阅读说明：本技术 一种工业化提纯三尖杉宁碱的方法 (Method for industrially purifying cephalomannine ) 是由 杨青春 滕院 赵晓怡 陶琪 普倩 于 2021-05-26 设计创作，主要内容包括：本发明公开了一种工业化提纯三尖杉宁碱的方法,先通过对三尖杉宁碱原料拌料、晾料制成料胶,再通传统的过柱层析方法即可得到三尖杉宁碱半成品,最后重结晶得到三尖杉宁碱成品。本发明有效提高了三尖杉宁碱的含量(≥98.5％),大幅度降低了其中杂质数量、含量,使三尖杉宁碱产品质量得到保障,同时使提纯三尖杉宁碱时分离纯化效果好、收率高,三尖杉宁碱的总收率达≥80％、上样量大、可回收反复进行料胶拌料、三尖杉宁碱杂质数量和含量大幅减少或降低,不仅保证三尖杉宁碱含量的同时还保证了三尖杉杂质最低水平。(The invention discloses a method for industrially purifying cephalomannine, which comprises the steps of firstly mixing cephalomannine raw materials, airing the mixture to prepare a material adhesive, then obtaining a semi-finished product of the cephalomannine by a traditional column chromatography method, and finally recrystallizing to obtain a finished product of the cephalomannine. The method effectively improves the content of the cephalomannine (more than or equal to 98.5 percent), greatly reduces the quantity and the content of impurities in the cephalomannine, ensures the quality of cephalomannine products, simultaneously has good separation and purification effect and high yield when the cephalomannine is purified, has the total yield of the cephalomannine of more than or equal to 80 percent, has large sample loading amount, can recycle and repeatedly carry out material-glue mixing, greatly reduces or reduces the quantity and the content of the cephalomannine impurities, not only ensures the content of the cephalomannine, but also ensures the lowest level of the cephalomannine impurities.)

1. The method for industrially purifying cephalomannine is characterized by comprising the following preparation steps:

(1) a cephalomannine crude product with the cephalomannine content of 7-35 percent is used as a raw material product;

(2) stirring materials by using industrial-grade silica gel, wherein the weight ratio of the stirred materials to the glue is 1: 1.3-2;

(3) industrial-grade silica gel is used as a stationary phase, and the weight material-to-gel ratio of a sample to a silica gel total packed column is 1:3.7 +/-0.5;

(4) acetone: petroleum ether 1000:400(V/V) or Rf 0.1-0.2 as mobile phase;

(5) separating and purifying cephalomannine by silica gel column chromatography, preparing raw materials and silica gel into a sizing agent, placing the sizing agent on the top end of a clean silica gel column, and loading the sample by a dry method;

(6) when the mobile phase is eluted, the flow velocity of effluent of the chromatographic column is 150-200L/h;

(7) collecting eluate by TLC spot plate;

(8) separating cephalomannine sections, and concentrating to obtain cephalomannine semi-finished product;

(9) and (3) passing the cephalomannine semi-finished product through acetone: recrystallizing petroleum ether at a ratio of 1: 0.6-1 (V/V) to obtain the cephalomannine finished product.

2. The method for industrial purification of cephalomannine according to claim 1, characterized in that the industrial grade silica gel has a particle size of 200-300 meshes.

3. The method for the industrial purification of cephalomannine according to claim 1, characterized in that the mobile phase is acetone or petroleum ether.

4. The method for the industrial purification of cephalomannine according to claim 1, characterized in that the mobile phase ratio is acetone: petroleum ether is 1000:400 or Rf: 0.1-0.2 (V/V).

5. The method for industrially purifying cephalomannine according to claim 1, characterized in that the weight ratio of the sample to the total silica gel column is 1:3.7 plus or minus 0.5.

6. The method for industrially purifying cephalomannine according to claim 1, characterized in that the weight-to-rubber ratio of the stirred materials is 1: 1.3-2.

7. The method for industrial purification of cephalomannine according to claim 1, characterized in that the flow rate of the effluent of the chromatographic column is 150-200L/h.

8. The method for the industrial purification of cephalomannine according to claim 1, characterized in that the solvents used for the recrystallization, namely acetone: and (3) petroleum ether is 1: 0.6-1 (V/V).

Technical Field

The invention relates to the technical field of separation and purification of plant extracts, in particular to a method for industrially purifying cephalomannine.

Background

The paclitaxel is a natural secondary metabolite separated and purified from the bark of a gymnosperm yew, and has good anti-tumor effect through clinical verification, and particularly has special effects on ovarian cancer, uterine cancer, breast cancer and the like with high incidence rate of cancer, and is the most popular anti-cancer drug in the international market in recent years and is considered to be one of the most effective anti-cancer drugs for human in the future 20 years;

in recent years, the population and the cancer incidence of the earth are explosively increased, the demand of paclitaxel is also obviously increased, the paclitaxel required by clinical and scientific research is mainly extracted from taxus chinensis, because the content of the paclitaxel in a plant body is quite low (only 0.069% of the bark of the short-leaf taxus chinensis with the highest content is acknowledged), about 13.6kg of bark can extract 1g of paclitaxel, 3-12 taxus chinensis trees with more than one hundred years are required for treating an ovarian cancer patient, and thus, a large amount of cutting of the taxus chinensis is caused, so that the precious tree species are endangered and killed, in addition, the resources of the taxus chinensis are poor, the taxus plants grow slowly, and great difficulty is caused in further development and utilization of the paclitaxel;

although chemical synthesis is completed, the required conditions are strict, the yield is low, the expenditure is high, and the method has no industrial significance, the existing semi-synthesis method of the taxol is mature, and is considered as an effective way for expanding the taxol source except artificial planting, the semi-synthesis method can utilize plant resources to a greater extent, but the method is not essentially different from a method for directly extracting the taxol, a large amount of taxus chinensis trees are consumed, the problem of shortage of the plant sources cannot be fundamentally solved, obviously, the taxol extraction from taxus chinensis plant tissues is greatly limited, and the method has very important significance for searching a new way for obtaining the taxol;

cephalomannine is a taxol analog, the anticancer spectrum of which is similar to that of taxol, and can also be used for synthesizing taxol, the cephalomannine is a natural taxane substance contained in taxus, the cephalomannine is an initial raw material or a precursor for preparing semi-synthetic taxol, the two substances have similar structures, only the N position of a side chain is slightly different, the cephalomannine naturally exists in the taxus and has far higher content than the taxol, the method is simple and high in synthesis yield, the cephalomannine is hydrolyzed to prepare 10-deacetyl baccatin III, and the 10-deacetyl baccatin III is condensed with the side chain to prepare semi-synthetic taxol, docetaxel and other anticancer taxanes, and the cephalomannine product further expands the sources of the taxol, docetaxel, the cabazitaxel and realizes resource recycling.

Disclosure of Invention

The invention aims to solve the defects in the prior art, and provides an industrial method for purifying cephalomannine, which has the advantages that the separation and purification effect of the cephalomannine is good, the yield is high, the total yield of the cephalomannine is more than or equal to 80 percent, the sample loading amount is large, the material mixing and stirring can be recycled, the quantity and the content of cephalomannine impurities are greatly reduced or reduced, the content of the cephalomannine is ensured, and the lowest level of the cephalomannine impurities is also ensured.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for industrially purifying cephalomannine comprises the following preparation steps:

(1) a cephalomannine crude product with the cephalomannine content of 7-35 percent is used as a raw material product;

(2) stirring materials by using industrial-grade silica gel, wherein the weight ratio of the stirred materials to the glue is 1: 1.3-2;

(3) industrial-grade silica gel is used as a stationary phase, and the weight material-to-gel ratio of a sample to a silica gel total packed column is 1:3.7 +/-0.5;

(4) acetone: petroleum ether 1000:400(V/V) or Rf 0.1-0.2 as mobile phase;

(5) separating and purifying cephalomannine by silica gel column chromatography, preparing raw materials and silica gel into a sizing agent, placing the sizing agent on the top end of a clean silica gel column, and loading the sample by a dry method;

(6) when the mobile phase is eluted, the flow velocity of effluent of the chromatographic column is 150-200L/h;

(7) collecting eluate by TLC spot plate;

(8) separating cephalomannine sections, and concentrating to obtain cephalomannine semi-finished product;

(9) and (3) passing the cephalomannine semi-finished product through acetone: recrystallizing petroleum ether at a ratio of 1: 0.6-1 (V/V) to obtain the cephalomannine finished product.

Preferably, the industrial grade silica gel has a particle size of 200-300 meshes.

Preferably, the mobile phase is acetone or petroleum ether.

Preferably, the mobile phase ratio is acetone: petroleum ether is 1000:400 or Rf: 0.1-0.2 (V/V).

Preferably, the weight ratio of the sample to the total silica gel column is 1:3.7 +/-0.5.

Preferably, the weight material-to-glue ratio of the stirring material is 1: 1.3-2.

Preferably, the flow rate of the effluent of the chromatographic column is 150-200L/h.

Preferably, the solvents used for recrystallization are acetone: and (3) petroleum ether is 1: 0.6-1 (V/V).

Compared with the prior art, the invention has the beneficial effects that:

1. the separation and purification effect is good, and the cephalomannine crude product with the cephalomannine content of 7-35% is conveniently purified into the cephalomannine finished product with the cephalomannine content of more than or equal to 98.5%.

2. The yield is high, and the total yield of the cephalomannine is more than or equal to 80 percent.

3. The sample loading amount is large, and the weight ratio of the sample to the silica gel total column is 1:3.7 +/-0.5.

4. The silica gel is repeatedly used for a plurality of times, the material and the glue at the top can be recycled and repeatedly mixed when the column chromatography is finished, the lower layer clean silica gel can be repeatedly used for more than 8 times after the column is washed, and the material and the glue can be mixed for use after the repeated use is finished.

5. The amount and content of cephalomannine impurities are greatly reduced or lowered, so that the minimum level of the cephalomannine impurities is ensured while the content of the cephalomannine is ensured.

Drawings

FIG. 1 is a diagram of the structure of the paclitaxel molecule of the present invention;

FIG. 2 is a molecular structure diagram of cephalomannine in the present invention;

FIG. 3 is a molecular structure diagram of 10-deacetylbaccatin III in the present invention.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, but rather should be construed as broadly as the present invention is capable of modification in various respects, all without departing from the spirit and scope of the present invention.

It will be understood that when an element is referred to as being "secured to" another element, it can be directly on the other element or intervening elements may also be present. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or intervening elements may also be present. The terms "vertical," "horizontal," "left," "right," and the like as used herein are for illustrative purposes only and do not represent the only embodiments.

Example 1: a method for industrially purifying cephalomannine comprises the following specific operations:

(1) 500g of cephalomannine crude product with the cephalomannine content of 12.6 percent in the raw material sample, wherein the effective amount of the cephalomannine is 63 g;

(2) dissolving a cephalomannine sample in 4L of acetone, weighing 650g of industrial-grade silica gel, adding the industrial-grade silica gel, stirring the materials, and drying to obtain 1149g of material gel;

(3) weighing 1100g of industrial-grade silica gel, filling the industrial-grade silica gel into a glass chromatographic column with the diameter of 150mm and the height of 1000mm, and knocking the outer wall by a rubber hammer to fill the silica gel in the column;

(4) pouring 1149g of easily-dried material glue into a chromatographic column, placing the chromatographic column above clean silica gel, and knocking the outer wall by using a rubber hammer to fill the silica gel and the material glue in the column;

(5) preparing a mobile phase according to acetone and petroleum ether 1000:400(V/V) or Rf 0.1-0.2, and performing column chromatography;

(6) collecting according to 2000 ml/bottle, collecting 1-6 bottles of eluate as part I, basically containing no cephalomannine, and performing centralized treatment to recover the solvent;

(7) 7-9 bottles of eluate are part II, TLC monitoring is carried out, a small amount of cephalomannine exists, the cephalomannine is combined, concentrated and recovered, the concentrate is 13.8g, the content of the cephalomannine is 3.3 percent, namely the effective amount of the cephalomannine is 0.455g and accounts for 0.7 percent of the cephalomannine in the raw materials;

(8) taking 10-19 bottles of eluate as part III, monitoring by TLC, and collecting a large amount of cephalomannine, mixing, concentrating and recovering the solvent to obtain 70.7g of concentrate, wherein the content of cephalomannine is 85.9 percent, namely the effective amount of the cephalomannine is 60.7g and accounts for 96.3 percent of the cephalomannine in the raw materials;

(9) 20-24 bottles of eluate are part IV, TLC monitoring is carried out, a small amount of cephalomannine exists, the mixed concentrated solutions are concentrated and the solvent is recovered, the concentrate is 56.3g, the content of the cephalomannine is 3.2 percent, namely the effective amount of the cephalomannine is 1.8g, and the cephalomannine accounts for 2.9 percent of the cephalomannine in the raw materials;

(10) 70.7g of the third part concentrate is dissolved in 260ml of acetone solvent, 257ml of petroleum ether is added for recrystallization, the step is repeated for 3 times to obtain crystals, and 54.1g of cephalomannine sample is produced after drying, the cephalomannine content is 98.9 percent, namely 53.5g of the effective amount of the cephalomannine, and the crystal accounts for 84.9 percent of the cephalomannine in the raw materials, namely 84.9 percent of the total yield of the cephalomannine.

Example 2: a method for industrially purifying cephalomannine comprises the following specific operations:

(1) 650g of cephalomannine crude product with the cephalomannine content of 8.2 percent in the raw material sample, wherein the effective amount of the cephalomannine is 53.3 g;

(2) dissolving a cephalomannine sample in 5L of acetone, weighing 975g of industrial-grade silica gel, adding the industrial-grade silica gel, stirring the materials, and drying to obtain 1623g of material gel;

(3) weighing 1430g of industrial-grade silica gel, filling the industrial-grade silica gel into a glass chromatographic column with the diameter of 150mm and the height of 1000mm, and knocking the outer wall by a rubber hammer to fill the silica gel in the column;

(4) pouring 1623g of easily-dried material glue into a chromatographic column, placing the chromatographic column above clean silica gel, and knocking the outer wall by a rubber hammer to fill the silica gel and the material glue in the column;

(5) preparing a mobile phase according to acetone and petroleum ether 1000:400(V/V) or Rf 0.1-0.2, and performing column chromatography;

(6) collecting according to 2000 ml/bottle, taking 1-7 bottles of eluate as part I, basically not containing cephalomannine, and carrying out centralized treatment to recover the solvent;

(7) using 8-9 bottles of eluate as part II, monitoring by TLC, and collecting and concentrating a small amount of cephalomannine, and recovering solvent to obtain 20.3g of concentrate, wherein the content of cephalomannine is 2.9 percent, namely the effective amount of cephalomannine is 0.59g and accounts for 1.1 percent of cephalomannine in the raw materials;

(8) taking 10-18 bottles of eluate as part III, monitoring by TLC, wherein a large amount of cephalomannine exists, merging, concentrating and recovering the solvent, and obtaining 59.5g of concentrate with the cephalomannine content of 86.1 percent, namely 51.2g of the cephalomannine effective amount which accounts for 96.1 percent of the cephalomannine in the raw materials;

(9) using 19-23 bottles of eluate as part IV, monitoring by TLC, and collecting a small amount of cephalomannine, combining, concentrating and recovering the solvent, wherein the concentrate is 45.2g, and the content of the cephalomannine is 3.1 percent, namely the effective amount of the cephalomannine is 1.4g and accounts for 2.7 percent of the cephalomannine in the raw materials;

(10) dissolving 59.5g of the third part concentrate in 220ml of acetone solvent, adding 218ml of petroleum ether for recrystallization, repeating the step for 3 times to obtain crystals, drying to obtain a cephalomannine sample of 46.2g, wherein the cephalomannine content is 98.8 percent, namely the effective amount of the cephalomannine is 45.6g, and the cephalomannine accounts for 85.5 percent of the cephalomannine in the raw materials, namely the total yield of the cephalomannine is 85.5 percent.

Example 3: a method for industrially purifying cephalomannine comprises the following specific operations:

(1) 580g of cephalomannine crude product with 27.2 percent of cephalomannine content in the raw material sample, wherein the effective amount of the cephalomannine is 157.8 g;

(2) dissolving a cephalomannine sample in 4L of acetone, weighing 870g of industrial-grade silica gel, adding the industrial-grade silica gel, stirring the materials, and drying to obtain 1446g of material gel;

(3) weighing 1276g of industrial-grade silica gel, filling the industrial-grade silica gel into a glass chromatographic column with the diameter of 150mm and the height of 1000mm, and knocking the outer wall by a rubber hammer to fill the silica gel in the column;

(4) 1446g of easily dried material glue is poured into a chromatographic column, the chromatographic column is placed above clean silica gel, and the outer wall is beaten by a rubber hammer to fill the silica gel and the material glue in the column;

(5) preparing a mobile phase according to acetone and petroleum ether 1000:400(V/V) or Rf 0.1-0.2, and performing column chromatography;

(6) collecting according to 2000 ml/bottle, collecting 1-5 bottles of eluate as part I, basically containing no cephalomannine, and performing centralized treatment to recover the solvent;

(7) taking 6-8 bottles of eluate as part II, monitoring by TLC, and collecting a small amount of cephalomannine, combining, concentrating and recovering the solvent to obtain 48.1g of concentrate, wherein the content of cephalomannine is 2.7 percent, namely the effective amount of cephalomannine is 1.3g and accounts for 0.8 percent of the cephalomannine in the raw materials;

(8) 9-17 bottles of eluate are part III, TLC monitoring is carried out, a large amount of cephalomannine exists, the cephalomannine is combined, concentrated and recovered, the concentrate is 175.3g, the content of the cephalomannine is 86.9 percent, namely the effective amount of the cephalomannine is 152.3g, and the cephalomannine accounts for 96.5 percent of the cephalomannine in the raw materials;

(9) taking 18-22 bottles of eluate as part IV, monitoring by TLC, and collecting a small amount of cephalomannine, mixing, concentrating and recovering the solvent to obtain 122.6g of concentrate, wherein the content of cephalomannine is 3.1 percent, namely the effective amount of cephalomannine is 3.8g and accounts for 2.4 percent of the cephalomannine in the raw materials;

(10) dissolving 175.3g of the third part concentrate in 650ml of acetone solvent, adding 645ml of petroleum ether for recrystallization, repeating the step for 3 times to obtain crystals, drying to obtain 136.6g of cephalomannine sample, wherein the cephalomannine content is 98.5 percent, namely the effective amount of 134.6g of cephalomannine accounts for 85.3 percent of the cephalomannine in the raw materials, namely the total yield of the cephalomannine is 85.3 percent. Example 4: a method for industrially purifying cephalomannine comprises the following specific operations:

(1) 610g of cephalomannine crude product with the cephalomannine content of 11.3 percent in the raw material sample, wherein the effective amount of the cephalomannine is 68.9 g;

(2) dissolving a cephalomannine sample in 5L of acetone, weighing 854g of industrial-grade silica gel, adding the industrial-grade silica gel, stirring the materials, and drying to obtain 1462g of material gel;

(3) weighing 1400g of industrial-grade silica gel, filling the industrial-grade silica gel into a glass chromatographic column with the diameter of 150mm and the height of 1000mm, and knocking the outer wall by a rubber hammer to fill the silica gel in the column;

(4) pouring 1462g of easily-dried material glue into a chromatographic column, placing the chromatographic column above clean silica gel, and knocking the outer wall by using a rubber hammer to fill the silica gel and the material glue in the column;

(5) preparing a mobile phase according to acetone and petroleum ether 1000:400(V/V) or Rf 0.1-0.2, and performing column chromatography;

(6) collecting according to 2000 ml/bottle, collecting 1-6 bottles of eluate as part I, basically containing no cephalomannine, and performing centralized treatment to recover the solvent;

(7) 7-9 bottles of eluate are part II, TLC monitoring is carried out, a small amount of cephalomannine exists, the cephalomannine is combined, concentrated and recovered, the concentrate is 33.3g, the content of the cephalomannine is 2.4 percent, namely the effective amount of the cephalomannine is 0.8g and accounts for 1.2 percent of the cephalomannine in the raw materials;

(8) taking 10-18 bottles of eluate as part III, monitoring by TLC, and collecting a large amount of cephalomannine, combining, concentrating and recovering the solvent, wherein the concentrate is 76.6g, and the content of the cephalomannine is 86.7 percent, namely the effective amount of the cephalomannine is 66.4g and accounts for 96.3 percent of the cephalomannine in the raw materials;

(9) using 19-25 bottles of eluate as part IV, monitoring by TLC, and collecting and concentrating a small amount of cephalomannine, and recovering solvent to obtain 45.5g of concentrate, wherein the content of cephalomannine is 3.3%, namely the effective amount of cephalomannine is 1.5g and accounts for 2.2% of cephalomannine in the raw materials;

(10) dissolving 76.6g of the third part concentrate in 285ml of acetone solvent, adding 270ml of petroleum ether for recrystallization, repeating the step for 3 times to obtain crystals, drying to obtain 60.1g of cephalomannine sample, wherein the cephalomannine content is 98.7 percent, namely the effective amount of the cephalomannine is 59.3g, and the cephalomannine accounts for 86.1 percent of the cephalomannine in the raw materials, namely the total yield of the cephalomannine is 86.1 percent.

Example 5: a method for industrially purifying cephalomannine comprises the following specific operations:

(1) 700g of cephalomannine crude product with the cephalomannine content of 7.6 percent in the raw material sample, wherein the effective amount of the cephalomannine is 53.2 g;

(2) dissolving a cephalomannine sample in 6L of acetone, weighing 910g of industrial-grade silica gel, adding the industrial-grade silica gel, stirring the materials, and drying to obtain 1608g of material gel;

(3) weighing 1680g of industrial-grade silica gel, filling the industrial-grade silica gel into a glass chromatographic column with the diameter of 150mm and the height of 1000mm, and knocking the outer wall by a rubber hammer to fill the silica gel in the column;

(4) 1608g of easily dried material gel is poured into the chromatographic column, is placed above the clean silica gel, and the outer wall is beaten by a rubber hammer to fill the silica gel and the material gel in the column;

(5) preparing a mobile phase according to acetone and petroleum ether 1000:400(V/V) or Rf 0.1-0.2, and performing column chromatography;

(6) collecting according to 2000 ml/bottle, taking 1-7 bottles of eluate as part I, basically not containing cephalomannine, and carrying out centralized treatment to recover the solvent;

(7) using 8-10 bottles of eluate as part II, monitoring by TLC, and collecting a small amount of cephalomannine, combining, concentrating and recovering the solvent, wherein the concentrate is 19.2g, and the content of the cephalomannine is 2.6 percent, namely the effective amount of the cephalomannine is 0.5g and accounts for 0.9 percent of the cephalomannine in the raw materials;

(8) using 11-19 bottles of eluate as part III, monitoring by TLC, and collecting and concentrating a large amount of cephalomannine, wherein the combined concentrated solution is used for recovering the solvent, the concentrate is 59.1g, and the content of the cephalomannine is 86.5 percent, namely the effective amount of the cephalomannine is 51.1g, and accounts for 96.1 percent of the cephalomannine in the raw materials;

(9) 20-24 bottles of eluate are part IV, TLC monitoring is carried out, a small amount of cephalomannine exists, the mixed concentrated solutions are concentrated and the solvent is recovered, 35.5g of concentrate is obtained, the content of the cephalomannine is 3.1 percent, namely the effective amount of the cephalomannine is 1.1g, and the cephalomannine accounts for 2.0 percent of the cephalomannine in the raw materials;

(10) dissolving 59.1g of the third part concentrate in 220ml of acetone solvent, adding 216ml of petroleum ether for recrystallization, repeating the step for 3 times to obtain crystals, drying to obtain a cephalomannine sample of 46.5g, wherein the cephalomannine content is 98.6 percent, namely the effective amount of the cephalomannine is 45.8g, and the cephalomannine accounts for 86.1 percent of the cephalomannine in the raw materials, namely the total yield of the cephalomannine is 86.1 percent.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.