Application and preparation method of tripterine

A technology for the use of tripterygium, which is applied in the field of tripterygium, can solve the problems of cumbersome extraction process, high cost, high equipment investment, etc., and achieve the effect of high product purity, high raw material utilization rate and low cost

Inactive Publication Date: 2012-02-15
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because tripteryglide has the highest content in the root bark, the existing technology extracts tripterygium mainly uses the root bark and discards the relatively small amount of cane part, which causes insufficient utilization of raw materials and increases the cost
[0011] The content of tripterine in plants is very low, basically around 0.1%, so it often requires a tedious extraction process
The extraction process of the prior art uses a large number of organic solvents such as dichloromethane, methanol, n-hexane, ethyl acetate, petroleum ether, and chloroform. The recovery steps of these solvents are cumbersome, require high equipment investment, and are likely to cause environmental pollution; In order to obtain a high-purity product of more than 98%, the prior art uses countercurrent chromatography, preparative high-performance liquid chromatography, and other large-scale equipment with high requirements for samples, which also increases the cost.

Method used

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  • Application and preparation method of tripterine
  • Application and preparation method of tripterine
  • Application and preparation method of tripterine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] FAAH, MGL enzyme inhibitory activity detection

[0036] Add 30 μL (1 mg / mL) protein into the injection bottle, then add 2 μL DMSO (blank control group) or different concentrations of tripterine and react at 37 degrees Celsius for 10 minutes. Then add 170 μL of buffer solution containing substrate AEA or 2-OG (in the experiment, 2-OG was used to replace 2-AG, which is difficult to detect quantitatively, and this substitution is well known and allowed in the art), and the concentration of AEA is 5 μM. After reacting at 37° C. for 30 minutes, 200 μL of methanol solution containing heptadecanoic acid (internal standard) was added to terminate the reaction. The production of arachidonic acid and oleic acid in the hydrolyzate was detected by liquid chromatography-mass spectrometry (LC-MS), and graphed with Graphpad Prism 5. In this way, the EC50 values ​​obtained by measuring tripterine to FAAH and MGL enzymes are 4.69 and 16.70 μM respectively (see figure 1 and figure 2 ...

Embodiment 2

[0038] LC-MS detection of endocannabinoid content in cells

[0039] Four hours after cell administration, cells were harvested with methanol / water (1:1, v:v), sonicated and extracted with chloroform. The lower organic layer was collected by centrifugation at 3000 rpm, dried with nitrogen gas, redissolved with 100 μL of 3:1 methanol / chloroform, and then detected the content of endocannabinoids by LC-MS. This experiment showed that after induction by tripterine, the expression of AEA in cells was significantly increased (see image 3 ).

Embodiment 3

[0041] Cytotoxicity test

[0042]Glioma cells were inoculated in a 96-well plate at a concentration of 5000 cells / well, and culture medium containing different concentrations of tripterygium wilfordii was replaced the next day after the cells adhered to the wall for 24 hours. Then add 10 μL of cell counting reagent (CCK-8), continue to incubate for 2 hours, and then measure the absorbance with a 450 nm microplate reader. The number of dead cells was calculated with the blank group as 100%, and the EC50 value of tripterine on various glioma cells was preliminarily predicted based on the curve. Experiments have shown that the EC50 values ​​of tripterine to C6, BT325, SHG44 and other glioma cells are between 2.0-3.5 μM, showing good cytotoxic activity (see Figure 4 ).

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Abstract

The invention discloses application and a preparation method of tripterine. The tripterine can be used for preparing the drug for treating brain glioma. The preparation method of the tripterine comprises the following steps: (1) mixing and crushing the above-ground vine part and underground root part of Tripterygium wilfordii, carrying out reflux extraction with ethanol, filtering and concentrating at reduced pressure; (2) ultrasonically mixing and suspending the concentrated ethanol extract with ethanol-water, standing still and pouring out supernatant; (3) separating the supernatant with adsorbent resin, carrying out sequential gradient eluting with ethanol-water, and collecting eluate; (4) concentrating the eluate at reduced pressure, purifying by silica gel column chromatography, recovering solvent and drying to obtain red powdery extract; and (5) dissolving the extract with hot ethanol to obtain saturated or unsaturated solution, standing to precipitate red crystals and drying. Compared with the prior art, the method has the advantages of high raw material utilization rate, environmental friendliness, simpleness and low cost and is suitable for industrial production; in addition the purity of the prepared product is high.

Description

technical field [0001] The invention relates to the use and preparation method of tripterine. Background technique [0002] 1 The use of tripterine [0003] Tripterygium wilfordii is a pentacyclic triterpenoid that exists in plants such as tripterygium wilfordii and southern snake vine of the Euonymus family. Its molecular formula is C 29 h 38 O, the structural formula is shown in the figure. Previous studies have shown that tripterygium has anti-oxidation, anti-rheumatism, anti-Alzheimer's effects, etc. Pharmacological studies in recent years have confirmed that tripterygne can induce apoptosis of cancer cells by affecting the proteasome of cancer cells. [0004] [0005] Glioma is the most common tumor in the brain, accounting for 35%-60% of all intracranial tumors. It is one of the intracranial malignant tumors with high malignancy, easy recurrence and poor prognosis. cytoplasmic cells. Malignant gliomas are still prone to recurrence even after comprehensive treat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/56A61P35/00C07J63/00
Inventor 傅瑾杨隆河邱彦陈玲朱程刚任杰刘祖国黄锐
Owner XIAMEN UNIV
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