 2 =0.9996), with a linear range of 0.5165 ng / ml to 206.5920 ng / ml.

[0150] V. Precision Examination

[0151] Accurately pipette 1 μl of the reference solution ST4 (concentration 5.1648 ng / ml), inject it 6 times consecutively, and perform the determination according to the planned chromatographic-mass spectrometry conditions. The results are shown in Table 4.

[0152] Table 4: Precision test results (peak area)

[0153]

[0154] The results showed good precision.

[0155] VI. Repeated Examination

[0156] Take raw water Pinellia ternata, ginger water Pinellia ternata, processed water Pinellia ternata, and clear water Pinellia ternata powder, and prepare 6 parallel test solutions for each according to the proposed sample pretreatment method. Analyze the samples according to the proposed chromatographic-mass spectrometry conditions. The results are shown in Table 5.

[0157] Table 5: Results of repeatability testing (content μg / g)

[0158]

[0159] The results showed that the method had good reproducibility.

[0160] VII. Stability Test

[0161] According to the proposed detection method, the reference standard ST4 and the test solutions of raw water Pinellia ternata, ginger water Pinellia ternata, processed water Pinellia ternata, and clear water Pinellia ternata were taken and stored at room temperature. The samples were injected at 0 h, 4 h, 8 h, 12 h, 16 h, and 24 h to evaluate the stability of the reference standard solution and the test solution during the detection process. The results are shown in Table 6.

[0162] Table 6: Stability test results (peak area)

[0163]

[0164] The results showed that the reference solution and the test solutions of raw Pinellia ternata, ginger-infused Pinellia ternata, processed Pinellia ternata, and purified Pinellia ternata had good stability within 24 hours.

[0165] 8. Accuracy (Recovery Rate) Examination

[0166] Nine portions of each of the following powders (passed through a No. 3 sieve): raw Pinellia ternata, ginger-infused Pinellia ternata, processed Pinellia ternata, and clear Pinellia ternata powder were accurately weighed. Based on the results of the repeatability test, three different concentrations of reference standards were added to the sample at ratios of 0.5:1, 1:1, and 1.5:1 to the amount of the analyte in the sample. Three parallel solutions of each concentration were prepared and the samples were analyzed under the proposed chromatographic-mass spectrometry conditions. The results are shown in Table 7-10.

[0167] Table 7: Results of Accuracy Study of Raw Pinellia ternata

[0168]

[0169] Table 8: Results of Accuracy Assessment of Ginger-infused Pinellia

[0170]

[0171] Table 9: Results of Accuracy Assessment of Pinellia ternata

[0172]

[0173] Table 10: Results of Accuracy Assessment of Qingshui Banxia

[0174]

[0175] The results showed that the method had good accuracy (recovery rate).

[0176] IX. Limit of Detection (LOD) and Limit of Quantification (LOQ)

[0177] Take 1 μl of a 0.1033 ng / mL reference standard solution and accurately pipette it. Perform the determination under the planned chromatographic-mass spectrometry conditions. The signal-to-noise ratio (S / N) is 42.1. Calculate the method detection limit and quantitation limit using the following formulas:

[0178] LOD (ng / g) = Standard concentration (ng / ml) × Sample dilution factor (20) × 3 / SN / Sample volume (0.5 g)

[0179] LOQ (ng / g) = Standard concentration (ng / ml) × Sample dilution factor (20) × 10 / SN / Sample volume (0.5 g)

[0180] The method detection limit is 0.3 ng / g, and the quantitation limit is 1 ng / g.

[0181] 10. Proportion of adulterated Pinellia ternata with water

[0182] To avoid false positives or misjudgments, the adulteration of Pinellia ternata with different proportions of water-processed Pinellia ternata was investigated. Different processed Pinellia ternata with different water proportions (3%, 5%, 10%, 20%, 50%, and 100%) were mixed evenly with the corresponding processed Pinellia ternata to obtain adulterated samples. A series of test solutions with different proportions of water-processed Pinellia ternata were prepared according to step (2) of Example 4. 1 μl of each solution was precisely pipetted and analyzed under the proposed chromatographic-mass spectrometry conditions. The results of the adulteration of each processed Pinellia ternata with the corresponding processed Pinellia ternata are shown in Tables 11-14. Figure 20-23 .

[0183] Table 11: Results of linear relationship investigation of different proportions of raw Pinellia ternata mixed with raw Pinellia ternata

[0184]

[0185] Table 12: Results of linear relationship investigation of different proportions of ginger-infused Pinellia ternata with ginger-infused Pinellia ternata

[0186]

[0187] Table 13: Results of linear relationship investigation of Pinellia ternata with different proportions in the water-soluble Pinellia ternata addition method

[0188]

[0189] Table 14: Results of linear relationship investigation of different proportions of Pinellia ternata mixed with Pinellia ternata.

[0190]

[0191] The results showed that in different processed products, the peak area of ​​methylguanidine dipeptide increased with the increase of the proportion of water-processed Pinellia ternata, and showed a good linear relationship. The linear regression equations R0... 2 Not less than 0.9995.

Claims

1. A methylguanidine dipeptide compound, characterized in that, Its chemical structural formula is as follows: 。 2. The method for synthesizing the methylguanidine dipeptide compound according to claim 1, characterized in that, Includes the following steps: (1) Dissolve L-ornithine hydrochloride and 1,2-dimethyl-2-isothiourea hydroiodide in an alkaline solution and heat at 70-110℃ overnight; (2) Adjust the pH of the solution to neutral, purify, then add water to dissolve, and adjust the pH of the solution to acidic, pass through ion exchange resin, rinse with pure water first, then wash with ammonia water, and freeze dry to obtain intermediate 1; (3) Add pure water and isopropanol to intermediate 1, and add alkaline solution to adjust the pH of the solution to be alkaline; (4) Add isovaleryl chloride to the solution and stir at room temperature for 2-4 hours; (5) Keep the pH of the solution alkaline, heat to 45-55℃, then adjust the pH of the solution to weakly acidic, cool to 5-15℃, collect the precipitate, and obtain the crude compound. (6) The crude compound was dissolved in a methanol-water mixture, then eluted by gradient liquid chromatography and dried to obtain the product.

3. The method for synthesizing the methylguanidine dipeptide compound as described in claim 2, characterized in that, In step (1), the molar ratio of L-ornithine hydrochloride and 1,2-dimethyl-2-isothiourea hydroiodide is 1:3-5; the alkaline solution is a 1M sodium hydroxide solution.

4. The method for synthesizing the methylguanidine dipeptide compound as described in claim 2, characterized in that, In step (2), dilute hydrochloric acid is used to adjust the pH of the solution; the purification process is as follows: after evaporating the solvent, methanol is added to dissolve it, the impurities are removed by filtration, and the methanol solvent is recovered; the pH of the solution is adjusted to 3-5; the concentration of ammonia is 0.2M.

5. The method for synthesizing the methylguanidine dipeptide compound as described in claim 2, characterized in that, In step (3), the ratio of intermediate 1, pure water and isopropanol is 9-11 mmol: 7-9 ml: 24-26 ml; sodium hydroxide solution is added to adjust the pH of the solution to 10-12.

6. The method for synthesizing the methylguanidine dipeptide compound as described in claim 2, characterized in that, In step (4), the molar ratio of isovaleryl chloride to intermediate 1 is 14-16:

1.

7. The method for synthesizing the methylguanidine dipeptide compound as described in claim 2, characterized in that, In step (5), after heating, concentrated hydrochloric acid is used to adjust the pH of the solution to 5-6; in step (6), the volume ratio of methanol to water in the methanol-water mixed solution is 1-2:1-2.

8. The method for synthesizing the methylguanidine dipeptide compound as described in claim 2, characterized in that, In step (6), during elution, the chromatographic column was a Waters Xbridge 19x250mm 10μm, mobile phase A was purified water, mobile phase B was acetonitrile, and the flow rate was 25ml / min. The gradient elution conditions were: 0 min, 95% of phase A and 5% of phase B; 1.5 min, 95% of phase A and 5% of phase B; 9.5 min, 80% of phase A and 20% of phase B. 9.7 min, Phase A 5%, Phase B 95%; 12.7 min, Phase A 5%, Phase B 95%; 13 min, Phase A 95%, Phase B 5%; 16 min, Phase A 95%, Phase B 5%.

9. The use of the methylguanidine dipeptide compound described in claim 1 as a reference standard in identifying adulterated Pinellia ternata and processed Pinellia ternata medicinal materials and slices.

10. The application of the methylguanidine dipeptide compound as described in claim 9 as a reference standard in identifying adulterated Pinellia ternata (Pinellia ternata) medicinal materials and processed slices, characterized in that, The specific testing methods are as follows: (1) Using methylguanidine dipeptide as a reference standard and a water-water-soluble organic solvent mixture as a solvent, a reference standard solution was prepared; (2) Crush the Pinellia ternata medicinal material or decoction pieces, sieve them, add water-water-soluble organic solvent mixture solution, extract by ultrasonication, filter, and obtain Pinellia ternata extract; (3) Chromatography-mass spectrometry was performed on the reference solution in step (1) and the Pinellia ternata extract in step (2), respectively; High performance liquid chromatography conditions: Column: Thermo Hypersil GOLD aQ C 18 Chromatographic column; injection volume: 1 μl; flow rate: 0.3 mL / min; column temperature: 35 °C; mobile phase A: acetonitrile; mobile phase B: 20 ​​mmol / L ammonium formate-10% methanol solution; elution program: 0 min, mobile phase A 5%, mobile phase B 95%; 2 min, mobile phase A 5%, mobile phase B 95%; 8 min, mobile phase A 22%, mobile phase B 78%. Mass spectrometry conditions: Triple quadrupole mass spectrometer, electrospray ionization (ESI) positive ion mode, multiple reaction monitoring (MRM); spray voltage: static; positive ion mode: 3500 V; ion transfer tube temperature: 280℃; nebulizer temperature: 280℃; sheath gas: 40 Arb; purge gas: 1 Arb; methylguanidine dipeptide mass spectrometry acquisition parameters: (4) Compare the chromatograms of the reference solution and the extract of Pinellia ternata or processed medicinal slices. If the chromatogram of Pinellia ternata or processed medicinal slices shows a chromatographic peak with the same retention time as the chromatogram of the reference solution, it is determined that the Pinellia ternata or processed medicinal slices contains adulterated water-processed Pinellia ternata; otherwise, it is determined that the Pinellia ternata is adulterated anhydrous Pinellia ternata. Among them, the peak area of ​​methylguanidine dipeptide increased with the increase of the proportion of water-infused Pinellia ternata, and showed a good linear relationship. 2 Not less than 0.99.

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