Method for rapidly separating high-purity cyclobuxine D and cyclovirobuxine D

Abstract

The invention discloses a method for rapidly separating high-purity cyclobuxine D and cyclovirobuxine D. The invention relates to the technical field of natural medicine extraction and separation, and discloses a method for simultaneously obtaining high-purity cyclobuxine D and cyclovirobuxine D at one time by selecting a boxaceae plant extract and utilizing a silver ion-silica gel column chromatography method for separation. The method is simple and easy to implement, is convenient to operate, saves time and cost, has better reproducibility, and can realize industrial large-scale production.

Description

Technical Field

[0001] The invention relates to the technical field of natural drug extraction and separation, and in particular to a method for rapidly separating and obtaining high-purity cyclobuxine D and cyclovibrobuxine D. Background Art

[0002] Buxus Buxus ) is an important plant in the Buxaceae family. There are more than 70 species in the world, about 17 species and 11 varieties in China, including Buxus sempervirens , B.papillos a, B.microphylla , B． hildebrandtii , B.hyrcana etc. As this genus of plants is rich in alkaloids, they are usually called Buxus alkaloids. Foreign scholars generally call this type of compound steroidal alkaloids. Buxus alkaloids are generally derivatives of 4,4,14-trimethylpregnane. From the biological source, this type of alkaloid should be derived from the tetracyclic triterpene nucleus. Therefore, Buxus alkaloids are also regarded as terpene alkaloids. Many Buxus plants are famous Chinese herbal medicines. For example, Huangyangning Tablets and Guan'an Tablets for the treatment of cardiovascular diseases were developed from Buxus.

[0003] Huangyangning Tablets are a drug independently developed by my country in the 1980s for the treatment of cardiovascular diseases. The main ingredient of Huangyangning Tablets is cyclovirobuxine D. Among the raw materials of Huangyangning Tablets currently produced by the process, cyclovirobuxine D is the alkaloid with the highest content besides cyclovirobuxine D.

[0004] Among them, cyclobuxine D, English name: Cyclobuxine D, molecular formula: C 25 H 42 N2O, CAS No.: 2241-90-9. Cyclovirobuxin D, English name: Cyclovirobuxin D, molecular formula: C 26 H 46 N2O, CAS number: 860-79-7. Figure 1 As shown, cycloboxine D and cycloviboxine D have the same parent nucleus, and their structures and physicochemical properties are extremely similar. The only difference is the dimethyl group at the 4th position (cycloviboxine D) or the double bond outside the ring at the 4th position (cycloboxine D).

[0005] In the existing separation technology, there are many separation cases for cyclovirobuxine D or cycloubuxine D, for example: In the Chinese patent authorization number CN 112521440 A "A method for purifying cyclobuxine D", a sample with a cyclobuxine D content of 15% is purified to 98.5% by accumulation, column chromatography purification and recrystallization. The specific extraction method refers to the patent authorization number CN 1182151 C to obtain Huangyangning, and then remove cyclovihuangbuxine D through multiple methanol recrystallizations to accumulate cyclobuxine D crude products. The crude cyclobuxine D is subjected to silica gel column chromatography (the mobile phase is methyl chloride: methanol: ammonia water = 70~20:1:1.05~0.2) to obtain cyclobuxine D purified solid, and then recrystallized with a mixed solvent of methyl chloride and acetonitrile to obtain cyclobuxine D with a purity of ≥98%.

[0006] Chinese patent authorization number CN 1319984 C "Method for purifying cyclovibrobuxine D" purifies cyclovibrobuxine D with a content of about 80% in the raw material drug of Huangyangning to 90% by alumina column chromatography; Chinese patent publication number CN 102993260 A "A new method for purifying cyclovibrobuxine D" uses a molecular imprinting stationary phase on the surface of mesoporous silica as a carrier to purify cyclovibrobuxine D in the raw material drug to 90%; Chinese patent publication number CN 1461751 A "Preparation of cyclovibrobuxine D and its dispersion preparation" uses a weak acid solution to extract the crude powder of small-leaved boxwood and its cognate plants, concentrates the filtrate to recover ethanol, dissolves the extract in water, adjusts the pH to 9 with ammonia water, extracts with chloroform, and then purifies it by column chromatography (chloroform: methanol = 9:1) to obtain cyclovibrobuxine D with a purity of more than 99.99%; Chinese patent authorization number CN 1064869 A "Separation and extraction process of cyclovibrobuxine and its tablets" and CN 1182151 C "Purification method of cyclovibrobuxine D and its preparation" Wet boxwood with ethanol and ammonia water, pH is alkaline, filter and remove the soaking liquid. Soak the filter residue in cyclohexane for extraction, concentrate and crystallize, and then wash with acetone to obtain boxwood (containing about 40% to 70% cyclovibrobuxine D). Boxwood is subjected to silica gel column chromatography (mobile phase is chloroform: n-hexane: methanol: triethylamine = 4:4:2:0.08), and high-purity cyclovibrobuxine D is obtained by segment collection.

[0007] The above methods have one thing in common, that is, they can only separate one of cyclovirobuxine D or cyclovirobuxine D. However, due to the similar properties of the two, there is currently no existing technology that can achieve the simultaneous separation of cyclovirobuxine D and cyclovirobuxine D at one time. These separate separation methods have the characteristics of complex separation methods, generally low purity, and great difficulty in realizing industrial preparation. Summary of the invention

[0008] The object of the present invention is to provide a method for quickly separating high-purity cyclobuxine D and cyclovibrobuxine D. The method is simple to operate and convenient to use, and can achieve separation of high-purity cyclobuxine D and cyclovibrobuxine D through one column chromatography. Meanwhile, the invention can realize industrial production.

[0009] The embodiment of the present invention is achieved as follows: A method for rapidly separating high-purity cyclobuxine D and cyclovibrobuxine D, comprising: Extracts of Buxaceae plants were selected and purified by chromatography using a silver ion-silica gel column to obtain high-purity cyclobuxine D and cyclovibuxine D.

[0010] The beneficial effects of the embodiments of the present invention are: The embodiment of the present invention provides a method for rapidly separating high-purity cyclobuxine D and cyclovibrobuxine D, which selects a plant extract of the Buxaceae family and uses a silver ion-silica gel column for chromatography purification to obtain high-purity cyclobuxine D and cyclovibrobuxine D. The method can simultaneously separate and obtain high-purity cyclobuxine D and cyclovibrobuxine D by only one column chromatography.

[0011] The method is simple, easy to operate, has good reproducibility, and the obtained compound has high purity. At the same time, it can realize industrial large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0013] Figure 1 The chemical structures of cyclovibrobuxine D and cyclovibrobuxine D; Figure 2 The HPLC chromatogram of the crude extract containing cyclobuxine D and cyclovibrobuxine D prepared in Example 1; Figure 3 The MS total ion current spectrum of the crude extract containing cyclobuxine D and cyclovibrobuxine D prepared in Example 1; Figure 4 The HPLC chromatogram of cyclobuxine D prepared in Example 1; Figure 5 The MS total ion current spectrum of cyclobuxine D prepared in Example 1; Figure 6 The H NMR spectrum of cyclobuxine D prepared in Example 1 ( 1 H-NMR); Figure 7 The carbon NMR spectrum of cyclobuxine D prepared in Example 1 ( 13 C-NMR); Figure 8 The mass spectrum (ESI+) of cyclobuxine D prepared in Example 1; Fig. 9 The HPLC chromatogram of cyclovirobuxine D prepared in Example 1; Fig.10 The MS total ion current spectrum of cyclovirobuxine D prepared in Example 1; Fig.11 The hydrogen nuclear magnetic resonance spectrum of cyclovibroboxine D prepared in Example 1 ( 1 H-NMR); Fig.12 The carbon NMR spectrum of cyclovibroboxine D prepared in Example 1 ( 13 C-NMR); Fig.13 This is the mass spectrum (ESI+) of cyclovirobuxine D prepared in Example 1. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0015] The following is a specific description of a method for rapidly separating high-purity cyclobuxine D and cyclovibrobuxine D according to an embodiment of the present invention.

[0016] The embodiment of the present invention provides a method for rapidly separating high-purity cyclobuxine D and cyclovibrobuxine D, which comprises: Extracts of Buxaceae plants were selected and purified by chromatography using a silver ion-silica gel column to obtain high-purity cyclobuxine D and cyclovibuxine D.

[0017] The method can simultaneously separate and obtain high-purity cyclobuxine D and cyclovibrobuxine D through only one column chromatography.

[0018] Furthermore, the silica gel of the silica gel column is 50-400 mesh. The silica gel column is eluted with an eluent containing silver ions to obtain a silver ion-silica gel column; the content of the silver ion-containing substance in the eluent is 1wt%-10wt%. Within the above ratio range, a better separation effect can be obtained.

[0019] The eluent includes solvent A and solvent B, wherein solvent A includes at least one of dichloromethane, chloroform, tetrahydrofuran and ethyl acetate; and solvent B includes at least one of methanol, ethanol, n-propanol and isopropanol. Preferably, solvent A is dichloromethane or chloroform, and solvent B is methanol. The volume ratio of solvent A to solvent B is (80-8):1.

[0020] Furthermore, the eluent also contains 1 vol% to 20 vol% ammonia water. 。

[0021] During the column chromatography process, TLC was used to monitor the separation. The TLC conditions were as follows: the thin layer plate was one of the silica gel G thin layer plate and the silica gel GF-254 thin layer plate; the developing solvent was dichloromethane or chloroform: methanol (containing 1-20% ammonia water) = (30-5): 1; the color developer was bismuth potassium iodide reagent.

[0022] The extract of the boxwood plant is obtained by crushing the root, stem and leaf of the boxwood plant and then extracting it with an organic solvent, wherein the organic solvent includes at least one of dichloromethane or chloroform. The plant raw material or the finished product slices can be used.

[0023] Optionally, when performing extraction, the solid-liquid ratio is controlled to be 1: (5-50), and extraction is performed 1-6 times. The extraction temperature is 15-60°C, and the duration of each extraction is 30-200 minutes. Under this condition, the extraction effect is better, and the effective components can be fully extracted.

[0024] The features and performance of the present invention are further described in detail below in conjunction with the embodiments. Example

[0025] This embodiment provides a method for rapidly separating high-purity cyclobuxine D and cyclovibrobuxine D, which comprises: S1. The roots, stems and leaves of the Buxaceae plant were crushed and extracted with dichloromethane (solid-liquid ratio 1:10) for 3 times at 40°C for 60 minutes each time. After the extraction, the residue was filtered out and concentrated to obtain the Buxaceae plant extract.

[0026] S2. Purify the boxwood plant extract obtained in step S1 by silver ion-silica gel column chromatography, and select 200-mesh silica gel as the column filling. Prepare an eluent according to the ratio of dichloromethane:methanol:ammonia water=20:1:0.1, and add 5wt% silver nitrate to the eluent.

[0027] S3. During the column chromatography process, TLC was used to monitor the separation. The TLC conditions were as follows: the thin layer plate was a silica gel G thin layer plate; the developing solvent was dichloromethane:methanol (containing 10% ammonia water) = 20:1; the color developer was bismuth potassium iodide reagent.

[0028] S4. Collect the fractions of cyclobuxine D and cyclovibuxine D respectively, and concentrate them under reduced pressure to obtain pure products of cyclobuxine D and cyclovibuxine D.

[0029] in, Figure 2 and Figure 3 The HPLC chromatogram and MS total ion current spectrum of crude extracts containing cyclobuxine D and cyclobuxine D are shown respectively. It can be seen that cyclobuxine D and cyclobuxine D cannot be completely separated under HPLC separation, and there is obvious overlap between the chromatographic peaks of cyclobuxine D (retention time 4.517) and cyclobuxine D (retention time 4.761).

[0030] Figure 4 and Figure 5 The HPLC chromatogram and MS total ion current spectrum of cyclobuxine D after separation and purification by the method of this embodiment are respectively shown. It can be seen that there are no impurity peaks around cyclobuxine D, and the separation purity is high.

[0031] Figure 6~Figure 8 The H NMR spectra of cyclobuxine D are shown respectively. 1 H-NMR), carbon nuclear magnetic resonance spectroscopy ( 13 C-NMR) and mass spectrometry (ESI+); the specific characterization is as follows: 1 H-NMR (600 MHz, CDCl3) δ : 4.80 (1H, s, H-22a), 4.58 (1H, s, H-22b), 4.10 (1H, ddd, J = 9.80, 7.20, 2.50 Hz, H-16), 2.88 (1H, dd, J = 11.60, 4.10 Hz,H-3), 2.51 (1H, m, H-20), 2.49 (3H, s, H3-25), 2.44 (3H, s, H3-24), 2.09 (3H, overlapped, H-1a / H2-2), 1.88 (1H, dd, J = 10.10, 1.88 Hz, H-15a), 1.69 (2H, overlapped, H-11a / 17), 1.61 (1H, dd, J = 7.15, 3.20 Hz, H-7a), 1.51 (1H, overlapped, H-8), 1.40 (1H, dd, J= 11.00 2.65 Hz, H-15b), 2.32 (1H, m, H-5), 1.17 (3H, overlapped, H2-6 / H-11b), 1.14 (3H, s, H3-21), 1.09 (3H, d, J = 6.20Hz, H3-23), 1.02 (1H, m, H-7b), 0.27 (1H, d, J = 4.40 Hz, H-19a), 0.05 (1H, d, J = 4.40 Hz, H-19b).

[0032] 13 C-NMR (150 MHz, CDCl3) δ : 154.1 (C-4), 101.0 (CH2-22), 78.7 (CH-16), 63.8 (CH-3), 62.0 (CH-17), 59.1 (CH-20), 47.8 (CH-8), 47.5 (C-14), 45.3 (C-13), 44.8 (CH2-15), 44.6 (CH-5), 34.7 (CH3-25), 34.7 (CH2-1), 33.9 (CH3-24), 32.4 (C-9), 32.0 (CH2-11), 31.8 (CH2-12), 28.0 (CH2-19), 26.9 (CH2-2), 25.5(CH2-6), 23.9 (CH2-7), 23.2 (C-10), 21.0 (CH3-21), 19.3 (CH3-18), 18.7 (CH3-23).

[0033] The mass spectrum (ESI+) showed a molecular ion peak of 387, which was consistent with the molecular weight of cyclobuxine D.

[0034] Fig. 9 and Fig.10 The HPLC chromatogram and MS total ion current spectrum of cyclovirobuxine D after separation and purification by the method of this embodiment are respectively shown. It can be seen that there are no impurity peaks around cyclovirobuxine D, and it also has a high separation purity.

[0035] Fig.11 and Fig.13 The nuclear magnetic resonance proton spectra of cyclovibroboxene D are shown respectively. 1 H-NMR), carbon nuclear magnetic resonance spectroscopy ( 13 C-NMR) and mass spectrometry (ESI+); the specific characterization is as follows: 1 H-NMR (600 MHz, CDCl3) δ : 4.09 (1H, ddd, J = 9.80, 7.20, 2.50 Hz, H-16), 2.48 (1H, m, H-20), 2.43 (6H, d, J = 1.20 Hz, H-25 / 26), 1.93 (1H, dd, J =11.0, 3.2 Hz, H-3), 1.60 (1H, m, H-17), 1.53 (1H, dd, J = 13.0, 4.5 Hz, H-8),1.08 (1H, br t, J = 6.40 Hz, H-21), 1.10 (1H, s, H-24), 1.28 (1H, dd, J = 12.0,4.0 Hz, H-5), 0.52 (1H, d, J = 4.25 Hz, H-19a), 0.30 (1H, d, J = 4.25 Hz, H-19b).

[0036] 13 C-NMR (150 MHz, CDCl3) δ : 78.8 (CH-16), 68.7 (CH-3), 62.2 (CH-17), 59.1 (CH-20), 48.7 (CH-5), 48.2 (CH-8), 47.5 (C-14), 45.2 (C-13), 44.8 (CH2-15), 40.0 (C-4), 35.8 (CH3-26), 34.0 (CH3-25), 32.7 (CH2-1), 31.9 (CH2-12), 30.5 (CH2-19), 27.0 (CH2-2), 26.9 (C-10), 26.3 (CH2-11), 26.3 (CH2-7), 25.9(CH3-23), 21.5 (CH2-6), 21.0 (CH3-21), 19.6 (C-9), 19.3 (CH3-18), 18.7 (CH3-24), 15.2 (CH3-22).

[0037] The mass spectrum (ESI+) showed a molecular ion peak of 403, which was consistent with the molecular weight of cyclobuxine D. Example

[0038] This embodiment provides a method for rapidly separating cyclobuxine D and cyclovibrobuxine D, which comprises: S1. The roots, stems and leaves of the Buxaceae plant were crushed and extracted with chloroform (solid-liquid ratio 1:30) for 2 times at 30°C for 120 minutes each time. After the extraction, the residue was filtered out and concentrated to obtain the Buxaceae plant extract.

[0039] S2. Purify the boxwood plant extract obtained in step S1 by silver ion-silica gel column chromatography, and select 100 mesh silica gel as the column filling. Prepare an eluent according to the ratio of chloroform: methanol: ammonia water = 15: 1: 0.15, and add 8wt% silver sulfate to the eluent.

[0040] S3. During the column chromatography process, TLC was used to monitor the separation. The TLC conditions were as follows: the thin layer plate was a silica gel G thin layer plate; the developing solvent was chloroform:methanol (containing 15% ammonia water) = 30:1; the color developer was bismuth potassium iodide reagent.

[0041] S4. Collect the fractions of cyclobuxine D and cyclovibuxine D respectively, and concentrate them under reduced pressure to obtain pure products of cyclobuxine D and cyclovibuxine D.

[0042] Comparative Example 1 This comparative example provides a method for separating cyclobuxine D and cyclovibrobuxine D, and its operation steps are basically the same as those in Example 1, except that no silver ion compound is added to the eluent.

[0043] Without adding silver ion compounds, the peak times of cyclovibrobuxine D and cyclovibrobuxine D basically overlap and cannot be separated.

[0044] Comparative Example 2 This comparative example provides a method for separating cyclobuxine D and cyclovibrobuxine D, and the operation steps are basically the same as those in Example 1, except that no ammonia water is added to the eluent.

[0045] Without the addition of ammonia, cyclovibrobuxine D and cyclovibrobuxine D showed obvious tailing, resulting in reduced separation purity.

[0046] In summary, the embodiment of the present invention provides a method for quickly separating cyclobuxine D and cyclovibuxine D, which selects a plant extract of the Buxaceae family and uses a silver ion-silica gel column for chromatography purification to obtain high-purity cyclobuxine D and cyclovibuxine D. The method can achieve high-purity cyclobuxine D and cyclovibuxine D at the same time through one column chromatography. The method is simple and easy to operate, has good reproducibility, and is easy to realize industrial large-scale production.

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for rapidly separating high-purity cyclobuxine D and cyclovibrobuxine D, characterized in that: include: By selecting extracts of Buxaceae plants and using silver ion-silica gel column chromatography to perform one-time separation and purification, high-purity cyclobuxine D and cyclovibrobuxine D can be obtained.

2. The method according to claim 1, characterized in that The silver ion-silica gel column is obtained by loading a substance containing silver ions on a silica gel column, and the substance containing silver ions includes at least one of silver nitrate, silver sulfate, silver fluoride and silver acetate.

3. The method according to claim 2, characterized in that The silica gel column is eluted with an eluent containing the substance containing silver ions to obtain the silver ion-silica gel column; the content of the substance containing silver ions in the eluent is 1wt%~10wt%.

4. The method according to claim 3, characterized in that The eluent comprises solvent A and solvent B, wherein solvent A comprises at least one of dichloromethane, chloroform, tetrahydrofuran and ethyl acetate; and solvent B comprises at least one of methanol, ethanol, n-propanol and isopropanol.

5. The method according to claim 4, characterized in that The volume ratio of the solvent A to the solvent B is (80~8):

1.

6. The method according to claim 5, characterized in that The eluent also contains 1 vol% to 20 vol% of aqueous ammonia.

7. The method according to claim 1, characterized in that The boxwood plant extract is obtained by crushing the roots, stems and leaves of the boxwood plant and then extracting them using an organic solvent, wherein the organic solvent includes at least one of dichloromethane and chloroform.

8. The method according to claim 7, characterized in that When extracting, control the solid-liquid ratio to 1: (5~50) and extract 2~5 times.

9. The method according to claim 8, characterized in that The extraction temperature is 15~60℃, and each extraction time is 30~200min.

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