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Method for separating phellodendrine glucuronic acid conjugate from urine

A technology of aldehyde acid conjugate and glucuronide is applied in the field of separation of metabolites of Phellodendron alkaloids in vivo, which can solve the problems of less Phellodendron alkaloids and no research report on the metabolism, and achieves the effects of simple operation, low cost and high efficiency.

Active Publication Date: 2019-06-28
GUANGDONG HOSPITAL OF TRADITIONAL CHINESE MEDICINE
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  • Claims
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Problems solved by technology

[0005] At present, there are few in vivo studies on phellodine, and there have been no research reports on its metabolism in vivo. Separating and extracting target metabolites from the body will help to further quantitatively study the changes and kinetics of phellodine in vivo, and clarify the role of phellodine in vivo. Metabolic processes and pathways, and a more comprehensive understanding of the mechanism of action of Phellodendronine

Method used

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  • Method for separating phellodendrine glucuronic acid conjugate from urine
  • Method for separating phellodendrine glucuronic acid conjugate from urine
  • Method for separating phellodendrine glucuronic acid conjugate from urine

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Embodiment 1

[0030] Embodiment 1: the separation of Phellodendronine glucuronide

[0031] Eighteen SD rats were intragastrically given the solution of cortex base at the dose of 80 mg / kg cortex base, once a day, collected urine samples from 0 to 24 hours, and administered continuously for two weeks. The collected urine samples were combined and centrifuged to remove the residue, and stored at -80°C for separation.

[0032] During the separation process, each elution fraction was analyzed using the established LC-MS n analysis by the method, and distinguish the metabolites M1, M2, and M3 according to the peak time, such as figure 1 . M1 is phellodine-2,11-O-D-glucuronide, M2 is phellodine-2-O-D-glucuronide, and M3 is phellodine-11-O-D-glucuronide.

[0033] 1.1 Preliminary separation of AB-8 macroporous adsorption resin:

[0034] A total of 4.5 L of urine samples were collected. Firstly, the urine samples were suction-filtered to remove impurities and precipitates, and then treated with ...

Embodiment 2

[0045] Embodiment 2: the preparation of phellodine glucuronide:

[0046] Preparation conditions for metabolite M1: use Phenomenes Polar-RP-80A semi-preparative column (10×250mm, 4μm), flow rate is 4mL / min; mobile phase is methanol: ultrapure water (5:95), injection time is 15min, the detection wavelength is 234nm, the injection volume is 80μL, the collection time is 4-4.5min, RT=4.25min, after preparation, all collected solutions are combined and concentrated to dryness, dissolved in 5mL ultrapure water, stored at -80℃, and finally used After freeze-drying, 10 mg of off-white powder was obtained.

[0047] Metabolite M2 preparation conditions: Phenomenes Polar-RP-80A semi-preparative column (10×250mm, 4μm), flow rate is 4mL / min; mobile phase is methanol: ultrapure water (25:75), and the injection time is 16min, the detection wavelength is 234nm, the injection volume is 80μL, the collection time is 12.6-13.1min, RT=12.8min, after preparation, all collected solutions are combine...

Embodiment 3

[0049] Implementation 3: Structural identification of Phellodendronine glucuronide conjugates:

[0050] Metabolite M1 is a white amorphous powder, the powder is dissolved in 40% acetonitrile water, and LC-MS n Analytical verification. by LC-MS n The quasi-molecular ion m / z 694[M+H] was obtained by a full-scan mass spectrometry + , which is 352 Da more than the relative molecular mass of the original drug Phellodendriline. Fragment ions m / z 518, m / z 342 and m / z 368 can be obtained in the secondary scanning mass spectrometry, and fragment ions m / z 342, m / z 368 and m / z 192 can be obtained in the tertiary scanning mass spectrometry. Determine m / z 694.23416[M+H] + The molecular formula is C 32 h 40 NO 16 , PDA absorption wavelength is 234nm, 284nm.

[0051] Take 8 mg of metabolite M1 powder and dissolve it in DMSO-d6 at 600M 1 H-NMR spectrum detection and under 150M conditions 13 C-NMR spectrum detection. 1 The high field region in the H-NMR spectrum gives δ3.20 (3H, s, ...

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Abstract

The invention discloses a method for separating phellodendrine glucuronic acid conjugate from urine. The method comprises the steps that a urine sample is subjected to AB-8 macroreticular resin separation twice, and an ODS open column is utilized for enriching metabolites; a Sephadex LH20 gel column is utilized for purification; obtained flow components are prepared into a target product by utilizing an Agilent 1200 type preparation solution, and LC-MSn analysis verification is utilized for identifying the structure of the product. According to the method, through separation and extraction, the in-vivo phellodendrine metabolite is obtained, the chemical structure of the in-vivo phellodendrine metabolite is determined, quantitative analysis is conducted, and feasibility conditions and experiment bases are provided for analysis of the fragmentation pattern and conversion relationship of the in-vivo phellodendrine metabolite and the in-vivo pharmacokinetic study and pharmacologic action mechanism study of the in-vivo phellodendrine metabolite. The method has the advantages of simple operation, low cost, high efficiency and the like.

Description

technical field [0001] The invention relates to a method for separating metabolites of phellodine. Background technique [0002] Cortex Phellodendron is the dry bark of Phellodendron chinense Schneid., a Rutaceae plant. Phellodendrine is one of the main chemical components in Phellodendron Phellodendron, which has remarkable pharmacological activities in terms of lowering blood pressure, anti-nephritis, inhibiting cellular immune response, and central nervous system inhibition. Phellodendrine also shows good antioxidant effect in vivo, which can reduce the expression of AKT, IKK, NF-KB phosphorylation and COX-2, down-regulate the increase of active oxygen and lipid peroxidation induced by AAPH, and improve the ROS-mediated inflammatory response. Phellodendronine is a tetrahydroisoquinoline type quaternary ammonium salt alkaloid, the molecular formula is C20H24NO4, the molecular weight is [M-H]+ m / z=342.17, and the structural formula is: [0003] [0004] Cortexine has ...

Claims

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Application Information

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IPC IPC(8): C07H7/027C07H1/06G01N30/88
Inventor 刘奕明林爱华邱全玉黎雄
Owner GUANGDONG HOSPITAL OF TRADITIONAL CHINESE MEDICINE
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