CN108191942B - Compound with neuroprotective effect, preparation method and application thereof - Google Patents

Abstract

The invention discloses a gynostemma pentaphylla extract compound with a neuroprotective effect, a preparation method thereof and application thereof in medicines, belonging to the field of phytochemistry]^‑And M/z 1107.6[ M-H]^‑Further enriching the concentrated part by column chromatography; purifying the enriched fraction to obtain M/z 977.5[ M-H ]]^‑Compounds I and II of (1), M/z 1107.6[ M-H]^‑Compound III of (1). Pharmacodynamic tests show that the compounds of the formulas (I), (II) and (III) have obvious neuroprotective effect.

Description

Compound with neuroprotective effect, preparation method and application thereof

Technical Field

The invention relates to saponin compounds, in particular to dammarane type saponin compounds extracted and separated from plant Gynostemma pentaphylla (Gynostemma pentaphylum); the invention also relates to a preparation method of the saponin compound and application of the saponin compound in medicine, belonging to the field of phytochemistry.

Background

Neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease and the like, are chronic progressive nervous system diseases caused by degeneration of neurons in specific regions of the brain, are one of the diseases with high incidence rate among the elderly all over the world, seriously endanger human health, oxidative stress participates in the occurrence and development of neurodegenerative diseases and is considered to be one of the main causes of neurodegenerative diseases, SY-SY5Y is a tumor cell with low degree of differentiation, shows moderate-level dopamine- β -hydroxylase activity, has similar cell morphology, physiological and biochemical functions to normal nerve cells, and hydrogen peroxide (H-H5Y) is used as a basic principle of the treatment of neurodegenerative diseases₂O₂) As an intermediate product of oxidative metabolism in vivo, it can penetrate cell membrane, and cause toxicity to various cells (particularly neuronal cells of brain tissue), resulting in cell death. H₂O₂The SH-SY 5Y-induced nerve cell damage model is a nerve damage model caused by classical oxidative stress and is used for research on neuroprotection.

Evidence indicates that a large amount of antioxidant components, such as vitamins, minerals and phenolic compounds, exist in natural products, and can effectively eliminate free radicals and prevent neurodegeneration. Gynostemma pentaphylla is a whole plant of Gynostemma pentaphylla (Gynostemma) Pentaphyllum (Thunb.) Makino in Cucurbitaceae (Cucurbitaceae), is distributed in mountain valley, hills and rocky mountain areas, is easy to realize mechanized large-scale planting, has abundant resources, has the efficacy superior to ginseng and is inferior to the price of tea. The saponin component is the main drug effect component in the gynostemma pentaphylla, and pharmacological research shows that the gynostemma pentaphylla saponin has various pharmacological activities including anti-inflammatory and antioxidant activities. Therefore, the saponin component with the neuroprotective effect is developed from the gynostemma pentaphylla, and has potential significance for preventing neurodegenerative diseases.

Disclosure of Invention

The present inventors isolated novel compounds having structural formulae (I), (II), (III) from dried whole herb of gynostemma pentaphyllum and studied their neuroprotective effects, thereby completing the present invention.

One object of the present invention is to provide compounds of the following formulae (I), (II), (III):

it is a further object of the present invention to provide a process for the preparation of the compounds of formula (I), (II), (III) above.

A process for preparing a compound of formula (I), (II), (III) above, comprising:

(1) extracting the whole plant of gynostemma pentaphylla with an organic solvent with higher polarity to obtain a gynostemma pentaphylla total extract;

(2) carrying out primary segmentation treatment on the gynostemma pentaphylla total extract to obtain different parts;

(3) l CMS iterative Trace detection on M/z 977.5[ M-H]^-And M/z 1107.6[ M-H]^-Further enriching the concentrated part with column chromatography;

(4) purifying the enriched fraction to obtain M/z 977.5[ M-H ]]^-Compounds I and II, m/z 1107.6[M-H]^-Compound III of (1).

In the preparation method, the organic solvent with higher polarity in the step (1) is preferably a methanol solution or an ethanol solution;

preliminary segmentation is carried out on the twisted-strand blues in the step (2), and the preferred segmentation method is macroporous resin or silica gel column chromatography or medium-pressure rapid preparation column chromatography; the corresponding mobile eluent is 0-100% methanol or ethanol, dichloromethane/methanol with different mixture ratio and 20-100% methanol in turn.

The further purification method in the step (4) is preferably HP L C system C18 column, and the mobile phase is chromatographic acetonitrile-water.

The compounds of the formulae (I), (II), (III) according to the invention have a neuroprotective effect, in particular on H₂O₂The induced SH-SY5Y nerve cell injury has stronger protective effect.

Drawings

FIG. 1 MS (A) and MS/MS (B) spectra of gypenoside J1

FIG. 2 MS (C) and MS/MS (D) spectra of gypenoside J2

FIG. 3 MS (E) and MS/MS (F) spectra of gypenoside J3

FIG. 4 bar graph of neuroprotective effects of gypenoside J1-J3 for each treatment vs blank control: "+" indicates p < 0.05, "+" indicates p < 0.01; each treatment group vs model group: "^#"denotes p < 0.05"^##"denotes p < 0.01; vs positive control group for each experimental group: "^&"denotes p < 0.05"^&&"denotes p < 0.01.

Detailed Description

Example 1 the preparation of the compounds (I), (II), (III) is as follows:

(1) soaking dried whole plant of Gynostemma pentaphyllum Makino in 80% methanol at a feed ratio of 1: 8(w/v) for 30min, and performing ultrasonic extraction for 30 min/time and 3 times; mixing the ultrasonic extracts, and concentrating under reduced pressure to obtain herba Gynostemmatis methanol extract.

(2) Suspending the extract of herba Gynostemmatis methanol extract with appropriate amount of water, adsorbing with macroporous resin HP20, and sequentially eluting with 0%, 20%, 50%, 70%, and 95% ethanol solution to obtain different elution parts.

(3) L CMS detection of different elution sites, M/z 977.5[ M-H [)]^-And M/z 1107.6[ M-H]^-Concentrating the components at 70% ethanol elution part, concentrating 70% ethanol eluate, mixing with 1: 1, subjecting to silica gel column chromatography, eluting with 10: 1-1: 1 volume ratio of dichloromethane/methanol, collecting eluate in segments to obtain 10 components GPF70A-J, L CMS tracking detection, M/z 977.5[ M-H]^-And M/z 1107.6[ M-H]^-The GPF70H part is distributed in a centralized way, a Biotage rapid separation preparative chromatograph is used for separation through a 340g C18 column, the flow rate is 50ml/min, 12CV is eluted by 40-95% methanol in a gradient way, and 56-57% methanol eluent is collected and concentrated under reduced pressure to obtain the GPF70H56-57 part.

(4) Subjecting GPF70H56-57 part to binary isocratic elution with Shimadzu L C-20AT semi-preparative HP L C system, BDS HYPERSI L C18 column (250mm × 10mm, 5 μ M) and 27% acetonitrile-water as mobile phase AT flow rate of 3ml/min, and collecting M/z 977.5[ M-H]^-And M/z 1107.6[ M-H]^-The tip of the peak is white powder solid compounds (I), (II) and (III) [ named as gypenoside J1(gypenoside J1), gypenoside J2(gypenoside J2), gypenoside J3(gypenoside J3)]. The structure identification data of gypenoside J1-J3 are shown in Table 1 below.

TABLE 1 NMR data for gypenoside J1-J3

Test example 1 neuroprotective Effect test of the Compound of the present invention, gypenoside J1-J3

1) Experimental Material

Cell: human bone marrow neuroblastoma cell line SH-SY5Y cell; positive control compound: vitamin C;

2) experimental methods

Will log phase growthSH-SY5Y cell according to 5 × 10³The density of each well is inoculated in a 96-well plate, the temperature is 37 ℃, and the CO content is 5 percent₂Incubating and growing for 24 hr under saturated humidity condition, removing original culture medium, adding 800 μmol/L H₂O₂Stimulating SH-SY5Y cells for 4h, adding gypenoside J1-J3 and vitamin C with concentration gradient (5, 10, 20, 40 mu mol/L), continuously incubating for 36h, detecting absorbance OD by MTT or CCK8 method, and calculating cell survival rate.

3) Test compounds: the compound prepared in example 1(gypenoside J1-J3).

4) The experimental results are shown in figure 4

The results show that the compounds (I), (II) and (III) have obvious neuroprotective effect.

Claims (3)

1. A compound of the formula (I), (II):

。

2. use of a compound of claim 1 for the preparation of a medicament for the treatment of a neurodegenerative disease.

3. Use according to claim 2, wherein the neurodegenerative disease is due to oxidative stress.

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