阅读说明：本技术 一种倍半萜烯异构体衍生物高效转化和高纯度单体制备方法及其在抗肿瘤药物中的应用 (High-efficiency conversion and high-purity monomer preparation method of sesquiterpene isomer derivative and application of sesquiterpene isomer derivative in antitumor drugs ) 是由 孙蓉 王岱杰 吴东进 宋祥云 杜航 黄娜娜 钟颖 李建超 郭崇 李晓骄阳 刘闰平 于 2021-10-09 设计创作，主要内容包括：本发明提供一种倍半萜烯异构体衍生物高效转化和高纯度单体制备方法及其在抗肿瘤药物中的应用,属于化合物制备和单体化合物分离技术领域。所述制备方法至少包括：将吉马酮转化为芹子烯,并使用高速逆流色谱和制备型HPLC分离提取,获得芹子烯三种构型单体；所述高速逆流色谱中溶剂系统为含Ag～(+)的正己烷/甲醇/水。本发明提供的制备方法具有简单、分离度好、纯度高等优点,为倍半萜烯异构体衍生物等相关化合物的提取分析提供了新的解决方法,具有良好的实际应用之价值。(The invention provides a method for preparing high-efficiency conversion and high-purity monomers of sesquiterpene isomer derivatives and application of the sesquiterpene isomer derivatives in antitumor drugs, and belongs to the technical field of compound preparation and monomer compound separation. The preparation method at least comprises the following steps: converting germacrone into apiene, and separating and extracting by using high-speed counter-current chromatography and preparative HPLC to obtain three configuration monomers of the apiene; the solvent system in the high-speed counter-current chromatography contains Ag + N-hexane/methanol/water. The preparation method provided by the invention has the advantages of simplicity, good separation degree, high purity and the like, and provides extraction analysis for related compounds such as sesquiterpene isomer derivatives and the likeThe new solution is provided, and the method has good practical application value.)

1. A method for preparing high-efficiency conversion and high-purity monomers of sesquiterpene isomer derivatives, which is characterized by at least comprising the following steps:

converting germacrone into apiene, and separating and extracting by using high-speed counter-current chromatography and preparative HPLC to obtain three configuration monomers of the apiene; the solvent system in the high-speed counter-current chromatography contains Ag⁺N-hexane/methanol/water.

2. The method of claim 1, wherein the conversion of germacrone to celeriane is carried out by:

adding silica gel and phosphoric acid aqueous solution into the germacrone solution, and reacting at high temperature to obtain the germacrone;

preferably, the volume mass of the germacrone and the silica gel is 100-: 1-20 mg;

preferably, the concentration of the phosphoric acid aqueous solution is controlled to be 1-100%;

preferably, the specific temperature of the high-temperature condition is controlled to be 30-130 ℃;

preferably, the reaction time is 1-60 min.

3. The method of claim 2, wherein the reaction process and product are monitored by liquid chromatography under conditions comprising: the chromatographic column is C₁₈Column, mobile phase: methanol-water (95:5, v/v); detection wavelength: 200-220nm (preferably 206 nm); flow rate: 0.5-5mL/min (preferably 1.0 mL/min).

4. The preparation method of claim 1, wherein the separation and extraction by using high-speed countercurrent chromatography and preparative HPLC is as follows:

in HSCCC separation, an upper phase of n-hexane/methanol/silver nitrate solution is selected as a mobile phase, a lower phase is selected as a stationary phase, the volume ratio of the n-hexane/methanol/silver nitrate solution is 10:9.5:0.5, wherein Ag in the silver nitrate solution is⁺The concentration is controlled to be 1-5 mol/L.

5. The preparation method according to claim 4, wherein the stationary phase is injected into the HSCCC separation column at a flow rate of 10-30 mL/min (preferably 20 mL/min); the HSCCC rotates clockwise at the speed of 600-800 rpm (preferably 800rpm) at the temperature of 25-35 ℃ (such as 30 ℃), the mobile phase is pumped into the HSCCC separation column at the flow rate of 1.0-5.0 mL/min (preferably 2.0mL/min) in a mode from the tail end to the head end; when the fluid mechanics balance is achieved, the sample solution is injected through an injection valve;

preferably, the sample solution is obtained by dissolving a sample obtained after conversion of germacrone into apiene in an upper phase and a lower phase of a n-hexane-methanol-silver nitrate solution.

6. The method of claim 4, wherein the HSCCC separation process further comprises: continuously monitoring the effluent of the column at 210nm with an ultraviolet-visible detector;

collecting the fraction every 1-6min (preferably 5min) with an autosampler;

after separation, blowing the solvent in the HSCCC separation column by using nitrogen; vacuum concentrating the blown solvent, and extracting the residue with n-hexane; discarding the upper layer, vacuum concentrating the lower aqueous phase to dryness, and recovering silver nitrate.

7. The method of claim 4, wherein the separation is further performed by preparative HPLC; the specific method comprises the following steps: with C₁₈Separating by using a semi-preparative chromatographic column, wherein the preparation liquid phase conditions are as follows: methanol-water (92: 8), flow rate: 5-20mL/min (preferably 10mL/min), and 50-200 μ L (preferably 100 μ L).

8. The method according to claim 7, wherein the effluent of the column is continuously monitored at 200-220nm (preferably 206nm) using a UV-visible detector; fractions were collected every 0.5-3min (preferably 1min) using an autosampler.

9. The apiene configuration monomer obtained by the preparation method of any one of claims 1-8, wherein the apiene configuration monomer is alpha-apiene, beta-apiene and gamma-apiene respectively.

10. The use of the apiene configuration monomer of claim 9 in the preparation of an anti-tumor medicament;

preferably, the tumor includes osteogenic sarcoma, liver cancer, lung cancer and breast cancer.

Technical Field

The invention belongs to the technical field of compound preparation and monomer compound separation, and particularly relates to a method for preparing high-purity monomer and high-efficiency conversion of sesquiterpene isomer derivatives and application of the sesquiterpene isomer derivatives in antitumor drugs.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Sesquiterpenes are fragrant molecules with tranquilizing and anti-inflammatory effects, have anti-inflammatory and antihistamine effects, and also have effects of relieving itching, resisting allergy, relieving pain, resisting spasm, regulating immunity, sterilizing, promoting qi circulation, removing blood stasis, and repairing skin. Sesquiterpenes also act on the limbic system of the brain through the blood-brain barrier, and in particular, affect the limbus of the pineal and pituitary glands, increasing brain supply. The sesquiterpene component is rich in plant essential oil such as Olibanum, herba Agastaches, and fructus Citri Limoniae.

The apiene is a typical sesquiterpene compound, has three configurations of alpha-, beta-and gamma-as shown in figure 1, is widely present in plants such as prodenia, curcuma aromatica, celery seeds, cyperus rotundus, black pepper, ginger, saussurea lappa and the like, and has the characteristics of resisting spasm, relieving pain, balancing central nervous system and assisting in determining. However, to date, no separation studies have been conducted on the three configurational monomeric components of apiene.

High-speed countercurrent chromatography (HSCCC), an unsupported liquid-liquid partition chromatography technique, has been widely used to purify bioactive components from natural products. Compared with silica gel column chromatography, HSCCC eliminates the problems of irreversible adsorption, peak tailing, inactivation and contamination of the solid support.

Disclosure of Invention

Based on the defects of the prior art, the invention provides a method for preparing high-efficiency conversion and high-purity monomers of sesquiterpene isomer derivatives and application of the sesquiterpene isomer derivatives in antitumor drugs. According to the invention, germacrone is used as a raw material, reaction parameters and conditions are optimized, so that apiene is generated efficiently, three configuration monomers of the apiene are further separated by HSCCC, a material basis is provided for further research on pharmacological properties of the isoditerpene isoterpenoid isomer derivatives of the apiene, research and development of related medicines and the like, and the method has good practical application value.

In a first aspect of the present invention, there is provided a process for the preparation of high efficiency conversion and high purity monomers of sesquiterpene isomer derivatives, the process at least comprising:

converting germacrone into apiene, and separating and extracting by using high-speed counter-current chromatography and preparative HPLC to obtain three configuration monomers of the apiene; the solvent system in the separation and extraction method is Ag-containing⁺N-hexane/methanol/water.

In a second aspect of the invention, the celery alkene configuration monomer obtained by the preparation method is provided; the described apiene configuration monomer is three kinds, respectively are alpha-apiene, beta-apiene and gamma-apiene.

In a third aspect of the invention, the preparation method and/or the application of the sesquiterpene isomer derivative in preparing antitumor drugs are provided.

It is noted that tumors are used in the present invention as known to those skilled in the art, and include benign tumors and/or malignant tumors. Benign tumors are defined as cellular hyperproliferation that fails to form aggressive, metastatic tumors in vivo. Conversely, a malignant tumor is defined as a cell with various cellular and biochemical abnormalities capable of forming a systemic disease (e.g., forming tumor metastases in distant organs).

Further, the tumors include, but are not limited to, osteogenic sarcoma, liver cancer, lung cancer, and breast cancer.

The beneficial technical effects of one or more technical schemes are as follows:

according to the technical scheme, the germacrone is converted into the apiene, and the three configuration monomers of the apiene are obtained by using high-speed countercurrent chromatography and preparative HPLC (high performance liquid chromatography) separation and extraction, and the application of the apiene in resisting tumors is further researched; the inhibition effect on human osteosarcoma derived from mesenchymal tissue, human hepatocellular carcinoma derived from epithelial tissue and mouse hepatocellular carcinoma is proved, and the apiene has potential wide anti-tumor effect.

The preparation method of the technical scheme has the advantages of simplicity, good separation degree, high purity and the like, provides a new solution for extraction and analysis of related compounds such as sesquiterpene isomer derivatives and the like, and has good practical application value.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application.

FIG. 1 is a chemical structural diagram of the raw material germacrone and the conversion product of the present invention;

FIG. 2 is a graph showing the effect of silica gel addition on conversion in an example of the present invention;

FIG. 3 is a graph showing the effect of phosphoric acid addition on conversion in examples of the present invention;

FIG. 4 is a graph of the effect of conversion time on conversion in an example of the present invention;

FIG. 5 is a graph showing the effect of reaction temperature on conversion in an example of the present invention;

FIG. 6 is a high-speed countercurrent chromatographic separation of the conversion product in the example of the present invention;

FIG. 7 is a preparative liquid chromatogram of the mixed components of gamma-apiene and alpha-apiene according to an embodiment of the present invention;

FIG. 8 is an HPLC chart of total converted matter and monomers (1: gamma-apiene; 2: beta-apiene; 3: alpha-apiene) in the example of the present invention.

FIG. 9 shows that the complete medium was used as a negative control in the present example, and the concentrations were 5, 10, 20, 40, 80, 160, 320, 640, 1000. mu.g/mL^-1The solution of the apiene can inhibit MG-63, Hep G2 and Hepa 1-6 cells. A. Inhibition of MG-63 by apiene solution, IC₅₀＝147.8μg·mL^-1(ii) a B. Inhibition of Hep G2 by apiene, IC₅₀＝237.1μg·mL^-1(ii) a C. Inhibition of Hepa 1-6 by Apitene, IC₅₀＝143.0μg·mL^-1。

Detailed Description

It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

In one exemplary embodiment of the present invention, there is provided a method for preparing a high-efficiency conversion and high-purity monomer of sesquiterpene isomer derivatives, comprising at least:

converting germacrone into apiene, and separating and extracting by using high-speed counter-current chromatography and preparative HPLC to obtain three configuration monomers of the apiene; the solvent system in the high-speed counter-current chromatography contains Ag⁺N-hexane/methanol/water.

The specific method for converting the germacrone into the apiene comprises the following steps:

adding silica gel and phosphoric acid aqueous solution into the germacrone solution, and reacting at high temperature to obtain the germacrone.

Wherein the volume mass of the germacrone and the silica gel is 100-: 1-20mg, such as 200 μ L: 4mg, 200. mu.L: 8mg, 200. mu.L: 12mg, 200. mu.L: 16mg and 200. mu.L: 20 mg;

the concentration of the phosphoric acid aqueous solution is controlled to be 1-100%, such as 1%, 10%, 20%, 40%, 60%, 80% and 100%.

The high temperature is controlled to 30-130 deg.C, such as 30, 50, 70, 90, 110 and 130 deg.C.

The reaction time is 1-60min, such as 1, 10, 20, 30, 40, 50 and 60 min.

The screening optimization conditions show that the conversion rate is highest and can reach 41.34% when the concentration of the phosphoric acid aqueous solution is 80%, the addition amount of silica gel is 8mg, the temperature is 70 ℃ and the reaction time is 60 min.

In another embodiment of the present invention, the above reaction process and product can be monitored by liquid chromatography under the following detection conditions: the chromatographic column is C₁₈Column, mobile phase: methanol-water (95:5, v/v); detection wavelength: 200-220nm (preferably 206 nm); flow rate: 0.5-5mL/min (preferably 1.0 mL/min).

In another embodiment of the present invention, the specific separation and extraction method using high-speed countercurrent chromatography and preparative HPLC comprises:

in HSCCC separation, an upper phase of n-hexane/methanol/silver nitrate solution is selected as a mobile phase, a lower phase is selected as a stationary phase, the volume ratio of the n-hexane/methanol/silver nitrate solution is 10:9.5:0.5, wherein Ag in the silver nitrate solution is⁺The concentration is controlled to be 1-5mol/L, such as 1, 2, 3, 4 and 5 mol/L. When the concentration of silver ions in water is kept at 3mol/L, 3 kinds of celery are addedK of a compound of a vinylidene monomer_DThe values almost conform to the gold rule. Therefore, an n-hexane-methanol-silver nitrate solution (3mol/L) (10:9.5:0.5, v/v) solvent system was finally selected for HSCCC separation.

In another embodiment of the invention, the stationary phase is injected into the HSCCC separation column at a flow rate of 10-30 mL/min (preferably 20 mL/min); the HSCCC rotates clockwise at the speed of 600-800 rpm (preferably 800rpm) at the temperature of 25-35 ℃ (such as 30 ℃), the mobile phase is pumped into the HSCCC separation column at the flow rate of 1.0-5.0 mL/min (preferably 2.0mL/min) in a mode from the tail end to the head end; when the fluid mechanics equilibrium is reached, the sample solution is injected through the injection valve.

The sample solution may be obtained by dissolving a sample obtained by converting germacrone into apiene in the upper phase and the lower phase of n-hexane-methanol-silver nitrate solution.

More specifically, the HSCCC separation process further comprises: continuously monitoring the effluent of the column at 210nm with an ultraviolet-visible detector;

collecting the fraction every 1-6min (such as 5min) with an autosampler;

after separation, blowing the solvent in the HSCCC separation column by using nitrogen; vacuum concentrating the blown solvent, and extracting the residue with n-hexane; discarding the upper layer, vacuum concentrating the lower aqueous phase to dryness, and recovering silver nitrate.

Experiments demonstrated that under optimized conditions, the crude sample was completely dissolved in the selected biphasic solvent system for HSCCC separation with the following parameters: the rotation speed is 800rpm, the two-phase solvent system is n-hexane-methanol-silver nitrate solution (3mol/L) (10:9.5:0.5, v/v), the flow rate of the mobile phase is 2.0mL/min, and the separation temperature is 25 ℃. Under the optimal conditions, the compound 2 (namely beta-apiene) and the compound 1+3 mixture (namely gamma-apiene and alpha-apiene) are obtained from the crude extract, and the stationary phase retention rate of the countercurrent chromatography is 51 percent.

Thus the mixture of compounds 1 and 3 was further separated using preparative HPLC; the specific method comprises the following steps: with C₁₈Separating by using a semi-preparative chromatographic column, wherein the preparation liquid phase conditions are as follows: methanol-water (92: 8), flow rate: 5-20mL/min (preferably 10mL/min), and 50-200 μ L (preferably 100 μ L). Detection by UV-VisThe column effluent was monitored continuously at 200-220nm, preferably 206 nm. Fractions are collected every 0.5-3min (e.g., 1min) using an autosampler.

In a second aspect of the invention, the celery alkene configuration monomer obtained by the preparation method is provided; the described apiene configuration monomer is three kinds, respectively are alpha-apiene, beta-apiene and gamma-apiene.

In a third aspect of the invention, the preparation method and/or the application of the sesquiterpene isomer derivative in preparing antitumor drugs are provided.

It is noted that tumors are used in the present invention as known to those skilled in the art, and include benign tumors and/or malignant tumors. Benign tumors are defined as cellular hyperproliferation that fails to form aggressive, metastatic tumors in vivo. Conversely, a malignant tumor is defined as a cell with various cellular and biochemical abnormalities capable of forming a systemic disease (e.g., forming tumor metastases in distant organs).

Further, the tumors include, but are not limited to, osteogenic sarcoma, liver cancer, lung cancer, and breast cancer.

Tests prove that the celery alkene has the inhibiting effect on human osteosarcoma derived from mesenchymal tissues, human hepatocellular carcinoma derived from epithelial tissues and mouse hepatocellular carcinoma. The result indicates that the apiene not only has good inhibition effect on specific tumors, but also has a wide anti-tumor spectrum.

The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Examples

One-factor experiment for converting germacrone into apiene isomer

1. The process flow comprises the following steps:

accurately sucking 200 mu L of germacrone crude sample into a test tube with a plug by using a pipette, adding different amounts of silica gel (200 meshes and 300 meshes) as catalysts for reaction, and reacting for a certain time at a certain temperature in different pH environments. After the reaction, the reaction solution was transferred to a centrifuge tube, centrifuged at 8000r/min for 10min, and the supernatant was precisely measured at 10. mu.L, dissolved and diluted in 1mL of methanol, and then detected by HPLC.

2. The process optimization:

selecting different silica gel adding amounts (0, 4, 8, 12, 16 and 20mg), adding 5 mu L of phosphoric acid aqueous solutions with different concentrations (0%, 20%, 40%, 60%, 80% and 100%), different reaction times (10, 20, 30, 40, 50 and 60min) and different reaction temperatures (30, 50, 70, 90, 110 and 130 ℃) to perform single-factor experiments, detecting germacrone peak areas and total peak areas of three apiene by HPLC, calculating the reduction amount of the germacrone peak areas and the increase amount of the total peak areas of alpha-, beta-and gamma-apiene isomers, and comparing the influences of different conditions on conversion rate.

2.1 optimization of silica gel addition

A pipette gun was used to accurately pipette 200. mu.L of the crude germacrone into stoppered tubes, and different amounts of silica gel (0, 4, 8, 12, 16 and 20mg) were added to each tube, followed by 5. mu.L of 80% aqueous phosphoric acid solution and reaction at 90 ℃ for 30 min. After the reaction, the reaction solution was transferred to a centrifuge tube, centrifuged at 8000r/min for 10min, and the supernatant was precisely measured at 10. mu.L, dissolved and diluted in 1mL of methanol, and then detected by HPLC. As a result, it was found (FIG. 2) that the conversion rate increased gradually and the conversion rate decreased continuously when the amount of silica gel added was 8mg, and therefore the amount of silica gel added was 8mg, which was the optimum amount for the single factor.

2.2 optimization of different pH environments

mu.L of the germacrone crude sample was accurately pipetted into a stoppered test tube by using a pipette, 5. mu.L of phosphoric acid aqueous solutions (0%, 20%, 40%, 60%, 80% and 100%) at different concentrations were added, and 8mg of silica gel was added and reacted at 90 ℃ for 30 min. After the reaction, the reaction solution was transferred to a centrifuge tube, centrifuged at 8000r/min for 10min, and the supernatant was precisely measured at 10. mu.L, dissolved and diluted in 1mL of methanol, and then detected by HPLC. The results show (FIG. 3) that the conversion is highest when the amount of phosphoric acid added is 80%, and therefore the optimum amount of phosphoric acid added is 80% for one factor.

2.3 optimization of reaction time

Accurately sucking 200 μ L of germacrone crude sample into a stoppered test tube by using a pipette, adding 8mg of silica gel, adding 5 μ L of 80% phosphoric acid water solution, and reacting at 90 ℃ for 10, 20, 30, 40, 50 and 60min respectively. After the reaction, the reaction solution was transferred to a centrifuge tube, centrifuged at 8000r/min for 10min, and the supernatant was precisely measured at 10. mu.L, dissolved and diluted in 1mL of methanol, and then detected by HPLC. The results show (fig. 4) that at 50min of reaction, the conversion is highest and the conversion decreases for an extended period of time, so that the single-factor optimal reaction time is 50 min.

2.4 optimization of reaction temperature

Accurately sucking 200 mu L of germacrone crude sample into a test tube with a plug by using a pipette, adding 8mg of silica gel, adding 5 mu L of 80% phosphoric acid water solution, and reacting in oil bath pots at 30, 50, 70, 90, 110 and 130 ℃ for 50min respectively. After the reaction, the reaction solution was transferred to a centrifuge tube, centrifuged at 8000r/min for 10min, and the supernatant was precisely measured at 10. mu.L, dissolved and diluted in 1mL of methanol, and then detected by HPLC. The results show (FIG. 5) that the conversion is highest at a reaction temperature of 90 ℃ and therefore the optimum amount of addition for a single factor is 90 ℃.

3. Conditions of liquid phase detection

Mobile phase: methanol-water (95:5 v/v); detection wavelength: 206 nm; flow rate: 1 mL/min. Orthogonal experiments for conversion of damascone to celecoxib isomer

On the basis of a single-factor experiment, the increment of the total peak area of alpha-, beta-and gamma-celery alkene isomers is taken as a response value, and L is selected₉(3⁴) Orthogonal tables design the experiment, and the levels of each factor are shown in table 1.

TABLE 1 Table of levels of Experimental factors for conversion of germacrone to the Apitene isomer

TABLE 2 results of orthogonal experiments

The optimal factors are A2, B2, C1 and D3, namely 80 percent of phosphoric acid aqueous solution, 8mg of silica gel addition, 70 ℃ and 60min of reaction time.

The final conversion rate was 41.34% as verified under optimum conditions. The liquid chromatogram after conversion is shown in FIG. 6.

Separation experiment of HSCCC

1. Experimental methods

1.1 selection of two-phase solvent System

Selecting a suitable partition coefficient (K) of the target compound_DValue) is one of the most important factors for successful HSCCC separation. In the present study, the HPLC method is adopted to detect K of the target compound in different ratios of n-hexane/ethyl acetate/methanol/water (HEMAT) solvent systems_DValue, to obtain better separation effect, AgNO is added₃And (4) carrying out coordination effect separation.

K of the compound_DThe values were determined by HPLC. Firstly, preparing solvent systems with different proportions in a 5mL test tube and violently shaking, after full layering and balancing, respectively taking 2mL of the two-phase solvent system, adding 3mg of crude extract into the two-phase solvent system, violently shaking for 1min and fully layering, respectively taking 0.5mL of the upper phase and the lower phase, respectively putting the upper phase and the lower phase into a 4mL centrifuge tube, and respectively diluting with 2mL of methanol. The upper phase was analyzed by HPLC directly, and the lower phase was analyzed by HPLC after removal of silver ions by addition of sodium chloride. K_DThe value is defined as the peak area of the compound in the stationary phase divided by the peak area of the corresponding compound in the mobile phase, and is given by the formula:

K_D＝A_U/A_L

A_Uand A_LThe peak areas of the target compound in the stationary phase and the mobile phase.

1.2 preparation of solvent System and sample solution

In the HSCCC separation, a biphasic solvent system consisting of n-hexane/methanol/silver nitrate solution (3mol/L) (10:9.5:0.5, v/v) was placed in a separatory funnel. After vigorous shaking, the solution was allowed to stand for 5min, and after sufficient stratification, the upper and lower phases were separated. The upper phase (mobile phase) and the lower phase (stationary phase) were degassed by ultrasound prior to use. Sample solutions were prepared by dissolving 200mg of the transformation in 10mL of equal proportions of the upper and lower phases.

1.3 HSCCC separation Process

The stationary phase was first injected into the HSCCC separation column at a flow rate of 20.0 mL/min. The HSCCC apparatus was rotated clockwise at 800rpm at 30 ℃ and the mobile phase was pumped into the HSCCC separation column in tail-to-head mode at a flow rate of 2.0 mL/min. When hydrodynamic equilibrium was reached, 200mg of sample solution was injected through the injection valve. The effluent from the column was continuously monitored at 210nm with a uv-visible detector. Fractions were collected into 12mL tubes every 5min using an autosampler. After separation, the solvent in the HSCCC separation column was blown off with nitrogen. The blown-off solvent was concentrated under vacuum at 40 ℃ and the residue was extracted 3 times with an equal volume of n-hexane. Discarding the upper layer, vacuum concentrating the lower water phase at 40 deg.C to dry, and recovering silver nitrate.

1.4 semi-preparative liquid chromatography separation Process

With C₁₈Semi-preparative chromatography columns (5 μm, 10mm × 250mm, i.d.) were used for separation, preparing liquid phase conditions: methanol-water (92: 8), flow rate: 10mL/min, the sample size 100 uL. The effluent from the column was continuously monitored at 206nm with an ultraviolet-visible detector. Fractions were collected into 12mL tubes every 1min using an autosampler.

1.5 HPLC analysis and structural identification

The crude extract and CCC fractions were analyzed by HPLC chromatography. The chromatographic column is C₁₈Column (5 μm, 4.6 mm. times.250 mm, i.d.), mobile phase methanol and water (95:5, v/v), flow rate 1.0mL/min, wavelength 206nm, injection volume 5 μ L. The isolated compound was purified by ESI-MS,¹H-and¹³identification by C-NMR spectroscopy in CDCl₃As a solvent, chemical shifts (δ) are expressed in parts per million (ppm) and coupling constants (J) are expressed in Hz.

2. Results and discussion

2.1 screening of two-phase solvent systems

One of the most important steps in performing HSCCC separation is the selection of a suitable solvent system. According to Ito the golden rule, the partition coefficient of the target compound between the two phases should be in the range of 0.2-2.0 and the separation factor alpha value between the two compounds should be higher than 1.5. Too high K_DValues may result in prolonged elution times and too broad peaks, while too low a K_DValues may result in poor separation of peaks and may not achieve effective separation. The choice of a suitable biphasic system depends on the chemical properties of the compound of interest, such as sample polarity, solubility, ionic form and ability to form complexes. As the polarity of the target compound is smaller, n-hexane-ethyl acetate-methanol-water is found to be an ideal solvent system, and a wide polarity range can be covered by changing the volume ratio of the four solvents. The system was tested for different volume ratios and the K of the target compound was determined by HPLC_DThe value is obtained. However, the actual test results show that the K of the compound_DThe values are all too small to be separated by the conventional HSCCC method.

According to the Hongde rule, AgNO₃Silver ion (Ag) in (1)⁺) Is d¹⁰Form ([ Kr)]4d¹⁰) Occupying the empty s and p orbitals (5s,5px,5py and 5pz), can react with the bonding compound to form different stable complexes. Based on this phenomenon, silver ion chromatography is developed and applied to thin layer chromatography, high performance liquid chromatography, and supercritical fluid chromatography, and thus silver ion complex counter current chromatography is used in the present invention.

K of target compound in n-hexane-ethyl acetate-methanol-water (9:1:9:1, v/v) and (10:0:9.5:0.5, v/v) solvent systems and different concentrations of silver nitrate (0-3 mol/L)_DThe results are shown in Table 3. The results show that if [ Ag ] is not present in the solvent system⁺]All compounds elute quickly and cannot be isolated. With [ Ag ]⁺]The increase in concentration, increase in partition coefficient, and increase in elution time of all peaks. Apart from the separation factor (. alpha.) of Compounds 1 and 3₁₃) The variation is small and the separation coefficient between the remaining peaks is also improved. As shown in Table 3, for the HEMAT (9:1:9:1, v/v) solvent system, K for the compound_DToo small a value is difficult to retain effectively and the separation effect is too poor. For n-hexane-ethyl acetate-methanol-water (10:0:9.5:0.5, v/v) solutionAgent system, when the concentration of silver ion in water is kept at 3mol/L, K of 3 compounds_DThe values almost conform to the gold rule. Therefore, n-hexane-ethyl acetate-methanol-silver nitrate solution (3mol/L) (10:0:9.5:0.5, v/v) solvent system was finally selected for HSCCC separation.

TABLE 3 partition coefficient and separation factor for countercurrent chromatographic separation of apiene isomers

2.2 separation procedure of countercurrent chromatography

As shown in fig. 6, under optimized conditions, the crude sample (200mg) was completely dissolved in the selected biphasic solvent system (20mL) for HSCCC separation with the following parameters: the rotation speed is 800rpm, the two-phase solvent system is n-hexane-methanol-silver nitrate solution (3mol/L) (10:9.5:0.5, v/v), the flow rate of the mobile phase is 2.0mL/min, and the separation temperature is 25 ℃. Under optimal conditions, compound 2(51.6mg), compound 1+3 mixture (75.5mg) were obtained from 200mg of crude extract, with a stationary phase retention of 51% by countercurrent chromatography.

The mixture of compounds 1 and 3 was further separated using preparative HPLC (fig. 7) to give compound 1(28.2mg) and compound 3(14.9 mg).

The liquid phase purity is shown in fig. 8, and the purity of compounds 1-3 is 97.4%, 97.1% and 96.3%, respectively.

Tetraselinene and apiecene antitumor test

1. Materials and methods

1.1. Experimental methods and reagents

All cell lines were purchased from Shanghai bioscience research institute, high-glucose DMEM medium, Fetal Bovine Serum (FBS) from Gibco, USA, PBS, DMSO solvent (99% or more, Reagent grade), pancreatin cell digest (0.25% pancreatin, phenol red), penicillin-streptomycin solution (100X) from Beyotime, China, CCK-8 kit from Vazyme, China, culture bottles, multi-well plates, and other cell culture plastics from Corning, USA.

1.2 cell culture

Three tumor cell lines were used in this study: MG-63 (human osteosarcoma), Hep G2 (human hepatocellular carcinoma), Hepa 1-6 (mouse hepatocellular carcinoma). All cells were cultured in a constant temperature incubator (37 ℃, 5% CO) using complete medium (89% high-glucose DMEM medium, 10% FBS, 1% penicillin-streptomycin solution)₂) And replacing the complete culture medium once a day, and carrying out passage or experiment when the cells grow to 80%.

1.3 CCK-8 assay to assess the Effect of Apitene on proliferation of various tumor cells

Preparing a apiene solution. Weighing 100mg of the above separated apiene, dissolving in 100uL DMSO solution, supplementing complete culture medium to 10mL, and making into 10 mg. multidot.mL^-1The mother liquor of the apiecene. In the subsequent work, a proper amount of the apiene mother liquor is diluted in a complete culture medium to prepare apiene solutions with different concentrations.

CCK-8 is adopted to detect the influence of the apiene on the proliferation condition of each tumor cell. Tumor cells were treated with 10⁴The density of each well was inoculated into a 96-well plate, incubated in an incubator for 24 hours, after which the culture medium in each well was replaced with complete medium and a solution of apiecene (5, 10, 20, 40, 80, 160, 320, 640, 1000. mu.g.mL) at different concentrations^-1) Incubate for 24 hours, making 6 replicate wells per concentration. After 24h of incubation, 10uL of CCK-8 detection reagent was added to each well and incubated for 1h at 37 ℃. The absorbance at 450nm was then measured with a microplate reader and data analyzed using GraphPad Prism 8.0.2.

2. Results and discussion

2.1 the apiene can inhibit MG-63, Hep G2, and Hepa 1-6 cell proliferation

And (3) carrying out cell activity detection on the MG-63, Hep G2 and Hepa 1-6 cells by using the separated apiene solution. The concentrations used in the experiments were 5, 10, 20, 40, 80, 160, 320, 640, 1000. mu.g.mL^-1The cells were treated with the apiecene solution and cultured in complete medium as a negative control. The results show (as shown in FIG. 9) that the celery alkene has inhibition effect on three tumor cells, and the IC of MG-63, Hep G2 and Hepa 1-6₅₀147.8, 237.1 and 143.0. mu.g/mL, respectively^-1。

Experimental results show that the apiene has inhibition effects on human osteosarcoma derived from mesenchymal tissues, human hepatocellular carcinoma derived from epithelial tissues and hepatocellular carcinoma in mice. Therefore, the apiene is suggested to have potential wide anti-tumor effect.

The research explores the influence of the apiene on the proliferation of three tumor cell lines, and the result shows that the apiene can inhibit the proliferation of tumor cells in vitro, and the semi-inhibition concentrations of the apiene to MG-63, Hep G2 and Hepa 1-6 are 147.8, 237.1 and 143.0 mu G/mL respectively^-1。

It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.