CN112142816A

CN112142816A - Triterpenoid saponin compound with neuroprotective effect, and preparation method and application thereof

Info

Publication number: CN112142816A
Application number: CN201910559450.7A
Authority: CN
Inventors: 朴香兰; 邢韶芳; 刘慧�
Original assignee: Minzu University of China
Current assignee: Minzu University of China
Priority date: 2019-06-26
Filing date: 2019-06-26
Publication date: 2020-12-29

Abstract

Triterpene saponin compounds with neuroprotective effect, and its preparation method and application are provided. The invention discloses a gynostemma pentaphylla extract compound with a neuroprotective effect, a formula (I), (II) and (III) and a preparation method thereof, and application thereof in medicine, belonging to the field of phytochemistry. The preparation of the compounds of formula (I), (II), (III) is as follows: extracting the whole plant of gynostemma pentaphylla with an organic solvent with higher polarity to obtain a gynostemma pentaphylla total extract; carrying out primary segmentation treatment on the gynostemma pentaphylla total extract to obtain different parts; LC-MS repeated tracking detection on M/z 1125.6[ M-H ] in negative ion mode]^‑And M/z1109.5[ M-H]^‑Further enriching the concentrated part by column chromatography; purifying the enriched fraction to obtain M/z 1125.6[ M-H ]]^‑Compound I of (1), M/z1109.5[ M-H]^‑Compounds II and III of (1). Pharmacodynamic tests show that the compounds of the formulas (I), (II) and (III) have obvious neuroprotective effect.

Description

Triterpenoid saponin compound with neuroprotective effect, and 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

NerveDegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, etc., are chronic progressive neurological diseases caused by degeneration of neurons in specific regions of the brain, are one of the diseases with high incidence among the elderly worldwide, and seriously harm human health. Oxidative stress is involved in the occurrence and development of neurodegenerative diseases, and is considered to be one of the major causes of neurodegenerative diseases. SY-SY5Y cell is a tumor cell with low differentiation degree, shows moderate dopamine-beta-hydroxylase activity, and has similar cell morphology, physiological and biochemical functions to normal nerve cells; hydrogen peroxide (H)₂O₂) 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) LC-MS repeated tracking detection on M/z 1125.6[ M-H ] in negative ion mode]^-And M/z1109.5[ M-H]^-Further enriching the concentrated part with column chromatography;

(4) purifying the enriched fraction to obtain M/z 1125.6[ M-H ]]^-Compound I of (1), M/z1109.5[ M-H]^-Compounds II and II 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 an HPLC 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 mass spectrum of gypenoside Jp4

FIG. 2 mass spectrum of gypenoside Jp5

FIG. 3 mass spectrum of gypenoside Jp6

FIG. 4 Bar chart of neuroprotective effect of gypenoside Jp4-Jp 6.

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) LCMS detects different elution parts, and detects M/z 1125.6[ M-H ]]^-And M/z1109.5[ M-H]^-Concentrating the components at the 70% ethanol elution part, concentrating the 70% ethanol eluate, mixing the concentrated 70% ethanol eluate according to the ratio of 1: 1, performing silica gel column chromatography, eluting with dichloromethane/methanol at the volume ratio of 10: 1-1: 1, and collecting the eluate in sections to obtain 10 components GPF 70A-J. LCMS tracking detection, M/z 1125.6[ M-H]^-And M/z1109.5[ M-H]^-The components are intensively distributed at the GPF70H part, 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) Performing binary isoconcentration elution on GPF70H56-57 part with Shimadzu LC-20AT semi-preparative HPLC system, BDS HYPERSIL C18 column (250mm × 10mm, 5 μ M), 27% acetonitrile-water as mobile phase AT flow rate of 3ml/min, and collecting M/z 977.5[ M-H]^-And M/z 1125.6[ M-H]^-The tip of the peak is white powder solid compounds (I), (II) and (III) [ named as gypenoside Jp4(gypenoside Jp4), gypenoside Jp5(gypenoside JPS), gypenoside Jp6(gypenoside Jp6)]. The structure identification data of gypenoside Jp4-Jp6 are shown in tables 1-3 below.

TABLE 1 NMR data for Compound Jp4

Table 1 NMR spectroscopic data for compound Jp4

TABLE 2 NMR data for Compound Jp5

Table 2 NMR spectroscopic data for compound Jp5

TABLE 3 NMR data for Compound Jp6

Table 5 NMR spectroscopic data for compound Jp6

Test example 1 neuroprotective Effect test of the Compound of the present invention, gypenoside Jp4-Jp6

1) Experimental Material

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

2) experimental methods

SH-SY5Y cells in logarithmic growth phase are added according to the ratio of 5 multiplied by 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 24h under the condition of saturated humidity, and removing the original culture medium; adding 800 mu mol/L H₂O₂Stimulating SH-SY5Y cells for 4h, adding gypenoside Jp4, Jp5, Jp6 and vitamin C with concentration gradient, continuously incubating for 36h, detecting the light absorption value OD by an MTT or CCK8 method, and calculating the cell survival rate.

3) Test compounds: the compound prepared in example 1 (gypenoside Jp4-Jp 6).

4) The experimental results are shown in figure 4

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

Claims

1. A process for preparing a compound of the following formulae (I), (II) and (III), comprising:

(4) purifying the enriched fraction to obtain M/z 1125.6[ M-H ]]^-Compound I of (1), M/z1109.5[ M-H]^-Compounds II and III of (1).

2. A method according to claim 1, characterized in that: soaking the dried whole plant of the gynostemma pentaphylla in 70-100% methanol or ethanol solution for 30min-12h according to the feeding ratio of 1: 6-1: 15 in the step (1), carrying out ultrasonic extraction for 3 times and 10-30 min/time, and concentrating the extracting solution under reduced pressure to obtain the total extract of the gynostemma pentaphylla.

3. A method according to claim 1, characterized in that: the preferable segmentation method in the step (2) is that when macroporous resin is used for separation, 0-100% methanol or ethanol solution is used for elution; when the silica gel column is used for chromatographic separation, dichloromethane/methanol (10: 1 to 2: 1) with different mixture ratios is eluted; the medium pressure flash preparative column chromatography is eluted with 20-100% methanol solution.

4. A method according to claim 1, characterized in that: step (4) is preferably an HPLC system C18 column, and the mobile phase is chromatographic acetonitrile-water, wherein the concentration of the mobile phase acetonitrile is 20-30%, and binary isocratic elution is performed.

5. Use of a compound of claim 1 for the preparation of a medicament for the treatment of a neurodegenerative disease.

6. Use according to claim 5, wherein the neurodegenerative disease is due to oxidative stress.