CN114349623A - Enantiomer-isopimarane diterpene with nerve cell protection activity and preparation method and application thereof - Google Patents

Abstract

The invention discloses an enantiopimarane-type diterpene with nerve cell protection activity and a preparation method and application thereof. In the present invention, a novel enantiopimarane-type diterpene EP-1 with nerve cell protection activity is obtained by separating and obtaining from Euphorbia japonicus, and the in vitro activity test results show that the compound has good nerve cell protection activity, At the concentration of 50 μmol/L, EP-1 can increase the survival rate of induced injury PC12 cells by 23%, and can effectively inhibit the accumulation of ROS in PC12 cells induced by H ₂ O ₂ . EP‑1 has the potential to be a potential lead compound for the treatment of neurodegenerative diseases.

Description

A kind of enantiopimarane-type diterpene with neuroprotective activity and its preparation method and application

technical field

The invention belongs to the technical field of biochemical industry, and in particular relates to an enantiopimarane-type diterpene with nerve cell protection activity and a preparation method and application thereof.

Background technique

Neurodegenerative diseases are a class of diseases in which the structure or function of neurons gradually loses or even dies, leading to dysfunction, mainly including Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic side sclerosing sclerosis (ALS) etc. From 2015 to 2035, China will enter a stage of rapid aging, the elderly population will increase from 212 million to 418 million, and the proportion of the population will increase to 29%. Taking Parkinson's disease as an example, the incidence rate of Parkinson's disease in the elderly over 65 years old in my country is 1.7%, and the number of new patients is about 100,000 every year, showing a rapid growth trend. At present, the etiology of these diseases is still unclear and incurable, which seriously threatens human health and daily life.

In the past ten years, with the rapid development of knowledge and research methods in various disciplines such as molecular biology, neurobiology and behavioral science, many new discoveries have been made in the study of the pathogenesis of neurodegenerative diseases. The research results show that the mechanisms that cause nerve cell death mainly include oxidative stress mechanism, mitochondrial dysfunction mechanism, excitotoxicity mechanism, inflammatory mechanism and apoptosis mechanism. . These findings provide new ideas and targets for the search for new drugs for the treatment of neurodegenerative diseases. Among them, oxidative stress refers to the excessive production of highly active molecules such as reactive oxygen radicals (ROS) and reactive nitrogen radicals (RNS) in the body when the body is subjected to various harmful stimuli, and the degree of oxidation exceeds the scavenging ability of oxides. The system and antioxidant system are out of balance, resulting in accumulation of oxidation products, reduction of reductase activity, peroxidation of DNA/RNA, proteins, lipids and tissue damage. Nerve cells are particularly sensitive to oxidative stress, and oxidative damage to nerve tissue is found in neurodegenerative diseases. Therefore, how to protect the body from oxidative stress and metabolize the excess ROS generated in the body has become an important research direction for the treatment of neurodegenerative diseases.

Euphorbia pekinensis Rupr. is a perennial herb of the family Euphorbiaceae. Its root is used as medicine, and it has the functions of purging water and drinking, reducing swelling and dispelling knots. Literature reports indicate that diterpenoids are the main active components of this plant. The present invention separates and obtains a kind of enantio-isopremane type diterpene with nerve cell protection activity from Euphorbia japonica. The in vitro activity test results show that the compound has better nerve cell protection activity, and the concentration of 50 μmol/L EP-1 can increase the survival rate of induced injury PC12 cells by 23%, and can effectively inhibit the accumulation of ROS in the cells induced by H ₂ O ₂ in PC12 cells. It has the potential to be a lead compound for the treatment of neurodegenerative diseases.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a kind of plant source enantiomer-isopimazone type diterpene EP-1 with novel structure, preparation method and application thereof, specifically including the following content:

In a first aspect, the present invention provides an enantio-isopremane-type diterpene, the structure of which is shown in the following formula (I):

In a second aspect, the present invention provides the application of the enantio-isopimarane-type diterpene described in the first aspect above in the preparation of a medicament for treating neurodegenerative diseases.

Preferably, the neurodegenerative diseases include Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis.

In the third aspect, the present invention provides the preparation method of the enantiopimarane-type diterpene described in the first aspect, the method comprising the following steps:

(1) soaking with methanol after pulverizing the root of Euphorbia japonicus, the leaching solution is concentrated into a crude extract; the crude extract is dispersed in water, and extracted with ethyl acetate;

(2) The ethyl acetate extract was roughly segmented by macroporous resin, and the gradient was carried out with methanol/water with volume ratios of 0:100, 30:70, 50:50, 80:20, and 100:0 as the mobile phase. Elution was carried out with dichloromethane/ethyl acetate with a volume ratio of 5:1 for TLC detection, and the component EPR-3 with an R _f value of 0.5-0.6 was collected;

(3) The component EPR-3 of step (2) is separated by MCI column chromatography, and the volume ratios are 10:80, 20:80, 30:70, 40:60, 50:50, 60:40, 70 :30, 80:20, 90:10, 100:0 methanol/water was used as the mobile phase for gradient elution, dichloromethane/ethyl acetate with a volume ratio of 5:1 was used for TLC detection, and the R _f value was collected. 0.5-0.6 component EPR-3.5;

(4) The component EPR-3.5 described in step (3) was separated by silica gel column chromatography, using petroleum ether/ethyl acetate with a volume ratio of 10:1, 5:1, 3:1, 1:1, 1:2 Gradient elution was performed as the mobile phase, and TLC detection was performed with dichloromethane/ethyl acetate with a volume ratio of 5:1, and the fraction EPR-3.5.3 with an R _f value of 0.55-0.6 was collected;

(5) The component EPR-3.5.3 of step (4) was separated by silica gel column chromatography, and the volume ratios were 50:1, 30:1, 15:1, 8:1, 4:1, 2:1, 1:1 petroleum ether/ethyl acetate was used as the mobile phase for gradient elution, and the volume ratio of 5:1 dichloromethane/ethyl acetate was used for TLC detection, and the components _EPR- 3.5.3.2;

(6) The component EPR-3.5.3.2 of step (5) is purified by semi-preparative liquid chromatography, the mobile phase is methanol/water with a volume ratio of 49:51, the flow rate is 2.0 mL/min, and the detection wavelength is 200 -400 nm to obtain the target compound enantio-isopremane-type diterpene, t _R =51 min.

Preferably, in the step (2), the methanol is soaked for 3 times, and 40 L of methanol is soaked for 7 days each time.

Preferably, the liquid chromatograph of the semi-preparative liquid chromatography in the step (7) is Waters 1525, the detector is a Waters2998 photodiode array detector, and the chromatographic column is a Waters Sunfire C18 semi-preparative column, and the specification is 10 × 250mm.

The beneficial effects of the present invention are as follows: the present invention is isolated from the root of Euphorbia pekinensis Rupr. to obtain an enantio-isopremane-type diterpene EP-1 with a novel structure; the compound has better protection of nerve cells At 50 μmol/L, it can increase the survival rate of PC12 cells induced by injury by 23%, and can effectively inhibit the accumulation of ROS generated in PC12 cells induced by H ₂ O ₂ in cells; this can be used as a potential treatment The leading compound for neurodegenerative diseases is used for preparing medicines for the treatment of neurodegenerative diseases.

Description of drawings

Figure 1 ¹ HNMR spectrum of compound EP-1;

Figure 2 ¹³ CNMR spectrum of compound EP-1;

Figure 3 ¹ H- ¹ H COSY spectrum of compound EP-1;

Figure 4 HSQC spectrum of compound EP-1;

Fig. 5 HMBC spectrum of compound EP-1;

Figure 6 NOESY spectrum of compound EP-1;

Fig. 7 IR spectrum of compound EP-1;

Figure 8 HRESIMS spectrum of compound EP-1;

Figure 9 The effect of compound EP-1 on PC12 cell viability;

Figure 10 DCFH-DA fluorescent probe detects the changes of ROS content in PC12 cells after compound EP-1 treatment.

Detailed ways

The technical solutions of the present invention will be further described in detail below through specific embodiments.

In the following embodiments of the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or commonly used in the art.

In the following examples of the present invention, the methods, unless otherwise specified, are conventional methods in the art.

The preparation of embodiment 1 compound EP-1

The preparation process of compound EP-1 of the present invention is as follows:

(1) after pulverizing the dried root (16.5kg) of Euphorbia japonicus, soak and extract 3 times with methanol at room temperature, soak 7 days with 40L methanol each time, and concentrate under reduced pressure to obtain 3.6kg of total extract;

(2) the crude extract is dispersed in water and extracted with ethyl acetate to obtain ethyl acetate extract;

(3) The ethyl acetate extract was roughly segmented with macroporous resin, and the gradient was carried out with methanol/water with volume ratios of 0:100, 30:70, 50:50, 80:20, and 100:0 as the mobile phase. Elution, collect each elution fraction, perform TLC detection with dichloromethane/ethyl acetate with a volume ratio of 5:1, and collect the fraction EPR-3 with an R _f value of 0.5-0.6;

(4) The component EPR-3 is separated by MCI column chromatography, and the volume ratios are 10:80, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0 methanol/water as mobile phase for gradient elution, collect each elution fraction, perform TLC detection with dichloromethane/ethyl acetate with a volume ratio of 5:1, and combine Collect the fraction EPR-3.5 with an R _f value of 0.5-0.6;

(5) The component EPR-3.5 was separated by silica gel column chromatography, and the gradient washing was carried out with petroleum ether/ethyl acetate with a volume ratio of 10:1, 5:1, 3:1, 1:1, 1:2 as the mobile phase Detach, collect each elution fraction, perform TLC detection with dichloromethane/ethyl acetate with a volume ratio of 5:1, and collect the fraction EPR-3.5.3 with an R _f value of 0.55-0.6;

(6) Component EPR-3.5.3 was separated by silica gel column chromatography, using petroleum ether with a volume ratio of 50:1, 30:1, 15:1, 8:1, 4:1, 2:1, 1:1 Gradient elution was carried out with ethyl acetate/ethyl acetate as the mobile phase, and each elution fraction was collected and detected by TLC with dichloromethane/ethyl acetate with a volume ratio of 5:1, and the fractions with an R _f value of 0.55-0.6 were combined and collected. EPR-3.5.3.2;

(7) Component EPR-3.5.3.2 was purified by semi-preparative liquid chromatography, the mobile phase was methanol/water with a volume ratio of 49:51, the flow rate was 2.0 mL/min, and the detection wavelength was 200-400 nm to obtain the target compound EP-1, t _R =51 min.

The structure of the above target compound EP-1 was determined by means of various spectroscopic methods and literature comparison methods.

The nuclear magnetic data of EP-1 is shown in Table 1, and the results of ¹ HNMR spectrum, ¹³ CNMR spectrum, ¹ H- ¹ H COSY spectrum, HSQC spectrum, HMBC spectrum, NOESY spectrum, IR spectrum, and HRESIMS spectrum are shown in Figures 1-8 respectively. shown.

Table 1 NMR data of compound EP-1

Example 2 Neuroprotective activity of EP-1

After the well-grown PC12 cells in the logarithmic growth phase were digested, they were made into a cell suspension and seeded in a 96-well cell culture plate at 1×10 ⁴ cells/mL, with 100 μL of medium per well, at 37°C, 5% Incubate for 24h under the condition of _CO2 , suck away the old medium, set up blank control group, model group and sample experimental group respectively, in which the sample experimental group is added with fresh medium containing the test sample (EP-1), and the final The concentration was 50 μM, and an equal volume of fresh medium was added to the blank control group and the model group. After pre-cultivation for 24 h, the old medium was sucked away, and fresh medium containing hydrogen peroxide was added to the model group and the sample experimental group respectively. The final concentration of hydrogen peroxide was 500 μM, and the same volume of medium was added to the blank control group.

The cells were cultured for 6 h, and the cell viability was determined by MTT assay. The specific method is as follows: suck off the medium in the above-mentioned 96-well culture plate, add medium containing 0.5 mg/mL MTT to each well, and after culturing for 4 hours, add 100 μL triple lysis solution (10% SDS, 5% MTT to each well) isobutanol and 0.1% HCl). After overnight incubation, complete dissolution of the precipitate was observed and the absorbance at 570 nm was measured. After subtracting the blank, the cell survival rate was expressed as the percentage of the control group: cell growth survival rate (%) = (absorbance value of experimental group - absorbance value of blank)/(absorbance value of control group - absorbance value of blank group) × 100%

The results are shown in FIG. 9 . The results show that the enantio-isopremarane-type diterpene EP-1 can increase the survival rate of the induced damage PC12 cells by 23%.

Example 3 Determination of ROS index

The PC12 cells in good growth state were seeded in a 6-well plate at 1×10 ⁴ cells/mL, with 2 mL of medium per well, and cultured for 24 h at 37°C and 5% CO ₂ , and the old medium was aspirated. Set blank control group, model group and sample experimental group (low-dose group, high-dose group) respectively, wherein sample experimental group adds the fresh culture medium containing test sample (EP-1), and the final concentration of the sample is 20 μM (low-dose group). ), 40 μM (high dose group), blank control group and model group were respectively added with the same volume of culture medium. After 24 hours of pre-cultivation, the old medium was sucked away, and fresh medium containing hydrogen peroxide was added to the model group and the sample experimental group respectively. The final concentration of hydrogen peroxide was 500 μM, and an equal volume of medium was added to the blank control group. Continue to incubate for 6 h, aspirate the medium, wash with PBS, add serum-free medium containing DCFH-DA at a concentration of 10 μM to each well, and incubate for 30 minutes in an incubator. The medium was aspirated, washed with PBS, and imaged with a fluorescence microscope. The stronger the fluorescence intensity, the higher the level of intracellular ROS.

The results are shown in FIG. 10 . The results show that the enantio-isopremane-type diterpene EP-1 of the present invention can effectively inhibit the accumulation of ROS in PC12 cells induced by hydrogen peroxide.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still understand the foregoing embodiments. The technical solutions described are modified, or some technical features thereof are equivalently replaced. Any modification, equivalent replacement, improvement, etc. made within the content and principles of the present invention shall be included within the protection scope of the present invention.

Claims (6)

1. An ent-isopimarane diterpene, which has a structure represented by the following formula (I):

2. use of an ent-isopimarane diterpene according to claim 1 for the preparation of a medicament for the treatment of neurodegenerative diseases.

3. The use of claim 2, wherein the neurodegenerative disease comprises parkinson's disease, alzheimer's disease, huntington's disease, amyotrophic lateral sclerosis.

4. A process for the preparation of an ent-isopimarane-type diterpene according to claim 1, which comprises the steps of:

(1) pulverizing radix Euphorbiae Pekinensis, soaking in methanol, and concentrating the extractive solution to obtain crude extract; dispersing the crude extract in water, and extracting with ethyl acetate;

(2) the ethyl acetate extract was subjected to coarse fractionation using a macroporous resin and gradient elution with methanol/water as mobile phase in a volume ratio of 0:100, 30:70, 50:50, 80:20, 100:0, in a volume ratio of 5:1, performing TLC detection on dichloromethane/ethyl acetate, and collecting R_fA component EPR-3 having a value of 0.5 to 0.6;

(3) separating the component EPR-3 in the step (2) by MCI column chromatography, and using the bodyPerforming gradient elution by taking methanol/water with volume ratio of 10:80, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10 and 100:0 as a mobile phase, and performing gradient elution by taking methanol/water with volume ratio of 5:1, performing TLC detection on dichloromethane/ethyl acetate, and collecting R_fA component EPR-3.5 having a value of 0.5 to 0.6;

(4) and (3) performing silica gel column chromatography separation on the component EPR-3.5 in the step (3), performing gradient elution by using petroleum ether/ethyl acetate as a mobile phase in a volume ratio of 10:1, 5:1, 3:1, 1:1 and 1:2, and performing gradient elution by using petroleum ether/ethyl acetate as a mobile phase in a volume ratio of 5:1, performing TLC detection on dichloromethane/ethyl acetate, and collecting R_fComponent EPR-3.5.3 having a value of 0.55 to 0.6;

(5) and (3) performing silica gel column chromatography separation on the component EPR-3.5.3 in the step (4), performing gradient elution by using petroleum ether/ethyl acetate as a mobile phase in a volume ratio of 50:1, 30:1, 15:1, 8:1, 4:1, 2:1 and 1:1, and performing gradient elution by using petroleum ether/ethyl acetate as a mobile phase in a volume ratio of 5:1, performing TLC detection on dichloromethane/ethyl acetate, and collecting R_fComponent EPR-3.5.3.2 having a value of 0.55 to 0.6;

(6) purifying the component EPR-3.5.3.2 in the step (5) by semi-preparative liquid chromatography, wherein the mobile phase is methanol/water with the volume ratio of 49:51, the flow rate is 2.0 mL/min, the detection wavelength is 200-400nm, and the target compound, namely the enantiomer-isopimarane diterpene, t is obtained_R＝51min。

5. The method according to claim 4, wherein the methanol in the step (2) is soaked 3 times with 40L of methanol for 7 days.

6. The method of claim 4, wherein the semi-preparative liquid chromatography of step (7) is performed using a Waters 1525, a Waters2998 photodiode array detector, and a Waters Sunfire C18 semi-preparative column having a size of 10X 250 mm.

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