CN110540504B - Preparation method of ingenane diterpene and application thereof in pharmacy - Google Patents
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Abstract
The invention belongs to the field of traditional Chinese medicine pharmacy, and relates to a preparation method of ingenane diterpene and application of the ingenane diterpene in preparation of anti-HIV drugs. The invention enriches ingenane diterpenoid ingredients from Euphorbia (Euphorbia ebracteolta) of Euphorbia (Euphorbia) and performs content determination of main active ingredients and anti-HIV activity test, and the result shows that: with the increase of the content of the ingene diterpenoid ingredients, the anti-HIV activity is in a clear rising trend; when the content of ingenol-type total diterpene in the enriched component is more than 47%, the enriched component shows strong anti-HIV activity, EC50The value reaches 0.0063 mug/mL, and the cytotoxicity is low (CC)5014), Selection Index (SI) greater than 2000, with clear advantages compared to the positive control drug zidovudine. The invention establishes a method for rapidly enriching ingenane total diterpenes from euphorbia ebracteolata and the enriched ingenane total diterpenes can be used for preparing anti-HIV drugs.
Description
Technical Field
The invention belongs to the field of traditional Chinese medicine pharmacy, relates to a preparation method of ingene type total diterpenoids in Euphorbia radix and application thereof in pharmacy, and particularly relates to a method for enriching ingene type total diterpenoids from Euphorbia radix and application thereof in preparing anti-HIV drugs.
Background
Euphorbia ebracteolata Hayata is a perennial herb of Euphorbiaceae (Euphorbiaceae) and is mainly produced in Henan, Shandong, Jiangsu, Anhui, Zhejiang, Fujian and other provinces, and is used as one of the original plants of Chinese wolfsbane root, the root of which is used as the medicine. The Chinese stellera root is originally recorded in Shen nong Ben Cao Jing (Shen nong's herbal), and mainly treats edema, abdominal distension, phlegm, food, insect accumulation, heart and abdominal pain, cough and asthma, scrofula, subcutaneous nodule, mange and maggot killing, and in recent years, the Chinese stellera root is widely used for treating scrofula, dermatosis and chronic bronchitis, obtains better treatment effect and forms a fixed prescription and dosage form.
Modern pharmacological researches find that the euphorbia pekinensis has activities of bacteriostasis, tumor resistance and the like, and suggest that the euphorbia pekinensis has potential important medicinal value. In particular, in recent years, ingenane-type diterpenoids having anti-HIV activity, which have strong anti-HIV activity, low cytotoxicity, high therapeutic index and the like, have been studied and found in euphorbia pekinensis, but practice has shown that the diterpenoids have extremely low polarity, which is very similar to that of lipid compounds in euphorbia pekinensis, resulting in difficulty in separation from them, and therefore, the establishment of a method for rapidly enriching ingenane-type diterpenoids from euphorbia pekinensis has attracted attention of those skilled in the art.
Based on the current situation of the prior art, the inventor of the application intends to provide a method for rapidly enriching ingenane type total diterpenoid ingredients from euphorbia ebracteolata, and the method provides a foundation for developing high-efficiency and low-toxicity anti-HIV drugs.
Disclosure of Invention
The invention aims to provide a preparation method of ingenane total diterpenoids and application thereof in pharmacy, and particularly relates to a method for enriching ingenane total diterpenoids from euphorbia lunata and application thereof in preparing anti-HIV drugs.
The technical scheme adopted by the invention for realizing the purpose is as follows:
a method for preparing anti-HIV ingenane total diterpenes from Euphorbia radix, comprising the steps of:
(1) mixing Euphorbia radix with organic solvent at a proper ratio, extracting for several times, mixing extractive solutions, concentrating, recovering solvent, and evaporating to obtain extract; the organic solvent is selected from methanol, ethanol, acetonitrile or acetone; the extraction is selected from heating reflux extraction, flash extraction, ultrasonic extraction or percolation extraction;
(2) suspending the extract obtained in the step (1) with water, extracting for a plurality of times with an organic solvent, combining the extract liquor, concentrating, recovering the solvent and evaporating to dryness to obtain an extract; the organic solvent is selected from petroleum ether, cyclohexane, n-hexane or ethyl acetate;
(3) performing normal phase silica gel column chromatography or high speed reverse flow column chromatography on the extract obtained in the step (2) to obtain a product of the next step;
the method of the forward silica gel column chromatography comprises the following steps: taking the extract, and respectively using petroleum ether with 8-10 times of column volume: acetone ═ 10: 1,5: 1,3: 1 or petroleum ether: ethyl acetate ═ 5: 1,2: 1, eluting and collecting petroleum ether: acetone ═ 5: 1 or petroleum ether: ethyl acetate ═ 2: 1, elution portion;
the adopted high-speed countercurrent column chromatography method comprises the following steps: selecting petroleum ether: ethyl acetate: methanol: a water-4: 1:4:1 system, wherein an upper phase (petroleum ether-ethyl acetate) is a stationary phase, a lower phase (methanol-water) is a mobile phase, the upper phase and the lower phase are distributed under the conditions that the flow rate is 1-2 mL/min and the rotation speed of a main engine is 600-1000 r/min, the peak position of the ingenane diterpene compound is determined according to HPLC, the effluent liquid is collected, and the solvent is concentrated, recovered and evaporated to dryness;
(4) performing gel column chromatography on the product obtained in the step (3), eluting with 20 times of column volume of organic solvent (methanol: dichloromethane: 1), and collecting 13 th to 18 th column volumes of active components;
(5) and (3) continuously purifying the active component obtained in the step (4) by using reversed-phase preparative column chromatography, wherein an elution system is acetonitrile-water or methanol-water, and elution conditions are as follows:
(6) measuring the content of active ingredients such as ingenol-20-myristate (E1), ingenol-3-myristate (E2), ingenol-20-palmitate (E3) and ingenol-3-palmitate (E4) of each sample obtained in the steps (1) to (5) and testing the anti-HIV activity of each sample, wherein the result shows that the content of the ingenol-type total diterpenes in the extract obtained in the step (2) is 0.665%, and after enrichment, the content of the ingenol-type total diterpenes in the sample obtained in the step (5) reaches 76.32%; simultaneously, the anti-HIV activity is obviously enhanced, and the EC of the anti-HIV agent is50The value is increased from 0.066 mu g/mL of the initial extract to 0.0053 mu g/mL; when the content of ingenol-type total diterpene in the enriched component is more than 47%, the enriched component shows strong anti-HIV activity, EC50The value can reach 0.0063 mug/mL, and the cytotoxicity is low (CC)5014), Selection Index (SI) greater than 2000, can be used for preparationanti-HIV medicine.
The invention has the following beneficial effects:
the method for enriching the ingenane total diterpenoids is simple, convenient and rapid, and the obtained ingenane total diterpenoids have definite components, high content, strong anti-HIV activity and low toxicity, and are suitable for industrial preparation. The method can be used for large-scale enrichment of ingenane-type total diterpenoids in Euphorbia ebrata, and the obtained ingenane-type total diterpenoids can be used for preparing anti-HIV drugs.
Drawings
FIG. 1 is a flow chart of the enrichment process of example 5.
FIG. 2 shows the structure of ingenol-20-myristate (E1), ingenol-3-myristate (E2), ingenol-20-palmitate (E3) and ingenol-3-palmitate (E4).
FIG. 3 is a liquid chromatogram of the samples DFC-EEH, DFC-EEH-P, DFC-EEH-P2, DFC-EEH-P22, and DFC-EEH-P222 in example 5.
Detailed Description
Example 1: method for enriching ingenane type total diterpene in Euphorbia radix
Taking 100g of Euphorbia radix, pulverizing with a small pulverizer, adding 500mL of 95% ethanol, heating, refluxing and extracting for 3 times, each time for 1 hour, mixing the extractive solutions, concentrating, and evaporating to dryness to obtain 15.4g of extract; dispersing the extract in 250mL of warm water, and extracting with cyclohexane for 4 times, 250mL each time, to obtain 2.9g of cyclohexane extraction part; mixing the sample with 3.0g of silica gel (200-300 meshes), mixing the mixture with petroleum ether: acetone (10: 1; 5: 1; 3: 1) as an elution system, carrying out reduced pressure gradient elution by using a normal phase silica gel chromatographic column, and collecting petroleum ether: acetone ═ 5: 1, fraction 1400 mg; recovering solvent under reduced pressure, separating with Sephadex LH-20 gel column chromatography, eluting with methanol: carrying out isocratic elution on a dichloromethane-1: 1 system, combining the volumes of 13 th to 18 th columns, and collecting the solvent under reduced pressure to obtain 515mg of an active component; this fraction was purified by reverse phase high performance liquid chromatography using acetonitrile: water system, gradient elution, elution conditions are: 0-15 min: acetonitrile: 80% of water: 20 percent; 15-25 min: acetonitrile: 80% of water: 20% -85%: 15 percent; 25-65 min: acetonitrile: 85% of water: 15% -100%: 0%, 65-90 min: acetonitrile: 100% of water: 0 percent. Flow rate 10mL/min, detection wavelength 210nm, column temperature: room temperature; collecting the components in 25-65 min, recovering the solvent under reduced pressure, and separating to obtain the total diterpene with ingenane type 198 mg.
Example 2: method for enriching ingenane type total diterpene in Euphorbia radix
Taking 100g of euphorbia pekinensis roots, crushing the euphorbia pekinensis roots by using a small crusher, adding 500mL of acetonitrile, carrying out flash extraction for 6 times, each time for 1 minute, carrying out suction filtration to obtain filtrate, concentrating and evaporating to dryness to obtain 13.9g of extract; dispersing the extract in 250mL of warm water, and extracting with petroleum ether for 4 times, 250mL each time, to obtain 2.6g of petroleum ether extract part; suspending the above samples with a small amount of methanol, and eluting with high speed countercurrent chromatography for three times; the method comprises the following steps of (1) mixing petroleum ether: ethyl acetate: methanol: water (4: 1:4: 1) is used as an elution system, the above phase is used as a stationary phase, a lower phase solvent is used as a mobile phase, the flow rate is 2.0mL/min, the rotation speed is 900r/min, the detection wavelength is 210nm, the sample loading amount is about 860mg each time, the peak position of the ingene diterpene compound is determined according to HPLC, the effluent liquid in the peak position is collected, the solvent is concentrated and recovered, and the solvent is evaporated to dryness; recovering solvent under reduced pressure, separating with Sephadex LH-20 gel column chromatography, eluting with methanol: isocratic elution is carried out on a trichloromethane 1:1 system, the volumes of 13 th to 18 th columns are combined, and the solvent is collected under reduced pressure to obtain 550mg of an active component; this fraction was purified by reverse phase high performance liquid chromatography with methanol: water system, gradient elution, elution conditions are: 0-15 min: methanol: 85% of water: 15 percent; 15-25 min: methanol: 85% of water: 15% -95%: 5 percent; 25-65 min: methanol: 95% of water: 5% -100%: 0%, 65-90 min: methanol: 100% of water: 0 percent; flow rate 10mL/min, detection wavelength 210nm, column temperature: room temperature; collecting the components at 25-65 min, recovering solvent under reduced pressure, and separating to obtain total diterpene 231 mg.
Example 3: method for enriching ingenane type total diterpene in Euphorbia radix
100g of Euphorbia radix is taken, crushed by a small crusher, added with 500mL of methanol, ultrasonically extracted for 3 times, each time for 30 minutes, filtered, concentrated and evaporated to dryness to obtain 13.1g of extract. Dispersing the extract in 250mL of warm water, and extracting with ethyl acetate for 4 times, 250mL each time, to obtain 3.0g of ethyl acetate extract part; mixing the sample with 3.0g of silica gel (200-300 meshes), mixing the mixture with petroleum ether: ethyl acetate (5: 1; 2: 1; 1: 1) was used as the elution system, and the pressure gradient elution was carried out using a normal phase silica gel column, and petroleum ether was collected: ethyl acetate ═ 2: 1 fraction 1670 mg. Recovering solvent under reduced pressure, separating with Sephadex LH-20 gel column chromatography, eluting with methanol: carrying out isocratic elution on a dichloromethane-1: 1 system, combining the volumes of 13 th to 18 th columns, and collecting the solvent under reduced pressure to obtain 510mg of an active component; this fraction was purified by reverse phase high performance liquid chromatography using acetonitrile: water system, gradient elution, elution conditions are: 0-15 min: acetonitrile: 80% of water: 20 percent; 15-25 min: acetonitrile: 80% of water: 20% -85%: 15 percent; 25-65 min: acetonitrile: 85% of water: 15% -100%: 0%, 65-90 min: acetonitrile: 100% of water: 0 percent; flow rate 10mL/min, detection wavelength 210nm, column temperature: room temperature; collecting the components in 25-65 min, recovering the solvent under reduced pressure, and separating to obtain 200mg of the ingene-type total diterpene part.
Example 4: method for enriching ingenane type total diterpene in Euphorbia radix
Taking 100g of euphorbia pekinensis roots, crushing by using a small crusher, adding 500mL of acetone, percolating and extracting for 3 times for 4 hours each time, combining filtrates, concentrating and evaporating to dryness to obtain 12.7g of extract; dispersing the extract in 250mL of warm water, and extracting with n-hexane for 4 times, 250mL each time, to obtain 2.5g of n-hexane extract; the above samples were suspended with a small amount of methanol and eluted in three portions by high speed counter current chromatography with petroleum ether: ethyl acetate: methanol: water (4: 1:4: 1) is used as an elution system, the upper phase is used as a stationary phase, a lower phase solvent is used as a mobile phase, the flow rate is 2.0mL/min, the rotating speed is 900r/min, the sample amount is about 830mg each time, and the components are collected according to the chromatogram in the example 1; recovering solvent under reduced pressure, separating with Sephadex LH-20 gel column chromatography, eluting with methanol: carrying out isocratic elution on a dichloromethane-1: 1 system, combining the volumes of 13 th to 18 th columns, and collecting the solvent under reduced pressure to obtain 550mg of an active component; this fraction was purified by reverse phase high performance liquid chromatography with methanol: water system, gradient elution, elution conditions are: 0-15 min: methanol: 85% of water: 15 percent; 15-25 min: methanol: 85% of water: 15% -95%: 5 percent; 25-65 min: methanol: 95% of water: 5% -100%: 0%, 65-90 min: methanol: 100% of water: 0 percent; flow rate 10mL/min, detection wavelength 210nm, column temperature: room temperature; collecting the components in 25-65 min, recovering the solvent under reduced pressure, and separating to obtain 215mg of the ingene-type total diterpene part.
Example 5: method for enriching ingenane type total diterpene in Euphorbia radix
Pulverizing Euphorbiae radix 1000g with medium-sized pulverizer, adding 5000mL 95% ethanol, heating and reflux extracting for 3 times, each for 1 hr, filtering, mixing extractive solutions, concentrating, and evaporating to obtain extract 128.4g (DFC-EEH). Dispersing the extract in 2500mL warm water, extracting with petroleum ether for 4 times (2500 mL each time) to obtain 28.0g (DFC-EEH-P) of petroleum ether extract part; mixing the sample with 30.0g of silica gel (200-300 meshes), mixing the mixture with petroleum ether: acetone (10: 1; 5: 1; 3: 1) as an elution system, carrying out reduced pressure gradient elution by using a normal phase silica gel chromatographic column, and collecting petroleum ether: acetone ═ 5: fraction 16.4g of 1 (DFC-EEH-P2); recovering solvent under reduced pressure, separating with Sephadex LH-20 gel column chromatography, eluting with methanol: the dichloromethane-1: 1 system is eluted isocratically, the 13 th to 18 th column volumes are combined, the solvent is collected under reduced pressure to obtain 5.0g of active component (DFC-EEH-P22), and the component is purified by reversed phase medium pressure column chromatography, and the mixture is purified by acetonitrile: gradient elution is carried out by a water system, and the elution conditions are as follows: 0-45 min: acetonitrile: 80% of water: 20 percent; 45-65 min: acetonitrile: 80% of water: 20% -85%: 15 percent; 65-135 min: acetonitrile: 85% of water: 15% -100%: 0%, 135-180 min: acetonitrile: 100% of water: 0 percent; flow rate 20mL/min, detection wavelength 210nm, column temperature: and (4) room temperature. Collecting fractions at 65-135 min, recovering solvent under reduced pressure, and separating to obtain 1.67g of ingene-type total diterpene (DFC-EEH-P222).
Example 6: content determination of each sample in ingenol type total diterpene enrichment method in Euphorbia ebracteolata
Taking the key active components DFC-EEH, DFC-EEH-P, DFC-EEH-P2, DFC-EEH-P22 and DFC-EEH-P222 obtained in example 5 to prepare 0.5mg/mL samples respectively, taking ingenol-20-myristate (E1), ingenol-3-myristate (E2), ingenol-20-palmitate (E3) and ingenol-3-palmitate (E4) as standard substances, measuring the content of four main diterpenes in each sample by using a UPLC-MS method, and semiquantifying the total ingenol type diterpenes; the content determination method adopts a standard curve method, and the liquid chromatogram conditions are as follows: 0-3 min, acetonitrile: water-40%: 60 percent; 3-15 min, acetonitrile: water-40%: 60% -55%: 45%, 15-45 min, acetonitrile: 55% of water: 45% -80%: 20%, 45-60 min, acetonitrile: 80% of water: 20% -100%: 0 percent and the flow rate is 0.3 mL/min; the column temperature is 25 ℃; the detection wavelength is 210 nm; the contents of the respective samples are shown in table 1.
TABLE 1 yield (%) of each sample in the method for enriching ingenane-type total diterpenes in Euphorbia radix and the content (%)% of diterpene components therein
Remarking: NF means not detected; the yield represents the extract extraction rate or the yield of each step of sample compared with the previous step of sample.
Example 7: anti-HIV Activity test of samples from the method for enriching the ingenane-type Total diterpenes in Euphorbia lunata
The key active ingredients obtained in example 5, DFC-EEH-P, DFC-EEH-P2, DFC-EEH-P22, and DFC-EEH-P222, were used for anti-HIV activity (EC)50) Testing and cytotoxic Activity (CC)50) Testing and calculating therapeutic index (SI, CC)50/EC50). The anti-HIV activity test method comprises infecting MT with HIV-1NL4-3 strain4Cells (infection times are 0.001), drugs with different concentrations are added at the same time, a fresh matrix containing drugs with proper concentrations is added after 48 hours of infection to maintain the normal growth of the cells, a P24ELISA kit is adopted to analyze the replication condition of the virus after 4 days, and zidovudine (AZT) is used as a positive drug in the experiment; the cytotoxic effect of the above active site on MT4 cells is determined by measuring ATP content in metabolized active cells by cell survival experiment(ii) the survival rate of the cell; the results show (as shown in table 2): when the content of the ingenane total diterpenes in the enriched component is more than 47%, the sample shows extremely strong anti-HIV activity, EC50The value can reach 0.0063 mug/mL, and the cytotoxicity is low (CC)5014), Selection Index (SI) greater than 2000, EC of the sample after further enrichment50The value can reach 0.0053 mu g/mL, CC50The Selection Index (SI) is greater than 2000, 11.
TABLE 2 anti-HIV Activity and cytotoxicity test results of samples in the ingenane-type Total diterpene enrichment method
Claims (5)
1. A method for preparing ingenol type total diterpene, which comprises the following steps:
(1) mixing Euphorbia radix with organic solvent such as methanol, ethanol, acetonitrile or acetone at a proper ratio, extracting for several times, mixing extractive solutions, and recovering solvent to obtain extract;
(2) suspending the extract obtained in the step (1) with water, extracting for several times with organic solvent petroleum ether, cyclohexane, n-hexane or ethyl acetate, combining the extracts, and recovering the solvent to obtain an extract;
(3) separating the extract obtained in the step (2) by normal phase silica gel column chromatography or high speed reverse flow column chromatography, collecting and combining the eluent containing the ingenane diterpenoid components to obtain the ingenane diterpenoid crude extract;
(4) and (3) further separating the ingenane diterpene crude extract obtained in the step (3) by gel column chromatography, eluting by using a methanol-dichloromethane or methanol-trichloromethane mixed solvent, collecting and combining eluates containing ingenane diterpene components, and obtaining the ingenane total diterpene.
2. The method of claim 1, wherein said extraction of step (1) is selected from the group consisting of heated reflux extraction, flash extraction, ultrasonic extraction, and percolation extraction.
3. The process of claim 1, wherein the normal phase silica gel column chromatography involved in step (3) is performed under the following conditions: petroleum ether-acetone as 10: 1. 5: 1. 3: 1 or petroleum ether-ethyl acetate in a ratio of 5: 1. 2: 1. 1:1, and collecting petroleum ether-acetone 5: 1 or petroleum ether-ethyl acetate 2: 1, elution fraction.
4. The process of claim 1, wherein the separation conditions of the high-speed countercurrent column chromatography involved in step (3) are: adopting a petroleum ether-ethyl acetate-methanol-water system mixed in a ratio of 4:1:4:1, wherein the petroleum ether-ethyl acetate in the upper phase is used as a stationary phase, and the methanol-water in the lower phase is used as a mobile phase; determining chromatographic peak containing the ingenane diterpenoid according to the ultraviolet spectrum or mass spectrum of the effluent, and collecting and combining corresponding effluent.
5. Use of a crude ingenol-type diterpene extract or total ingenol-type diterpene obtained by the method of claim 1 for the preparation of a medicament against HIV.
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