Preparation method and application of triterpenoid with immunosuppressive activity

Abstract

The invention relates to a preparation method of ISO Ailanthus altissima triterpenes ingredients (isomalabaricanetriterpenes) stellettin B with strong immunosuppressive activity, and application thereof in the treatment aspects of organ transplantation rejection reaction and autoimmune diseases as an immunosuppressor. The compound is shown in a structural formula 1. An experiment shows that the compound has strong activity of restraining mice spleen lymphocyte transformation; and the EC50 value achieves 14nM. A lead compound is provided for developing a novel immunosuppressor; development and utilization of marine medicinal resources are facilitated.

Description

technical field

[0001] The invention relates to a preparation method of stellettin B, an isotonic triterpenoid component, and its application as an immunosuppressant in the treatment of organ transplantation anti-rejection and autoimmune diseases. Background technique

[0002] The treatment of organ transplantation rejection and autoimmune diseases is one of the current clinical problems. Although there have been a lot of progress in recent years, the currently available immunosuppressants are still far from meeting the clinical needs. Searching for new immunosuppressants from natural products has always been a hot spot in the development of new drugs. According to reports, there are 5 categories of immunosuppressants currently on the market and under research abroad, many of which are natural products or semi-synthetic products derived from natural raw materials. The successful development of drugs such as sirolimus and mycophenolic acid shows that it is still important to...

Examples

Embodiment 1

[0015] Embodiment 1: Preparation of stellettin B

[0016] Sponge jaspis stellifera (wet weight 5 kg) was pulverized and homogenized, extracted by cold soaking with 40 liters of 95% ethanol for 24 hours, and the filter residue was extracted by cold soaking with 30 liters of 95% ethanol for 24 hours, and then filtered with 30 liters of 95% ethanol. % ethanol cold immersion extraction for 24 hours, combined the filtrate extracted three times, and concentrated under reduced pressure to extractum shape to obtain 162 grams of total extract;

[0017] The total extract was washed with CHCl 2 and H 2 O for partition extraction, CHCl 2 The layer was concentrated under reduced pressure to obtain CHCl 2 layer extract about 53 grams. CHCl 2 The layer extract was subjected to column chromatography crude separation with normal phase silica gel (200-300 mesh), and the mobile phase was dichloromethane: ethyl acetate gradient elution to obtain 15 fractions, marked as Fr.A~Fr.O;

[0018] F...

Embodiment 2

[0022] Embodiment 2: In vitro drug efficacy test of stellettin B

[0023] Mouse spleen lymphocytes were divided into 2 × 10 6 Individual / mL cell density, inoculated in 96-well cell culture plate, select the concentration of stellettin B that embodiment 1 obtains, add stellettin B solution respectively, establish blank zero group (only add culture fluid 200 μ l / hole), lymphocyte growth control group (negative control without drug solution) (adding 200 μl / well of cell suspension), ConA induction control group (adding 200 μl / well of cell suspension and ConA with a final concentration of 5 μg / mL), each test drug + ConA stimulation Groups (adding 200 μl / well of cell suspension, respectively containing different test drugs with different final concentrations and ConA with a final concentration of 5 μg / mL) each set up three replicate wells, and placed them in a 5% CO2 incubator at 37°C for 48 hours. After 48 hours, 20 μl of MTT working solution was added to continue culturing for 4...

technical field

[0001] The invention relates to a preparation method of stellettin B, an isotonic triterpenoid component, and its application as an immunosuppressant in the treatment of organ transplantation anti-rejection and autoimmune diseases. Background technique

[0002] The treatment of organ transplantation rejection and autoimmune diseases is one of the current clinical problems. Although there have been a lot of progress in recent years, the currently available immunosuppressants are still far from meeting the clinical needs. Searching for new immunosuppressants from natural products has always been a hot spot in the development of new drugs. According to reports, there are 5 categories of immunosuppressants currently on the market and under research abroad, many of which are natural products or semi-synthetic products derived from natural raw materials. The successful development of drugs such as sirolimus and mycophenolic acid shows that it is still important to...

Examples

Embodiment 1

[0015] Embodiment 1: Preparation of stellettin B

[0016] Sponge jaspis stellifera (wet weight 5 kg) was pulverized and homogenized, extracted by cold soaking with 40 liters of 95% ethanol for 24 hours, and the filter residue was extracted by cold soaking with 30 liters of 95% ethanol for 24 hours, and then filtered with 30 liters of 95% ethanol. % ethanol cold immersion extraction for 24 hours, combined the filtrate extracted three times, and concentrated under reduced pressure to extractum shape to obtain 162 grams of total extract;

[0017] The total extract was washed with CHCl 2 and H 2 O for partition extraction, CHCl 2 The layer was concentrated under reduced pressure to obtain CHCl 2 layer extract about 53 grams. CHCl 2 The layer extract was subjected to column chromatography crude separation with normal phase silica gel (200-300 mesh), and the mobile phase was dichloromethane: ethyl acetate gradient elution to obtain 15 fractions, marked as Fr.A~Fr.O;

[0018] F...

Embodiment 2

[0022] Embodiment 2: In vitro drug efficacy test of stellettin B

[0023] Mouse spleen lymphocytes were divided into 2 × 10 6 Individual / mL cell density, inoculated in 96-well cell culture plate, select the concentration of stellettin B that embodiment 1 obtains, add stellettin B solution respectively, establish blank zero group (only add culture fluid 200 μ l / hole), lymphocyte growth control group (negative control without drug solution) (adding 200 μl / well of cell suspension), ConA induction control group (adding 200 μl / well of cell suspension and ConA with a final concentration of 5 μg / mL), each test drug + ConA stimulation Groups (adding 200 μl / well of cell suspension, respectively containing different test drugs with different final concentrations and ConA with a final concentration of 5 μg / mL) each set up three replicate wells, and placed them in a 5% CO2 incubator at 37°C for 48 hours. After 48 hours, 20 μl of MTT working solution was added to continue culturing for 4...

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