A kind of kirenol derivative, its preparation method and application

Abstract

The present invention discloses a kirenol derivative, a preparation method and an application thereof. The derivative can be used to identify three original plants of Siegesbeckia orientalis L. Siegesbeckia orientalis L. does not contain any of the above kirenol derivatives; Siegesbeckia pubescens Makino contains 16-O-malonyl kirenol (Compound 1) and 15-O-malonyl kirenol (Compound 2); Siegesbeckia virgata Candolle contains 16-O-malonyl kirenol (Compound 1), 15-O-malonyl kirenol (Compound 2) and 15,16-di-O-malonyl kirenol (Compound 3). By comparing the presence or absence of three kirenol derivatives, rapid and accurate identification of three original plants of Siegesbeckia orientalis L. can be successfully achieved.

Description

Technical Field

[0001] The present invention relates to the field of Chinese medicine chemistry, and particularly relates to a kirenol derivative, a preparation method thereof, and the use of this derivative for identifying three original plants of Siegesbeckiae Herba. Background Art

[0002] Siegesbeckiae Herba is a commonly used Chinese medicine, first recorded in the Newly Revised Materia Medica in the Tang Dynasty. The 1963 edition of the Chinese Pharmacopoeia only included the aerial parts of Siegesbeckia pubescens Makino, but the pharmacopoeias from 1977 onwards expanded the medicinal sources to include Siegesbeckia orientalis L. and Siegesbeckia glabrescens Makino as the original plants of Siegesbeckiae Herba. Currently, the Chinese medicine Siegesbeckiae Herba used clinically includes the dried aerial parts of these three plants. Siegesbeckiae Herba has the effects of dispelling wind-dampness, strengthening tendons and bones, promoting joint movement, and detoxifying, and is mainly used to treat rheumatic arthralgia, weakness of tendons and bones, soreness and weakness of the waist and knees, limb paralysis, hemiplegia, rubella and damp sores, etc.

[0003] The three original plants of Siegesbeckiae Herba are widely distributed throughout China. It is very difficult to distinguish the appearance traits of the decoction pieces of the three original plants of Siegesbeckiae Herba, and it is necessary to combine microscopic characteristics and plant morphology for identification. Currently, the medicinal materials are sourced from the wild, and commercial Siegesbeckiae Herba often consists of a mixture of several original plants. Different original plants are one of the main factors affecting the quality of medicinal materials and Chinese medicine preparations. The phenomenon that the Chinese Pharmacopoeia includes different original plants of the same variety has attracted great attention. The current "Classification and Requirements for Submission of Chinese Medicine Registration Materials" requires the clarification of the original plants of the Chinese medicinal materials in the prescription to ensure the quality and stability of the preparations. The number of Chinese medicine preparations composed of Siegesbeckiae Herba has increased significantly, and the research on the basic substances of its original plant pharmacodynamic effects has become even more urgent.

[0004] In the Chinese Pharmacopoeia, the quality standards of Siegesbeckiae Herba and its formulated preparations, Xitong Pills and Xitong Capsules, respectively use thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) to identify and determine the content of kirenol as the main quality control measures. Although some literature has used the HPLC fingerprint method and in vitro anti-inflammatory model to analyze and compare the chemical composition differences and in vitro anti-inflammatory activities of the methanol extracts of the three original plants of Siegesbeckiae Herba, the fingerprint results only show that there are significant differences in the chemical compositions of the three original plants, and no differential components of the three original plants of Siegesbeckiae Herba have been found. Summary of the Invention

[0005] In order to solve the problems existing in the identification of the three original plants of Siegesbeckiae Herba, the present invention provides a kirenol derivative, a preparation method thereof, and an application, and uses this kirenol derivative to identify the three original plants of Siegesbeckiae Herba.

[0006] The kirenol derivative for identifying the three original plants of Siegesbeckiae Herba provided is of the structure of Formula I:

[0007]

[0008] Among them, for Compound 1, the substituent R1 is a hydrogen atom, and the substituent R2 is a malonyl group;

[0009] For Compound 2, the substituent R1 is a malonyl group, and the substituent R2 is a hydrogen atom;

[0010] For Compound 3, the substituent R1 is a malonyl group, and the substituent R2 is a malonyl group;

[0011] Among them, Compound 1 is 16-O-malonyl kirenol, Compound 2 is 15-O-malonyl kirenol, and Compound 3 is 15,16-di-O-malonyl kirenol, and Compound 3 is a new compound.

[0012] A preparation method of a kirenol derivative of Formula I structure includes:

[0013] (1) Preparation of Compounds 1 and 2: Siegesbeckia pubescens is extracted with water and concentrated to obtain an aqueous extract. The extract is separated by a D101 macroporous adsorption resin column and eluted with an ethanol-water system in a gradient manner. The 50% ethanol elution fraction is collected and loaded onto a polyamide column, eluted with water. The water elution fraction is separated by silica gel column chromatography (dichloromethane-methanol system) to obtain three parts A, B, and C in sequence. Part C is chromatographed on a C-18 reversed-phase silica gel column with a methanol-water system in a gradient manner, and further chromatographed on a polyamide column, eluted with water and freeze-dried to obtain a mixture of Compounds 1 and 2. The mixture of Compounds 1 and 2 is prepared by high-performance liquid column, and the eluents containing Compounds 1 and 2 are collected using SPE cartridges respectively, dried with nitrogen, and eluted with acetonitrile to obtain Compounds 1 and 2.

[0014] (2) Preparation of Compound 3: Siegesbeckia virgaurea is crushed and percolated with 60% ethanol (600 L) at room temperature. The ethanol extract is concentrated under reduced pressure to obtain an ethanol extract. The extract is loaded onto a macroporous adsorption resin column and eluted successively with water, 30%, 50%, and 75% ethanol. The 75% ethanol elution fraction is collected, loaded onto a silica gel column, eluted with dichloromethane-methanol (4 - 6:1, further preferably 5:1, v / v), and then chromatographed on a C-18 reversed-phase silica gel column, eluted with a formic acid aqueous solution (0.05 - 0.3%, further preferably 0.1%)-acetonitrile (4:1) to obtain Compound 3.

[0015] The kirenol derivative obtained according to the above preparation method can be used to identify three original plants of Siegesbeckiae Herba, and the specific steps include:

[0016] (1) Preparation of reference solution: Accurately weigh 2 mg of the mixture of 16 - O - malonyl kirenol and 15 - O - malonyl kirenol, and 1 mg of 15,16 - di - O - malonyl kirenol. Dissolve them with ultrapure water and prepare standard solutions with a concentration of 1 mg / mL respectively, thus obtaining the reference solutions.

[0017] (2) Preparation of test solution: Take 500 mg of Siegesbeckia herb powder (passed through 40 - mesh sieve) in a volumetric flask, accurately measure 10 mL of 70% methanol into the volumetric flask, sonicate for 60 min (power 250 W, frequency 35 kHz), shake well, and let it stand to room temperature. Take an appropriate amount and transfer it to a centrifuge tube, centrifuge for 10 min (rotation speed 12000 rpm), and take the supernatant, thus obtaining the test solution.

[0018] (3) Chromatographic conditions: Chromatographic column: ACQUITY UPLC@HSS T3 column (1.8 μm, 2.1×100 mm); column temperature 30 °C; mobile phase: A: 0.1% formic acid aqueous solution, B: acetonitrile; gradient elution program is shown in Table 4, mobile phase flow rate 0.5 mL / min, injection volume 3 μL.

[0019] Table 4: UPLC - QTOF - MS gradient elution conditions

[0020]

[0021] (4) Results:

[0022] None of the above three kirenol derivatives were detected in the 13 batches of Siegesbeckia samples; 16 - O - malonyl kirenol (Compound 1) and 15 - O - malonyl kirenol (Compound 2) were detected in all 6 batches of Siegesbeckia pubescens; 16 - O - malonyl kirenol (Compound 1), 15 - O - malonyl kirenol (Compound 2) and 15,16 - di - O - malonyl kirenol (Compound 3) were detected in all 7 batches of Siegesbeckia virgata. Thus, it can be seen that by comparing the presence or absence of the three kirenol derivatives, rapid and accurate identification of the three original plants of Siegesbeckia herb can be successfully achieved.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] It overcomes the identification problems of the three original plants of Siegesbeckia herb and provides a preparation method of kirenol derivatives for identifying the three original plants of Siegesbeckia herb. Description of the drawings

[0025] Figure 1 For the structures of the three kirenol derivatives;

[0026] Figure 2 For the TIC diagrams of the three kirenol derivatives;

[0027] Figure 3 TIC chromatograms of 13 batches of Siegesbeckia orientalis samples (without three kirenol derivatives);

[0028] Figure 4 TIC chromatograms of 7 batches of Siegesbeckia pubescens samples (compounds 1 and 2 present);

[0029] Figure 5 TIC chromatograms of 6 batches of Siegesbeckia virgata samples (compounds 1, 2 and 3 present). Detailed implementation mode

[0030] The following further illustrates the substantial content of the present invention in conjunction with examples, but the content of the present invention is not limited thereto.

[0031] Example 1: Preparation and structural identification of compounds 1 and 2

[0032] 1. Preparation of compounds 1 and 2: Siegesbeckia pubescens (15 kg) was extracted three times with 10 times the amount of water for 1 hour each time. The water extract was concentrated to obtain 2.24 kg of water extract paste, which was separated by D101 macroporous adsorption resin column chromatography and eluted with an ethanol-water system (0, 30%, 50%, 70% ethanol) to obtain 4 fractions: D water, D30, D50, and D70. D50 (92.5 g) was separated by polyamide column chromatography and eluted with water to obtain a total of 4 fractions. The first fraction was separated by silica gel column chromatography (dichloromethane-methanol system) to obtain three fractions: A, B, and C. Fraction C was further separated by C-18 column chromatography with a methanol-water system and a 10%-100% gradient elution, and then by polyamide column chromatography, eluted with water and freeze-dried to obtain a mixture of 16-O-malonyl kirenol and 15-O-malonyl kirenol (209 mg). The mixture of compounds 1 and 2 was prepared on a high-performance liquid column (Kromasil 100-5C18, 250*4.6 mm, E72710), with 23% acetonitrile: 0.1% formic acid aqueous solution = 23:77 as the mobile phase. The eluates containing compounds 1 and 2 were collected separately using SPE cartridges, dried with nitrogen, and eluted with acetonitrile to obtain compounds 1 and 2.

[0033] 2. Structural identification of compounds 1 and 2:

[0034] 2.1 Compound 1: HR-ESI-MS: 423.2397 ([C 23 H 36 O7-H] - ; calculated value 423.2383). 13 C NMR and 1The 1H NMR data are shown in Table 1. Compared with the NMR signals of kamebakaurinol in the literature [Liu K, Roder E. Diterpene aus Siegesbeckia glabrescens. Planta Med. 1991; 57: 395 - 396.], compound 1 exhibited a set of malonyl signals [δ C 167.22 (s), 41.05 (t), 167.89 (s), and δ H 3.38 (2H, s)], and high-resolution mass spectrometry also showed the presence of malonyl with m / z 337.2388 [M - H - malonyl] - . Compared with the NMR data of kamebakaurinol, the signals of C-16 and H-16 in compound 1 shifted downfield, and in the HMBC spectrum, correlations were observed between δ H 4.05 and 4.28 (each 1H, H-16) and the signal of C1' of malonyl (δ C 167.22), indicating that the malonyl group was linked to the C-16 hydroxyl group of the parent nucleus. Therefore, the structure of compound 1 was identified as 16-O-malonyl kamebakaurinol ( Figure 1 ).

[0035] Table 1: 1H NMR 1 and a 13C NMR 13 data (δ in ppm, J in Hz, CD3CN). b 600 MHz.

[0036]

[0037] a 150 MHz. b

[0038] 2.2 Compound 2: HR-ESI-MS: 423.2395 ([C 23 H 36 O7H] - ; calcd 423.2383). 13 13C NMR and 1 1H NMR data are shown in Table 2. Compared with the NMR signals of kamebakaurinol in the literature [Liu K, Roder E. Diterpene aus Siegesbeckia glabrescens. Planta Med. 1991; 57: 395 - 396.], compound 2 exhibited a set of malonyl signals [δ C167.15(s),41.20(t),168.08(s),andδ H 3.44 (2H, s)], and high-resolution mass spectrometry also showed the presence of malonyl m / z 337.2385 [MH-malonyl] - Compared with the NMR data of nonanol, the signals of C-15 and H-15 of compound 2 shifted to the downfield, and δ H 5.00 (H-15) and C1' (δ C 167.15) signal, indicating that the malonic acid monoacyl group is linked to the C-15 hydroxyl group of the parent nucleus. Therefore, the structure of compound 2 can be identified as 15-O-malonic acid monoacyl nonanol ( Figure 1 ).

[0039] Table 2: Compound 2 1 H NMR a and 13 C NMR b Data (δ in ppm, J in Hz, CD3CN).

[0040]

[0041] a 600 MHz. b 150MHz.

[0042] Example 2: Preparation and structural identification of compound 3

[0043] 1. Preparation of compound 3:

[0044] 30.0 kg of the medicinal material of Siegesbeckia pubescens was crushed and extracted by filtration with 60% ethanol (600 L) at room temperature. The ethanol extract was concentrated under reduced pressure to obtain an alcohol extract, which was applied to a macroporous adsorption resin column and eluted with water, 30%, 50% and 75% ethanol in sequence to separate it into AD components. Under UPLC-ESI-QTOF-MS analysis, component B (50.0 g) was analyzed by uplc on a silica gel column (200-300 mesh; dichloromethane-methanol = 5:1, v / v) to obtain subcomponents B1-B2. B2 was eluted with a 0.1% formic acid aqueous solution-acetonitrile system (4:1) through a C-18 reverse phase silica gel column, concentrated under reduced pressure, and freeze-dried to obtain compound 3.

[0045] 2. Structural identification of compound 3:

[0046] Compound 3: white amorphous powder, HR-ESI-MS: 509.2386 ([C 26 H 38 O 10H] - ; Calculated value 509.2387), melting point 102 - 106 °C, UV(MeOH) λ max = 202.6 (logε = 4.11). IR(KBr) 2943, 1716, 1320, 1202, 1153, 1019, 966 cm -1 . 13 C NMR and 1 H NMR data are shown in Table 3. Compared with the nuclear magnetic resonance signals of kamebakaurinol in the literature [Liu K, Roder E. Diterpene aus Siegesbeckia glabrescens. Planta Med. 1991; 57: 395 - 396.], compound 3 has two malonyl groups [δ C 167.31 (s), 41.26 (t), 167.81 (s), δ H 3.33 (2H, d, J = 2.5 Hz), and δ C 167.33 (s), 41.51 (t), 168.07 (s), δ H 3.41 (2H, d, J = 4.5 Hz)]. High - resolution mass spectrometry also shows the presence of fragment ion peaks at m / z 423.2692 [M - H - malonyl] - , 337.2384 [M - H - 2malonyl] - , further indicating the presence of two malonyl groups. Compared with the nuclear magnetic data of kamebakaurinol, the chemical shifts of H - 15 and H - 16 of compound 3 shift downfield, and in the HMBC spectrum, correlations can be seen between δ H 5.20 (H - 15) and δ C 167.31 (C1' of one of the malonyl groups); δ H 4.24 and 4.39 (each 1H, H - 16) and δ C 167.33 (C1" of the other malonyl group), indicating that these two malonyl groups are respectively attached to the C - 15 and C - 16 hydroxyl groups of compound 3. Therefore, the structure of compound 3 can be identified as 15,16 - di - O - malonyl kamebakaurinol ( Figure 1 ).

[0047] Table 3: 1 H NMR a and 13 C NMR b data (δ in ppm, J in Hz, CD3CN).

[0048]

[0049]

[0050] a 500 MHz. b 125MHz.

[0051] Example 3: Identification of Three Original Plants of Siegesbeckia orientalis L.

[0052] The three original plants of Siegesbeckia orientalis L. were identified by whether the above three kirenol derivatives peaks were contained in the UPLC-QTOF-MS chromatogram.

[0053] 1. Instrument and Equipment

[0054] Liquid chromatography: Waters ACQUITY UPLC system; Mass spectrometry: Waters SYNAPT G2 QTOF system.

[0055] 2. Preparation of Reference Substance Solution: Accurately weigh 2 mg of the mixture of 16-O-malonyl kirenol and 15-O-malonyl kirenol, and 1 mg of 15,16-di-O-malonyl kirenol, dissolve with ultrapure water, and prepare standard substance solutions with a concentration of 1 mg / mL respectively, then it is obtained.

[0056] 3. Preparation of Test Solution: Take 500 mg of Siegesbeckia orientalis L. medicinal powder (passed through 40-mesh sieve) in a volumetric flask, accurately measure 10 mL of 70% methanol in the volumetric flask, ultrasonicate for 60 min (power 250 W, frequency 35 kHz), shake well, and let stand to room temperature. Take an appropriate amount and put it into a centrifuge tube, centrifuge for 10 min (rotation speed 12,000 rpm), take the supernatant, then it is obtained.

[0057] 4. Chromatography and Mass Spectrometry Conditions: Chromatographic column: ACQUITY UPLC@HSS T3 column (1.8 μm, 2.1×100 mm); Column temperature 30 °C; Mobile phase: A: 0.1% formic acid aqueous solution, B: acetonitrile; Gradient elution program is shown in Table 4, mobile phase flow rate 0.5 mL / min, injection volume 3 μL. Electrospray ionization source (ESI), negative ion mode, capillary voltage -3.0 kV; Source temperature 120 °C, drying gas flow rate: nitrogen, 500 L / h; Drying gas temperature 350 °C; Collision gas flow rate: argon, 50 L / h; Cone orifice voltage 30 V.

[0058] Table 4: UPLC-QTOF-MS Gradient Elution Conditions

[0059]

[0060] 5. Results

[0061] As Figure 2As shown, peak 1: 16-O-malonyl kirenol, t R = 19.84 min, compound; (peak 2: 15-O-malonyl kirenol, t R = 19.98 min, compound 2; peak 3: 15,16-di-O-malonyl kirenol, t R = 21.88 min, compound 3.

[0062] As Figure 2 , 3 , 4, and 5 show, none of the 13 batches of Siegesbeckia orientalis contain the above three kirenol derivatives; all 6 batches of Siegesbeckia pubescens contain 16-O-malonyl kirenol (compound 1) and 15-O-malonyl kirenol (compound 2); all 7 batches of Siegesbeckia virgata contain 16-O-malonyl kirenol (compound 1), 15-O-malonyl kirenol (compound 2), and 15,16-di-O-malonyl kirenol (compound 3). Thus, it can be seen that by comparing the presence or absence of the three kirenol derivatives, rapid and accurate identification of the three original plants of Siegesbeckiae Herba can be successfully achieved.

Claims

1. A method for preparing a kirenol derivative, characterized in that, The following steps are involved: (1) Preparation of compounds 1 and 2: Extracting Siegesbeckia sibiricum with water, concentrating to obtain a water extract, separating through a macroporous adsorption resin column, eluting with an ethanol-water system gradient, collecting the 45-55% volume percent ethanol elution part onto a polyamide column, eluting with water, and separating the water elution part through a silica gel column chromatography to obtain three parts A, B and C in sequence; Part C was chromatographed on a C-18 reverse phase silica gel column using a methanol-water system gradient elution, and further chromatographed on a polyamide column, eluted with water and freeze-dried to obtain a mixture of compounds 1 and 2; The mixture of compounds 1 and 2 was prepared on a high performance liquid column, and the eluates containing compounds 1 and 2 were collected respectively, dried with nitrogen, and eluted with acetonitrile to obtain compounds 1 and 2; (2) Preparation of compound 3: The herb of Siegesbeckia pubescens was crushed, and then extracted by filtration with an ethanol aqueous solution of 55% to 70% by volume. The ethanol extract was concentrated under reduced pressure to obtain an alcohol extract, which was applied to an adsorption resin column and eluted with water and ethanol of different volume percentages in sequence. The 75% ethanol elution portion was collected and applied to a silica gel column, eluted with dichloromethane-methanol, and then passed through a C-18 reverse phase silica gel column, and eluted with an aqueous formic acid solution-acetonitrile to obtain compound 3; The compounds 1, 2 and 3 are nonanol derivatives of formula I: Wherein, the substituent R1 of compound 1 is a hydrogen atom, and the substituent R2 is a malonic acid monoacyl group; In compound 2, the substituent R1 is a malonic acid monoacyl group, and the substituent R2 is a hydrogen atom; In compound 3, the substituent R1 is a malonic acid monoacyl group, and the substituent R2 is a malonic acid monoacyl group.

2. The method for preparing kirenol derivatives according to claim 1, characterized in that, In step (1), silica gel column chromatography separation adopts a dichloromethane-methanol system.

3. The method for preparing the kirenol derivative according to claim 1, wherein In step (2), the volume ratio of dichloromethane to methanol in dichloromethane-methanol is 4 to 6:

1.

4. The method for preparing kirenol derivatives according to claim 1, characterized in that, In step (2), the mass percentage of the formic acid aqueous solution is 0.05-0.3%.

5. Use of kirenol derivatives in the identification of three original plants of Siegesbeckia orientalis L., characterized in that, The nonanol derivative is a structure of Formula I: Wherein, the substituent R1 of compound 1 is a hydrogen atom, and the substituent R2 is a malonic acid monoacyl group; In compound 2, the substituent R1 is a malonic acid monoacyl group, and the substituent R2 is a hydrogen atom; In compound 3, the substituent R1 is a malonic acid monoacyl group, and the substituent R2 is a malonic acid monoacyl group.

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