阅读说明：本技术 巨大戟二萜化合物及其提取方法与应用 (Euphorbia joridoides diterpene compound and extraction method and application thereof ) 是由 占扎君 章璇 章乐 马列峰 单伟光 于 2020-12-11 设计创作，主要内容包括：如式1或式2所示的巨大戟二萜化合物,化合物1、2的安全性良好,其对不同细胞的细胞毒均较小(IC_(50)>20μM),具有良好的安全性；当给予不同剂量的化合物1、2时,能有效增强阿霉素对MCF-7/Adr的抑制,逆转倍数可达6.5至21.5不等。上述两种新型二萜能明显逆转耐药肿瘤细胞MCF-7/Adr的耐药性,增强肿瘤细胞对化疗药物的敏感性,在抗肿瘤多药耐药中具有较好的应用前景。(The ingenol compound shown in formula 1 or formula 2 has good safety of compound 1 and 2, and has small cytotoxicity (IC) to different cells 50 >20 mu M), has good safety; when different doses of the compounds 1 and 2 are administered, the effect can be effectively increasedThe inhibition of strong adriamycin on MCF-7/Adr has the reversal multiple of 6.5 to 21.5. The two novel diterpenoids can obviously reverse the drug resistance of drug-resistant tumor cells MCF-7/Adr, enhance the sensitivity of the tumor cells to chemotherapeutic drugs and have better application prospect in anti-tumor multi-drug resistance.)

1. An ingenol compound represented by formula 1 or formula 2,

2. the method for extracting ingenol compounds represented by formula 1 or formula 2 as claimed in claim 1, characterized in that the method comprises the steps of:

(1) pulverizing radix kansui root tuber, extracting with 95% ethanol water solution at room temperature, mixing extractive solutions, and concentrating under reduced pressure to obtain radix kansui root tuber extract;

(2) dissolving the euphorbia kansui root tuber extract obtained in the step (1) with water A, extracting twice with petroleum ether A, and combining organic phases; the volume of the water A is 1.95-2 mL/g based on the mass of the euphorbia kansui root tuber extract; the volume ratio of the petroleum ether A to the water A used in each extraction is 1: 1;

(3) concentrating the organic phase obtained in the step (2) under reduced pressure, performing silica gel column chromatography, and performing gradient elution with mixed solution of petroleum ether and ethyl acetate in volume ratios of 20:1, 15:1, 10:1, 5:1, 2:1, 1:1 and 0:1 in sequence, wherein each gradient is eluted by 2 column volumes respectively;

(4) taking the elution part of the mixed solution of petroleum ether and ethyl acetate with the volume ratio of 2:1 in the step (3), concentrating under reduced pressure and evaporating to dryness, taking MCI as a filler, sequentially taking methanol aqueous solutions with volume fractions of 45%, 55%, 65%, 75%, 85% and 95% and methanol as eluents for gradient elution, and respectively collecting the elution parts;

(5) combining the eluted parts of the 85% methanol aqueous solution in the step (4), concentrating under reduced pressure, drying, performing 200-mesh 300-mesh silica gel column chromatography, sequentially performing first gradient elution by using a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 5:1, 4:1, 3:1, 2:1 and 1:1, respectively collecting first gradient eluates, performing TLC detection on the first gradient eluates by using a mixed solution of petroleum ether and acetone in a volume ratio of 2:1 as a developing agent, combining the first gradient eluates with an Rf value of 0.3-0.4, evaporating under reduced pressure to obtain Fr.A, performing 500-mesh silica gel column chromatography on the Fr.A, performing second elution by using a mixed solution of petroleum ether and acetone in a volume ratio of 5:1, collecting second eluates, combining the second eluates with an Rf value of 0.3, and evaporating to obtain a compound 2;

mixing the first gradient eluates with Rf value of 0.5, evaporating to dryness under reduced pressure to obtain Fr.B, subjecting Fr.B to semi-preparative chromatography with 85% methanol water solution as mobile phase and C-18ODS (octadecylsilane chemically bonded silica) as chromatographic column, and collecting t_RThe peak at 21min was evaporated to dryness to afford compound 1.

3. The method for extracting ingenol compounds represented by formula 1 or 2 according to claim 2, wherein the step (1) is carried out by extracting with 95% ethanol aqueous solution 3 times for 7 days.

4. The method for extracting ingenol compounds represented by formula 1 or formula 2 according to claim 2, wherein the volume of the ethanol aqueous solution extracted at each time in step (1) is 2L/kg based on the mass of the euphorbia kansui root.

5. The method for extracting ingenol compounds represented by formula 1 or formula 2 according to claim 2, wherein the volume of the water a in step (2) is 2mL/g based on the mass of the euphorbia kansui root extract.

6. The method for extracting ingenol compounds represented by formula 1 or formula 2 according to claim 2, wherein: the silica gel column in the step (3) is a 100-200-mesh silica gel column.

7. The use of the ingenol compound of formula 1 or 2 according to claim 1 in the preparation of a medicament for reversing tumor resistance.

8. The use of claim 7, wherein: the drug resistance is the drug resistance of the tumor to the adriamycin.

9. The use of claim 7, wherein: the tumor is MCF-7 cell.

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to two novel ingenol compounds, an extraction method thereof and application of the ingenol compounds serving as active ingredients in preparation of a medicine for reversing multidrug resistance of tumor cells.

Background

Cancer is one of the major diseases affecting human health, and with the aging trend of the population and the rapid increase of the population, the incidence and death of cancer are rapidly increased globally. Cancer is becoming a significant cause of death in the population and will be a significant obstacle to increasing life expectancy in humans. Among existing cancer treatment methods, chemotherapy is one of important methods. However, once tumor cells acquire resistance during the use of chemotherapeutic drugs, the effect of tumor chemotherapy is significantly reduced. Statistically, during cancer chemotherapy, about 90% of patients fail to achieve the expected therapeutic effect due to the development of multidrug resistance (MDR). The search for low toxicity to effectively reverse the drug resistance of tumor cells and enhance the sensitivity of tumor cells to chemotherapeutic drugs is more and more prominent in the demand of achieving better treatment and prognosis effects. Therefore, it is important to search for tumor MDR inhibitors.

Among the anti-tumor multi-drug resistance mechanisms, ABC transporter (ATP-binding cassette transporters super family) mediated tumor multi-drug resistance is the most common, wherein P-glycoprotein (P-gp) is the main target point for generating multi-drug resistance and is also the current research hotspot. Tumor multidrug resistance reversal agents with P-gp as a target point are divided into the following categories according to the development sequence: the first generation P-gp inhibitor represented by verapamil (verapamil) has low specific binding rate and strong toxic and side effects on human bodies; the second generation P-gp inhibitor which is represented by VX-710 and is modified on the basis of the structure of the first generation inhibitor interferes the normal metabolism in vivo and limits the clinical application. The third generation P-gp inhibitor is a representative drug, namely tariguidar, which has high affinity to P-gp protein, but the drugs have poor absorption and poor clinical effect. Therefore, a new structure is urgently needed for inhibitors against P-gp.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides two novel ingenol compounds, an extraction method thereof and application of the ingenol compounds in preparation of a medicine for reversing multi-drug resistance of tumor cells by using pharmaceutically acceptable salts or esters thereof as active ingredients.

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

an ingenol compound represented by formula 1 or formula 2,

in particular to ingenol A (1) and kansuinol B (2) with the effect of reversing multidrug resistance of tumor cells, the structures of which are respectively shown as formula 1 and formula 2, and the ingenol is ingenol type diterpene of which the side chain contains 2, 3-dimethylbutyryl (the C-3 or C-20 position is substituted by 2, 3-dimethylbutyryl).

The invention also provides an extraction method of the ingenol compound shown in the formula 1 or the formula 2, the method adopts ethanol extraction, and the extract is obtained by decompressing and concentrating the extract; sequentially extracting the obtained extract, performing silica gel column chromatography and MCI column chromatography, and separating to obtain the ingenol compound shown in formula 1 or formula 2, which specifically comprises the following steps:

(1) pulverizing radix kansui root tuber, extracting with 95% ethanol water solution at room temperature, mixing extractive solutions, and concentrating under reduced pressure to obtain radix kansui root tuber extract; the Euphorbia kansui is purchased from Bozhou in 2017 in 4 months, is produced in Shanxi mare, is identified as Euphorbia kansui, and is deposited in natural drug institute of pharmacy of Zhejiang industry university with the number of No. 20170422.

(2) Dissolving the euphorbia kansui root tuber extract obtained in the step (1) with water A, extracting with petroleum ether A for two times, and combining organic phases; the volume of the water A is 1.95-2 mL/g, preferably 2mL/g, based on the mass of the euphorbia kansui root tuber extract; the volume ratio of the petroleum ether A to the water A used in each extraction is 1: 1; the water A is water, and the water A is used for convenience of description and has no special meaning; the petroleum ether A is the petroleum ether, and the petroleum ether A is used for convenience of description and has no special meaning;

(3) concentrating the organic phase obtained in the step (2) under reduced pressure, performing silica gel column chromatography, and performing gradient elution with mixed solution of petroleum ether and ethyl acetate in volume ratios of 20:1, 15:1, 10:1, 5:1, 2:1, 1:1 and 0:1 in sequence, wherein each gradient is eluted by 2 column volumes respectively;

(4) taking the elution part of the mixed solution of petroleum ether and ethyl acetate with the volume ratio of 2:1 in the step (3), concentrating under reduced pressure and evaporating to dryness, taking MCI as a filler, sequentially taking methanol aqueous solutions with volume fractions of 45%, 55%, 65%, 75%, 85% and 95% and methanol as eluents for gradient elution, and respectively collecting the elution parts;

(5) combining the eluted parts of the methanol water solution with the volume fraction of 85 percent in the step (4), concentrating under reduced pressure, drying, performing 200-mesh 300-mesh silica gel column chromatography, sequentially performing first gradient elution with mixed solution of petroleum ether and ethyl acetate at volume ratio of 5:1, 4:1, 3:1, 2:1 and 1:1, respectively collecting first gradient eluates, performing TLC detection on the first gradient eluent by using a mixed solution of petroleum ether and acetone with a volume ratio of 2:1 as a developing solvent, merging the first gradient eluent with an Rf value of 0.3-0.4, performing reduced pressure evaporation to obtain Fr.A, performing secondary elution on the Fr.A through a 500-mesh silica gel column, then performing secondary elution by using a mixed solution of petroleum ether and acetone with a volume ratio of 5:1, collecting the secondary eluent, merging the secondary eluent with an Rf value of 0.3, and performing evaporation to obtain a compound 2;

mixing the first gradient eluates with Rf value of 0.5, evaporating to dryness under reduced pressure to obtain Fr.B, subjecting Fr.B to semi-preparative chromatography with 85% methanol water solution as mobile phase and C-18ODS (octadecylsilane chemically bonded silica) as chromatographic column, and collecting t_RThe peak at 21min was evaporated to dryness to afford compound 1.

Preferably, step (1) is carried out by leaching 3 times with 95% by volume of an aqueous ethanol solution for 7 days.

Further preferably, the volume of the ethanol water solution extracted in each time in the step (1) is 2L/kg based on the mass of the euphorbia kansui root tuber

Preferably, the silica gel column in the step (3) is a 100-200-mesh silica gel column,8cm。

the invention also provides application of the ingenuity diterpenoid compound shown in the formula 1 or the formula 2 in preparation of a medicine for reversing tumor multidrug resistance.

Preferably, the resistance is tumor resistance to doxorubicin.

Preferably, the tumor is an MCF-7 cell.

Compared with the prior art, the invention has the beneficial effects that: the compounds 1 and 2 are safe and have low cytotoxicity (IC) to different cells₅₀>20 mu M), has good safety; when different doses of the compounds 1 and 2 are administered, the inhibition of MCF-7/Adr by adriamycin can be effectively enhanced, and the reversion multiple can reach 6.5 to 21.5. The two novel diterpenoids can obviously reverse the drug resistance of the drug-resistant tumor cell MCF-7/Adr, enhance the sensitivity of the tumor cell to chemotherapeutic drugs and have better application prospect in anti-tumor multi-drug resistance.

The specific implementation mode is as follows:

the invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.

Example 1

(1) Soaking pulverized radix kansui (5kg) powder in 10L 95 vol% ethanol at room temperature for 3 times (7 days each time), mixing extractive solutions, and concentrating to obtain 491g extract. The Euphorbia kansui is purchased from Bozhou in 2017 in 4 months, is produced in Shanxi mare, is identified as Euphorbia kansui, and is deposited in natural drug institute of pharmacy of Zhejiang industry university with the number of No. 20170422.

(2) 491g of euphorbia kansui root extract obtained in the step (1) is added with 1L of water to be dissolved, extracted twice by petroleum ether, 1L of petroleum ether is used each time, and the organic phase is concentrated under reduced pressure to obtain an organic phase (108 g).

(3) Taking the organic phase in the step (2), adopting silica gel of 100-200-mesh silica gel as column chromatography packing column for chromatography, wherein the column volume is 500mL, and performing gradient elution by using petroleum ether/ethyl acetate mixed solution, wherein the gradient elution step comprises the following steps: sequentially using petroleum ether/ethyl acetate with the volume ratio of 20: 1; 15: 1; 10: 1; 5: 1; 2: 1; 1: 1; 0:1 of mixed solution, and eluting 1000mL of mixed solution respectively in each gradient;

(4) taking an elution part with the volume ratio of petroleum ether to ethyl acetate being 2:1 in the step (3), concentrating under reduced pressure and evaporating to dryness, and taking MCI with the volume of 250mL of column volume as column chromatography packing, wherein the volume percentages are 45 percent respectively; 55 percent; 65 percent; 75 percent; 85 percent; 95 percent; and (3) performing gradient elution by using 100% methanol aqueous solution, eluting two column volumes by each gradient, and collecting and combining solutions obtained by eluting with 85% methanol aqueous solution in volume fraction. Concentrating the solution under reduced pressure, drying, and performing 200-300-mesh silica gel column chromatography in sequence at a volume ratio of 5: 1; 4: 1; 3: 1; 2: 1; eluting petroleum ether/ethyl acetate eluent with the ratio of 1:1, carrying out TLC detection by using a developing agent of petroleum ether/acetone 2:1, detecting spots under 254nm ultraviolet, spraying 10% sulfuric acid ethanol on TLC, heating for color development, combining fractions with similar Rf values and similar color development, combining parts with Rf values of 0.3-0.4, carrying out reduced pressure evaporation to obtain Fr.A, combining parts with Rf values of 0.5, and carrying out reduced pressure evaporation to obtain Fr.B.

(5) Taking Fr.A in the step (4), eluting with 200mL of 500-mesh silica gel column volume and using petroleum ether/acetone solution 5:1 as an elution condition, detecting by TLC with a developing agent of petroleum ether/acetone 2:1, detecting spots under 254nm ultraviolet, spraying 10% sulfuric acid ethanol on TLC, heating for color development, combining fractions with Rf of 0.3, and evaporating to obtain a compound 2(22.3 mg); Fr.B fraction was subjected to semi-preparative chromatography (mobile phase: 85% methanol/water by volume fraction) on a column of C-18ODS (octadecylsilane chemically bonded silica), and t was collected_RPeak 21min, evaporated to dryness to afford compound 1(4.9 mg);

physicochemical Properties and Pop data of Compound 1 and Compound 2

Compound 1: colorless wax; HR-ESI-MS ═ M/z 469.2539([ M + Na ]]⁺Calculated value of 469.2561), the molecular formula was determined to be C₂₆H₃₈O₆(ii) a Optically active [ alpha ]]²⁰ _D+14 (c-0.118, MeOH); infra-red (KBr) v_max 3411,2929,1710cm^-1. The hydrogen and carbon spectra are shown in Table 1.

Compound 2: colorless wax; HRESI-MS [ M + Na ]]⁺469.2539 (calculated 469.2561) and was identified as C₂₆H₃₈O₆(ii) a Optically active [ alpha ]]²⁰ _D＝-46(c＝0.118,CHCl₃) (ii) a Infrared (KBr) v_max 3435, 2960,1725cm^-1. The hydrogen and carbon spectra are shown in Table 1.

TABLE 1 Nuclear magnetic data for Compounds 1 and 2

¹H-NMR:600MHz；¹³C-NMR 150 MHz; solvent: CDCl₃

Example 2 reversal of P-gp mediated multidrug resistance in tumor cells by Compounds

The compounds 1 and 2 prepared in example 1 were prepared into 20 μ M high-concentration solutions using cell culture solutions, and the inhibitory effect on the adriamycin-resistant MCF-7/Adr cell line was evaluated, and the results showed that the cell numbers of the drug-added groups (compounds 1 and 2) were 141% and 153% of the blank control, respectively, after the addition of the above two compounds, confirming that the two compounds have no significant toxicity to the MCF-7/Adr cells, and are consistent with the proliferation-promoting effect of the diterpene compounds linked with short chain branches on the cells at high concentration in the literature.

Logarithmic growth MCF-7/Adr cells were selected and seeded in 96-well plates at a cell density of approximately 7X 10³Per well, compound (1 or 5 μ M) and doxorubicin (0.25,0.5,1,2,4,8, 16,32 μ M) with a series of concentration gradients were administered in the experimental group, verapamil (5 μ M) and doxorubicin (4,8,16,32,64,128 μ M) were administered in the positive control group, doxorubicin (4,8,16,32,64,128 μ M) was administered in the negative control group, 3 wells in each group were inoculated into 96-well plates and incubated in the incubator for 48 h. Determination of the IC of Adriamycin on MCF-7/Adr₅₀(Adr), IC of Adriamycin after addition of test substance or reversal agent positive control drug₅₀(P-gp inhibitor + ADR). Computing IC₅₀(ADR) and IC₅₀The ratio of Pgp inhibitor + ADR is the reversal fold. The results are shown in Table 2.

Experimental results show that in this evaluation method, the pseudoolivine diterpenes (compounds 1 and 2) have significant activity in reversing tumor multidrug resistance at both low concentrations (1 μ M) and high concentrations (5 μ M), with effective concentrations (1 μ M,5 μ M) well below cytotoxicity (neither compound 1 nor compound 2 is able to inhibit cell proliferation at 20 μ M concentrations).

TABLE 2 cytotoxic and Reversal abilities of Compounds 1 and 2 in MCF-7/ADR

The result shows that the adriamycin-resistant human breast cancer cell strain MCF-7/Adr is used as an active screening cell strain. Preliminary studies found that compounds 1,2 had no inhibitory effect on the cells at a concentration of 20 μ M, demonstrating negligible intrinsic toxicity. MTT method for determining IC of adriamycin on cells when compound 1 and compound 2 are combined with adriamycin, adriamycin is singly administered, and positive control verapamil is combined with adriamycin₅₀The reversal indexes of the two compounds on the cell are examined, and the reversal effects of the two compounds on the multidrug resistance of the tumor are explored. Further studies in experiments to reverse multidrug resistance showed that compounds 1 and 2 were present at low concentrations (1. mu.M) andthe inhibition effect of adriamycin on MCF-7/Adr cells can be effectively improved under high concentration (5 mu M), and the reversion index (reverse fold) can reach 6.5 to 21.5.

The research results show that the compounds 1 and 2 can reverse P-gp mediated tumor cell multidrug resistance and enhance the curative effect of chemotherapy drugs.