阅读说明：本技术 一种β榄香烯的制备方法 (Preparation method of β elemene ) 是由 黄璐琦 柯潇 高乾善 王晓 宋祥云 王岱杰 杨健 郭娟 王雅南 石章飞 于 2019-01-10 设计创作，主要内容包括：本发明提供一种β榄香烯的制备方法,通过分离生物发酵液中的吉玛烯A,随后进行热转化,精馏,得到高纯度的β榄香烯产物。本发明制备方法(3)工艺简便,成本低、效率高,可操作性强,适于产业应用。(The invention provides a preparation method of β elemene, which comprises the steps of separating germacrene A in biological fermentation broth, then carrying out thermal conversion and rectification to obtain a high-purity β elemene product.)

1. A process for preparing β elemene includes such steps as separating the fermented liquid containing germacrene A, extracting the solid with alcohol-contained solvent, separating, extracting the solid with alcohol-contained solvent or alkane, mixing the two extracts, washing, drying, concentrating, and heating to obtain β elemene.

2. The method of β elemene according to claim 1, wherein the alcohol-containing solvent is a lower alcohol or an alcohol solution, preferably a C1-C3 alcohol or an alcohol solution, more preferably one or more selected from methanol, absolute ethanol, 95% ethanol, propanol, and isopropanol.

3. The method for preparing β elemene according to claim 1 or 2, wherein the alcohol-containing solvent is used in an amount of 0.5 times or more, preferably 0.5-1.25 times the volume of the fermentation broth.

4. The β elemene preparation method as claimed in claim 1, wherein the solvent of the second extraction is preferably alkane selected from n-hexane, cyclohexane, petroleum ether, n-dodecane.

5. The method of β elemene according to claim 1 or 4, wherein the solvent used in the second extraction is 0.25 times or more, preferably 0.375-0.75 times of the volume of the fermentation broth.

6. The method of β elemene as claimed in any one of claims 1 to 5, wherein the extraction solution obtained by alcohol-containing solvent extraction is concentrated, and the extraction solutions are combined after alkane back-extraction.

7. The method as claimed in claim 7, wherein the alkane is selected from n-hexane, petroleum ether, cyclohexane.

8. The method for preparing β elemene according to claim 1, wherein the heating conversion temperature is 100-180 ℃, preferably 120-150 ℃, and the heating time is preferably 1 h.

9. The method of claim β elemene as claimed in any one of claims 1-8, wherein β elemene obtained by heat conversion is purified by rectification under reduced pressure.

10. The preparation method of β elemene as claimed in claim 9, wherein the vacuum distillation process comprises using a stainless steel mesh θ ring as column packing, the number of plates is 10-15, the temperature of the bottom of the column is 100-130 ℃, the reflux ratio is 0.5-3.0, and the fraction with boiling point of 75-76 ℃/85pa is cut off, and the boiling range is not more than 1 ℃.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a preparation method of β elemene.

Technical Field

Elemene is a sesquiterpene compound with anticancer activity extracted from curcuma zedoaria of Zingiberaceae in China, the chemical structure of the compound has 3 chiral carbon atoms, so 8 isomers exist theoretically, and α, β, gamma and four kinds of isomers are identified and reported in the literature at present, wherein, the trans isomer of β -elemene (β -elemene) is a main substance with anticancer activity, the chemical name of the compound is (1S,2S,4R) -1-methyl-1-vinyl-2, 4-diisopropenylcyclohexane, and the molecular structure is as follows:

the research shows that the β elemene can be used for treating various diseases such as adenocarcinoma, human glioma, liver cancer, lung cancer, colon cancer, gastric cancer, ovarian cancer and the like, and has the advantages of wide anticancer spectrum, definite curative effect, slight toxic and side effect, no bone marrow inhibition and the like.

At present, β elemene bulk drugs are mainly prepared by two ways of natural plant extraction and separation or chemical synthesis, chemical synthesis β elemene has some problems, such as complex process steps, low yield and difficulty in stereoisomer selection and control, so that only the level of literature research is remained at present, and the requirement of industrial large production cannot be met, elemene injection in China is mainly prepared by adopting a steam distillation method to obtain β elemene raw materials from plant volatile oil such as curcuma zedoary, citronella and the like, however, the elemene content in natural plants is generally low, a large amount of plant medicinal material resources are generally consumed, and simultaneously, complicated separation and purification steps are carried out to obtain elemene extracts meeting the purity requirement, for example, the elemene content in the zedoary volatile oil is about 5%, the elemene content in the natural plants is generally low, the volatile oil obtained by utilizing the steam distillation method to extract the elemene from curcuma zedoary, curcuma kwangsiensis and curcuma zedoary, the relative contents of the elemene extracted from the curcuma zedoary, the current situation of the traditional Chinese medicine sources and the extraction efficiency of the traditional Chinese medicines cannot be combined with the fevernal medical journal of Compositae [ 35 ].

The biosynthesis of β -elemene is to utilize the self-metabolic pathway of recombinant microorganism to carry out fermentation culture in proper fermentation liquor, so as to synthesize the germacrene A (Germacrene A), and then utilize the characteristic that the germacrene A can generate rearrangement reaction under the heating condition [ (+) -Germacrene A biosynthesis, Plant Physiol. (1998)117:1381-1392], so as to obtain β elemene, as shown in the attached figure 3.

The method for preparing β elemene through biological fermentation does not need to consume a large amount of plant resources, avoids complex chemical synthesis steps, has the advantages of low cost, high yield and the like, provides a new direction for industrial production of β elemene raw material medicines, and the germacrene A obtained through biological fermentation exists in culture medium fermentation liquor containing a large amount of hyphae and multiple nutrient components, and is separated as one of key steps for producing β -elemene.

Disclosure of Invention

In order to solve the above problems in the prior art, an object of the present invention is to provide a method for preparing β elemene.

A process for preparing β elemene includes such steps as separating the fermented liquid containing germacrene A, extracting the solid with alcohol-contained solvent, separating, extracting the solid with alcohol-contained solvent or alkane, mixing the two extracts, washing, drying, concentrating, and heating to transform it to β elemene.

In the β elemene preparation method, the alcohol-containing solvent is a lower alcohol or an alcohol solution, preferably a C1-C3 alcohol or an alcohol solution, and further preferably one or more of methanol, absolute ethanol, 95% ethanol, propanol and isopropanol, wherein the dosage of the extraction solvent A is 0.5 times or more, for example 0.75 times, 1 time, 1.25 times or more, and preferably 0.5-1.25 times of the volume of the fermentation liquor;

in the β elemene preparation method of the invention, the solvent for the second extraction is preferably alkane selected from one or more of n-hexane, cyclohexane, petroleum ether and n-dodecane, and the solvent for the second extraction is used in an amount of 0.25 times or more, for example, 0.375 times, 0.5 times, 0.625 times, 0.75 times, 1 time or more, preferably 0.375-0.75 times of the volume of the fermentation broth.

Preferably, in the β elemene preparation method, the extracting solution obtained by extracting the alcohol-containing solution is concentrated, alkane is used for back extraction, and then the extracting solutions are combined, wherein in the back extraction step, the alkane is preferably one or more of n-hexane, petroleum ether and cyclohexane.

Preferably, in the preparation method of β elemene, the heating conversion temperature is 100-;

the β elemene obtained by the heating conversion can be further purified by adopting a proper purification method, such as vacuum rectification and the like, wherein the preferable parameters of the vacuum rectification process in the invention are that stainless steel net theta ring is used as column packing, the number of tower plates is 10-15, the temperature of a tower kettle is 100-130 ℃, the reflux ratio is 0.5-3.0, and a fraction with the boiling point of 75-76 ℃/85pa is cut off, and the boiling range is not more than 1 ℃.

The invention has the advantages that:

(1) the method can effectively separate the germacrene A product in the biological fermentation liquid, and has high separation and extraction rate;

(2) β the conversion rate and recovery rate of elemene are high, which provides a new choice for the industrial production of β elemene bulk drugs;

(3) simple process, low cost, high efficiency and strong operability, and is suitable for industrial application.

Drawings

FIG. 1 is a 1H NMR spectrum of β elemene product prepared by the preparation method of the invention

FIG. 2 shows β elemene products prepared by the preparation method of the invention¹³C NMR spectrum

FIG. 3 shows the chemical reaction formula of heat conversion of Gimeran A to β elemene

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.

The preparation method of β elemene can be used for separating germacrene A in biological fermentation liquid and further carrying out thermal conversion to obtain β elemene, the inventor tries biological fermentation liquids obtained by different strains and/or different fermentation processes and is all applicable to the preparation method of the invention, and concretely, the germacrene A fermentation liquid prepared by the biological fermentation process disclosed in CN108060092A is taken as an example.

Method for detecting β elemene content in product

The preparation of test solution comprises diluting β elemene product with n-hexane 100 times and passing through organic nylon membrane (0.45 μm) to obtain test solution.

The preparation of the reference solution comprises precisely weighing 50mg of β -elemene standard (purchased from China institute for food and drug testing, Cat. No. 100268-201402) into a 50ml volumetric flask, and diluting with anhydrous ethanol to a constant volume to obtain the reference solution.

The detection conditions comprise Agilent 7890B, Agilent gas chromatography, a sample inlet temperature of 250 ℃, a sample volume of 1 mu L without flow division, a chromatographic column of Agilent HP-5ms (30m x 0.25mm), chromatographic conditions of 45 ℃, 1min, 10 ℃/min to 300 ℃, heat preservation at 300 ℃ for 5min, and an FID detector.

Method for detecting content of damascene A

Preparing a test solution:

(1) fermentation liquor: placing 20ml of fermentation liquor in a 50ml centrifuge tube, adding 5ml of n-dodecane, placing in a shaking table, shaking and extracting for more than 8 hours, then standing for 5min, placing 2ml of upper layer (containing an emulsifying layer) in the centrifuge tube, centrifuging at 12000rpm for 1min to separate an organic phase and a water phase, taking supernatant, diluting with n-hexane by 100 times, and passing through an organic nylon membrane (0.45 mu m) to serve as a test solution.

(2) Extraction or supernatant: a sample is diluted 10-50 times with n-hexane and passed through an organic nylon membrane (0.45 μm) as a test solution.

The preparation of the reference solution comprises the steps of carrying out rearrangement reaction on germacrene A in a sample solution to be tested at the temperature of 250 ℃ by using a gas chromatography injection port, and converting the germacrene A into β -elemene, so that β -elemene is adopted as the reference in the experiment, 50mg of β -elemene standard product (purchased from China food and drug testing institute, Cat. No. 100268-.

The detection conditions comprise Agilent 7890B, Agilent gas chromatography, a sample inlet temperature of 250 ℃, a sample volume of 1 mu L without flow division, a chromatographic column of Agilent HP-5ms (30m x 0.25mm), chromatographic conditions of 45 ℃, 1min, 10 ℃/min to 300 ℃, heat preservation at 300 ℃ for 5min, and an FID detector.

Thirdly, β elemene structure verification in the product

The compound has the following structure:

structure confirmation method and results:

the β -elemene product after rectification and purification is taken to be checked for NMR, MS. HNMR spectrum (figure 1) of the compound 1 shows that 24 hydrogen atoms exist,¹³c NMR spectrum (FIG. 2) shows the presence of 15 carbon atoms including 6 double-bonded C atoms (C:)_C150.4, 150.3, 147.7, 112.1, 109.9, 108.3), and MS data molecular ion peaks204([M]⁺) The combination indicates that the compound is a sesquiterpene compound with a molecular formula of C₁₅H₂₄. The compound also has a ring present, calculated on the basis of the degree of unsaturation.¹H NMR(CDC l3,600MH z):1.00(3H,s,H-15)、1.45(3H,m,H-2,3a)、1.64(1H,m,H-5a)、1.56 (2H,m,H-3b,5b)、1.71(3H,s,H-11)、1.76(3H,s,H-14)、1.91(1H,m,H-6)、2.01(1H,m,H-4)、 4.59(1H,s,H-10a)、4.82(1H,s,H-10b)、4.70(1H,s,H-13a)、4.72(1H,s,H-13b)、4.89(1H,d,J ＝12.6Hz,H-8a)、4.90(1H,d,J＝20.4Hz,H-8b)、5.82(1H,dd,J＝20.4、12.6Hz,H-7)。¹³C NMR (CDC l3,125MHz) 16.6(C-15), 21.1(C-14), 24.8(C-11), 26.8(C-5), 32.9(C-3), 39.8(C-1), 39.9(C-2), 45.7(C-6), 52.8(C-4), 108.3(C-13), 109.9(C-8), 112.1(C-10), 147.7(C-9), 150.3 (C-12), 150.4 (C-7). The data and literature [ chemical composition of elemene crude drug, Shenyang pharmaceutical university, 2009, 26(8) ]]The NMR and MS data of the compound 1(-) β -elemene are consistent, so that the compound is confirmed to be (-) β -elemene.

Test example 1

Taking three parts of a germlined gemmaline A fermentation liquor (with 8.50 g/L of germlinene A content) prepared by biosynthesis, respectively adopting a laboratory glass rectification device, an industrial 160L extraction tank and an industrial 100L single-effect concentrator to carry out distillation extraction, intermittently supplementing water, wherein the total extraction time is about 15 hours, separating an oil phase from the distilled liquid by a separating funnel or an oil-water separator, centrifuging the seriously emulsified part for 10 minutes by adopting a high-speed (12000r/min) centrifuge, recording the total volume of volatile oil, sampling and analyzing the germlinene A content, and calculating the extraction rate.

Adding anhydrous sodium sulfate into the volatile oil, drying, filtering to remove a drying agent, placing the volatile oil in an oil bath at 120 ℃, removing residual solvent under reduced pressure, heating to 150 ℃, keeping the temperature, reacting for 1 hour, rectifying and collecting β -elemene, weighing, sampling, analyzing the content, and calculating the recovery rate, wherein the experimental results are shown in table 1.

TABLE 1 effect of steam distillation extraction of fermentation broth

Note that GM-germacrene A (gemeneA), EM- β -elemene (elemene), the same as below

The GM extraction rate (GM content in volatile oil/GM content in fermentation liquid) is 100%

EM recovery ═ 100% (EM weight EM purity/GM content in fermentation broth)%

Experiments show that the fermentation liquor extracted by the distillation method has emulsification phenomena of different degrees, a large amount of foam is easily generated in the extraction process, the extraction process is seriously influenced, and meanwhile, the distillate contains more impurities and is easy to generate side reactions to generate new impurities in the long-time distillation process, so that the GM extraction rate and the β elemene recovery rate are lower.

Test example 2

Taking six parts of 400m L (the content of the germacrene A is 10.25 g/L) of germacrene A fermentation liquor prepared by biosynthesis, respectively adding 400ml of absolute ethyl alcohol, methanol, n-butyl alcohol, petroleum ether, n-dodecane and 95% ethyl alcohol plus n-hexane (1:1, v/v), shaking, stirring and extracting for 30min, standing and settling for 10 h for an un-emulsified sample, recording the volume of a supernatant, taking the supernatant, filtering, analyzing the content by using a gas chromatography method, and inspecting the difficulty of filtering operation by using a common filtering method in a laboratory.

TABLE 2 direct extraction of fermentation broth with organic solvent

Note: the extraction rate a is (GM content in supernatant a/GM content in fermentation broth) 100%

Experiments show that organic solvents such as n-butyl alcohol, petroleum ether, n-dodecane and a 95% mixed solvent (1:1, v/v) of n-hexane are adopted to extract fermentation liquor, emulsification phenomena of different degrees occur, an emulsion layer and an organic solvent layer are difficult to separate in a conventional filtration mode, although a high-speed (12000r/min) centrifugal device can be adopted in a laboratory to separate the emulsion layer, the volume of the fermentation liquor in industry is generally in the tonnage level, and at present, no high-speed centrifugal device capable of meeting the large-batch separation requirements exists temporarily.

When absolute ethyl alcohol and methanol are adopted for extraction, although emulsification does not occur, the extracting solution is difficult to filter, and the operability is poor.

Test example 3

Taking 6 parts of biosynthesis prepared germacrene A fermentation liquor 400m L (the germacrene A content is 10.25 g/L), centrifuging for 20min at 3000 revolutions by using a high-speed centrifuge, removing upper-layer liquid, adding 300ml of 95% ethanol, absolute ethanol, methanol, isopropanol, petroleum ether and n-dodecane into bottom solid sediment (namely hypha) respectively, shaking and stirring for 30min, filtering a settleable sample, centrifuging an emulsified sample, taking supernatant to analyze the germacrene A content, and calculating the extraction rate of the step, wherein the experimental results are shown in table 3.

TABLE 3 comparison of the effect of mycelium extraction with different solvents

Note: the extraction rate a is (GM content in supernatant a/GM content in fermentation broth) 100%

Test example 4

Taking 6 parts of biosynthetic germlined A fermentation liquor 40m L (the content of germlined A is 10.25 g/L), centrifuging for 20min at 3000 revolutions by using a high-speed centrifuge, removing an upper layer solution, adding 5, 10, 20, 30, 40 and 50ml of 95% ethanol into bottom solid sediment, shaking and stirring for 30min, centrifuging and settling, taking supernate to analyze the content of germlined A, and calculating the extraction rate a in the step, wherein the experimental result is shown in table 4.

TABLE 4 comparison of hypha extraction effect with different solvent dosage

Note: the extraction rate a is (GM content in supernatant a/GM content in fermentation broth) 100%;

test example 5

Centrifuging biosynthetic germacrene A fermentation liquor 2400m L (germacrene A content is 10.25 g/L) by a high-speed centrifuge, removing an upper layer solution to obtain a bottom solid sediment, adding absolute ethanol 2400m L into the bottom solid sediment, stirring and extracting for 1 hour, and filtering to obtain an ethanol extracting solution and solid hypha residues.

Dividing the mycelium residue into six parts, adding 200ml 95% ethanol, anhydrous ethanol, methanol, isopropanol, petroleum ether, and cyclohexane, shaking, stirring for 30min, centrifuging, collecting supernatant, and analyzing content by gas chromatography to calculate extraction rate b. The results are shown in Table 5.

TABLE 5 Effect of the second extraction experiment

Note: the extraction yield b is (GM content in supernatant b/GM content in fermentation broth) 100%

Test example 6

Centrifuging biosynthetic germacrene A fermentation liquor 2400m L (germacrene A content is 10.25 g/L) by a high-speed centrifuge, removing an upper layer solution to obtain a bottom solid sediment, adding absolute ethanol 2400m L into the bottom solid sediment, stirring and extracting for 1 hour, and filtering to obtain an ethanol extracting solution and solid hypha residues.

Dividing the mycelium residues into six parts, adding 50ml, 100 ml, 150 ml, 200ml, 250 ml and 300ml of petroleum ether respectively, shaking and stirring for 30min, centrifuging, sampling supernate, detecting and analyzing the content and calculating the extraction rate b in the step. The results are shown in Table 6.

TABLE 6 comparison of the effects of different solvent dosages in the second extraction process

Note: the extraction yield b is (GM content in supernatant b/GM content in fermentation broth) 100%

Test example 7

The ethanol extract of Experimental example 6 was divided into 3 parts, numbered 1, 2 and 3, and the ethanol was distilled off under reduced pressure until an oil-water separation interface appeared in the distillation flask, and then cooled to room temperature. And (3) removing the water layer from the No. 1, adding anhydrous sodium sulfate for drying, and filtering out a drying agent to obtain a germacrene A crude product No. 1. No. 2 and No. 3 are respectively added with 80ml of petroleum ether and cyclohexane, shaken for extraction, the water phase is separated, the organic phase is added with anhydrous sodium sulfate for drying, the drying agent is filtered out, and the solvent is evaporated under reduced pressure to obtain crude products of the germacrene A No. 2 and No. 3.

Sampling and weighing No. 1, No. 2 and No. 3 germlined A, respectively placing in an oil bath at 120 ℃, reducing pressure to remove residual solvent, heating to 150 ℃, preserving heat for reaction for 1 hour, weighing and sampling to analyze the germlined A and the converted β -elemene content, and calculating the conversion rate and the recovery rate, wherein the results are shown in Table 7.

TABLE 7 thermal conversion experiments for different extraction processes

Note: GM conversion (EM content in crude EM/GM content in crude GM) 100%;

the recovery rate of EM is (EM content in crude EM/GM content in extract) 100%