Cell culture and AMPK assay by Western blot analysisHPLC analysis of active compounds and myristicin under different extraction conditionsExtraction and isolation of active compounds on AMPK from Myristica fragransProcessing procedures for reducing myristicin contents from extracts of Myristica fragransAnimal experiments
Trang 1Le Thi Van Thu Advisor: Prof Oh Won-Keun Ph.D Department of Pharmacy,
Graduate School of Chosun University
October, 2009
Thesis for Master Degree
Trang 2 Metabolic syndrome
WHO definition of the metabolic syndrome, 1999
Glucose intolerance or diabetes mellitus and/or insulin resistance together with two or more of the other components listed below:
Impaired glucose regulation or diabetes;
Insulin resistance
Raised arterial pressure ≥ 140/90 mmHg;
Raised plasma triglycerides and/or low HDL-cholesterol
Central obesity
Trang 3 Consequences of the syndrome
Cardiovascular disease, hypertension and chronic renal disease
Diabetes and obesity
Trang 4 AMP activated protein kinase:
a target for total metabolic control
Structure and regulation of AMPK
Trang 5 Acyl CoA carboxylase and fat metabolism
Trang 6 Uses: spice, cosmetic,
drugs.
Part used: seed
Trang 7 Cell culture and AMPK assay by Western blot analysis
HPLC analysis of active compounds and myristicin under different extraction conditions
Extraction and isolation of active compounds on AMPK
from Myristica fragrans
Processing procedures for reducing myristicin contents
from extracts of Myristica fragrans
Animal experiments
Trang 8Mob Phase: EtOH:H2O
60% EtOH 80% EtOH 90% EtOH 100% EtOH Aceton
OP-CC (6 × 60 cm), Silica gel (63 - 200 µm)
Sol System: n-Hexan: Aceton (6:1 - 0:1)
Compound 6 (12 mg)
Compound 7 (8.5 mg)
Compound 1 (370 mg)
Compound 2 (28.9 mg) Compound 3 (12.7 mg) Compound 4 (6.7 mg) Compound 5 (7.8 mg)
OP-CC (5 × 60 cm)
RP ODS-A (12 nm) MeOH: H2O
Trang 9 Structure determination of tetrahydrofuran type-lignans from
Myristica fragrans
13C-NMR
Compound 1
Trang 10 Structure determination of tetrahydrofuran type-lignans from
Myristica fragrans
13C-NMR
Compound 2
Trang 11 Structure determination of tetrahydrofuran type-lignans from
Myristica fragrans
13C-NMR
Compound 3
Trang 12 Structure determination of tetrahydrofuran type-lignans from
Myristica fragrans
13C-NMR
Compound 4
Trang 13 Structure determination of tetrahydrofuran type-lignans from
Myristica fragrans
13C-NMR
Compound 5
Trang 14 Structure determination of tetrahydrofuran type-lignans from
Myristica fragrans
13C-NMR
Compound 6
Trang 15 Structure determination of tetrahydrofuran type-lignans from
Myristica fragrans
13C-NMR
Compound 7
Trang 16O HO
MeO
OH OMe
O
O HO
MeO
OH OMe
O HO
MeO
OH OMe
O
O O
2'
4' 5' 6'
2'' 3'' 4'' 5'' 6''
Fig 3 Chemical structures of compounds 1–7 isolated from Myristica fragrans
Trang 17 Processing procedures for reducing myristicin contents from extracts
Mob Phase: EtOH:H2O
60% EtOH 80% EtOH 90% EtOH 100% EtOH Aceton
Trang 18 Processing procedures for reducing myristicin contents from
extracts of Myristica fragrans
Step 1
0 50 100
Minutes c:\gilson\test\test-1.009\test-1.gdt : uv1 : Step 1: Inj Number: 1
c:\gilson\test\test-1.009\test-1.gdt : uv2 : Step 1: Inj Number: 1 pump a
pump b
3
1
2 4
5
6
7
Fig 18 A representative HPLC profile of some major lignans (1–6) and myristicin from
the total MeOH extract of Myristica fragrans with detections at 205 and 280 nm Key to peak identity: (1) macelignan, (2) meso-dihydroguaiaretic acid, (3) (±)-trans-
dehydrodiisoeugenol, (4) nectandrin B, (5) licarin A, (6) otobaphenol, and (7) myristicin
Chromatographic method used: 0-40 min (50-70% MeOH), 40-52 min (70-100% MeOH), 52-60 min (100% MeOH)
Trang 19 Processing procedures for reducing myristicin contents from
extracts of Myristica fragrans
Step 3
0 50 100
Fig 19 A representative HPLC profile of isolated lignans 1−7 from the 80%
EtOH fraction of the 30% EtOH-soluble extract of Myristica fragrans with
detections at 205 and 280 nm Key to peak identity: (1) tetrahydrofuroguaiacin B, (2) saucernetindiol, (3) verrucosin, (4) nectandrin B, (5) nectandrin A, (6)
fragransin C1, and (7) galbacin (For chromatographic protocol see Experimental
and Methods)
Trang 20Table 4 Contents of nectandrin B and myristicin in each solvent
fraction from 30% EtOH-soluble extract of M fragrans
Table 3 Contents of nectandrin B and myristicin in each solvent
extract of M fragrans
Trang 21 Effect of tetrahydrofuran type-lignans from Myristica fragrans
on AMPK activation
Fig 20 Stimulatory effects of isolated lignans 1−7 on AMP-activated protein
kinase (AMPK) activity (A) The stimulatory effects of isolated compounds 1−7
on AMPK activity at the concentration of 20 μg/mL (B) The stimulatory effects of g/mL (B) The stimulatory effects of
isolated compounds 1−7 on AMPK activity at the concentration of 5 μg/mL (B) The stimulatory effects of g/mL.
Trang 22 Animal experiment
Table 5 Characterization of ND mice as control with normal diet, and HFD
mice treated with or without THF for six weeks
Trang 23 Animal experiment
Trang 24 AMPK - a therapeutic target for the treatment of metabolic syndrome
seven 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignans were isolated
Tetrahydrofuran mixture exert anti-obesity effect in C57BL/6 models
Processing procedure to prepare a myristicin-free, lignan enriched fraction was established.