Supplemental Information Inhibitory Effects of Chung Hun Wha Dam Tang (chwdt) on High Fat Diet-Induced Obesity via amp-activated Protein Kinase activation




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Supplemental Information
Inhibitory Effects of Chung Hun Wha Dam Tang (CHWDT) on High Fat Diet-Induced Obesity via AMP-activated Protein Kinase activation

Md. Jamal Uddin1*, Yeonsoo Joe1*, Min Zheng 2, Sena Kim1, Hoyoung Lee3,

Tae-Oh Kwon4, and Hun Taeg Chung1

1School of Biological Sciences, 2School of Medical Sciences, University of Ulsan, 3Korea Institute of Oriental Medicine,Dept. Medical Research, Korea Institute of Oriental Medicine. 1672 Yuseongdae-ro, Yuseong-gu, Daejeon, 305-811, Republic of Korea 4College of Life Science and Natural Resources, Wonkwang University School of Medicine, Korea

Supplemental method

High Performance Liquid Chromatography Analysis

The filtered sample was used for HPLC analysis. All solvents (HPLC-grade water, acetonitrile, and methanol) used in this study were obtained from SK chemicals (Ulsan, Korea). The HPLC system (Hitachi Co. Japan) consisted of a pump (L-2130), autosampler (L-2200), column oven (L-2350), and diode array UV/VIS detector (L-2455). The output signal of the detector was recorded using an EZchrom Elite software for Hitachi. For separation of sample, an OptimaPak C18 column (5 μm, 100Å, 4.6 mm × 250 mm, RS tech, Korea) was used and UV wavelength was 254 nm.




Supplemental Table 1: Composition of CHWDT.

Name of Herbs

Scientific Name

Amount

Citrus Unshius Pericarpium

Citrus unshiu

3.75g

Pinelliae Tuber

Rhizoma pinelliae

3.75g

Poria Sclerotium

Pachyma hoelen rumphius

3.75g

Ponciri Fructus Immaturus

Poncirus trifoloata.

2.625g

Atractylodis Rhizoma Alba

Atractylodes japonica

2.625g

Cnidii Rhizoma

Cnidium officinale

1.875g

Scutellariae Radix

Scutellaria baicalensis

1.875g

Angelica Dahuricae Radix

Angelica dahurica

1.875g

Osterici Radix

Curcumae Longae Rhizoma

1.875g

Ginseng Radix

Panax ginseng

1.875g

Arisaematis Rhizoma

Arisaematis Rhizom

1.875g

Saposhnikoviae Radix

Ledebouriella seseloides

1.875g

Asiasari Radix et Rhizoma

Asiasarum sieboldi

1.125g

Coptidis Rhizoma

Coptis japonica

1.125g

Glycyrrhizae Radix

Glycyrrhiza glabra

1.125g

Zingiberis Rhizoma Crudus

Zingiber ojficinale

3 slice

*Yield after lipholization is 16.2g (46.29%) from 35g of above mixture.


Supplemental Table 2: Primer sequences of gene used in this study.


Primer

Mouse (sequences and size)

Human (sequences and size)

GAPDH

(f-aggccggtgctgagtatgtc,

r-tgcctgcttcaccttct; 530bp)



(f-ccacccatggcaaattccatggca,

r-tctagacggcaggtcaggtccacc; 520bp)



18s

(f-cagtgaaactgcgaatggct,

r-tgccttccttggatgtggta; 397bp)



-

SREBP1c

(f-aaacccgaagtggtggagac,

r-cggtgtgtacccgtagcatc; 305bp)



(f-aggactgtgctctgcgagtg,

r-ggctgggtcacacagttcag; 499bp)



FAS

(f-ttgctgccgtgtccttctac,

r-acgcgtgatggtctgtttgt; 455bp)



(f-acggaggccatatgcttctt,

r-acgtggacggatactttccc; 335bp)



CPT1

(f-gcagcattcttcgtgacgtt,

r-atgacctcctggcattctcc; 350bp)



(f-ctgagcacggcaagatgagt,

r-gtccagtttgcgcctgtaaa; 449bp)



ACO

(f-tggacagccaatgctggtat,

r-gctgcacgtagcttgtaggc; 329bp)



-

LCAD

(f-gaggtctgggaaaaagctgg,

r-ttcattccgttttccaccaa; 458bp)



-

PGC1α

(f-ggaactgcaggcctaactcc,

r-ttggagctgttttctggtgc; 495bp)



(f-ggaactgcaggcctaactcc,

r-cactgtccctcagttcaccg; 595bp)



G6Pase

(f-agactcccaggactggttca,

r-gtagaatccaagcgcgaaac; 601bp)



(f-tacgtcctcttccccatctg,

r-cctggtccagtctcacaggt; 240bp)



PEPCK

(f-actgttggctggctctcact, r-tgccttcggggttagttatg; 605bp)

(f-gagctgacggattcacccta,

r-ccactgccaaaggagatgat; 349bp)



TNFα

(f-agttctatggcccagaccct,

r-cggactccgcaaagtctaag; 463bp)



(f-aagcctgtagcccatgttgt,

r-cagatagatgggctcatacc; 365bp)



iNOS

(f-ccaccttggtgaagggactgagct,

r-gctgcggggagccattttggt; 381bp)



(f-cagtacgtttggcaatggagactgc,

r-ggtcacattggaggtgtagagcttg; 340bp)




Legend of the supplemental figures

Supplemental Figure 1. Structures and HPLC chromatograms of CHWDT. HPLC chromatogram of CHWDT extract and standard solution at 280nm.



Supplemental Figure 2. CHWDT increases cell viability in HepG2 cells.

Cells were incubated with CHWDT (0, 50, 100 and 200g/ml) for 24h. After 24h, MTT assay was performed to measure the cell viability. Data indicating mean ± SEM of three independent experiments with triplicate wells, p< 0.05 as *.








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