EBPα

Author’s Accepted Manuscript Gangjihwan, a polyherbal composition, inhibits fat accumulation through the modulation of lipogenic transcription factors SREBP1C, PPARγ and C/EBPα Jaewoong Jang, Yoonju Jung, Seyeon Chae, Soo Hyun Cho, Michung Yoon, Heejung Yang, Soon Shik Shin, Yoosik Yoon

PII: DOI: Reference:

www.elsevier.com/locate/jep

S0378-8741(17)31072-3 http://dx.doi.org/10.1016/j.jep.2017.08.024 JEP10994

To appear in: Journal of Ethnopharmacology Received date: 1 April 2017 Revised date: 20 August 2017 Accepted date: 20 August 2017 Cite this article as: Jaewoong Jang, Yoonju Jung, Seyeon Chae, Soo Hyun Cho, Michung Yoon, Heejung Yang, Soon Shik Shin and Yoosik Yoon, Gangjihwan, a polyherbal composition, inhibits fat accumulation through the modulation of lipogenic transcription factors SREBP1C, PPARγ and C/EBPα, Journal of Ethnopharmacology, http://dx.doi.org/10.1016/j.jep.2017.08.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Gangjihwan, a polyherbal composition, inhibits fat accumulation through the modulation of lipogenic transcription factors SREBP1C, PPARγ and C/EBPα

Jaewoong Janga, Yoonju Junga, Seyeon Chaea, Soo Hyun Chob, Michung Yoonc, Heejung Yangd*, Soon Shik Shine*, Yoosik Yoona*

a

Department of Microbiology, College of Medicine, Chung-Ang University, Seoul 06974, Republic of Korea

b

Department of Family Medicine, College of Medicine, Chung-Ang University Hospital, Seoul 06973, Republic of Korea

c

Department of Biomedical Engineering, Mokwon University, Daejon 35349, Republic of Korea

d

College of Pharmacy, Kangwon National University, Chuncheon 24341, Republic of Korea

e

Department of Formula Sciences and Research Center of Korean Medicine for Diabetes and Obesity, College of Korean Medicine, Dong-eui University, Busan 47227, Republic of Korea

*Corresponding author: Prof. Yoosik Yoon, Department of Microbiology, College of Medicine, Chung-Ang University, Seoul, 06974, Republic of Korea, Tel: +82-2-820-5767, Fax:+82-2-823-5423, E-mail: [email protected]

(Y. Yoon)

Prof. Soon Shik Shin, Department of Formula Sciences and Research Center of Korean

1

Medicine for Diabetes and Obesity, College of Korean Medicine, Dong-eui University, Busan 47227, Republic of Korea, Tel: +82-51-850-7414, Fax:+82-51-853-4036, E-mail: [email protected]

(S.S. Shin)

Prof. Heejung Yang, College of Pharmacy, Kangwon National University, Chuncheon, 24341, Republic of Korea, Tel: +82-33-250-6919, Fax:+82-33-259-5631, E-mail: [email protected] (H. Yang).

Contract/grant sponsor: Korean Health Technology R&D Project (HI15C0075), Ministry of Health and Welfare, Republic of Korea

SHORT TITLE:

Standardization and anti-obesity mechanism of polyherbal composition

ABBREVIATIONS: ACC1, acetyl-CoA carboxylase 1; C/EBPα, CCAAT/enhancer binding protein alpha; DF, Gangjihwan; DEG, differentially expressed gene; DMEM, Dulbecco’s modified Eagle medium; DMSO, dimethylsulfoxide; FAS, fatty acid synthase; HPLC, high performance liquid chromatography; PCR, polymerase chain reaction; PPARγ, peroxisome proliferatoractivated receptor gamma; SCD1, stearoyl-CoA desaturase 1; SD, standard deviation; SREBP1C, sterol regulatory element binding protein 1C

2

ABSTRACT

Ethnopharmacological relevance: Gangjihwan (DF) which is composed of Ephedra intermedia, Lithospermum erythrorhizon, and Rheum palmatum has been used for the treatment of obesity in traditional medical clinics in Korea. Aim of the study: This study was conducted to standardize DF and elucidate its mechanism of action for inhibiting fat accumulation in adipocytes and adipose tissues. Materials and Methods: The herbal ingredients of DF were extracted in water, 30% ethanol or 70% ethanol and freeze-dried followed by HPLC analyses. 3T3-L1 adipocytes and high-fat diet-induced obese mice were treated with each of the three DF preparations. Messenger RNA and protein expression levels were measured by real-time qPCR and Western blotting. RNA-Seq analyses were conducted to examine the effects of DF treatment on whole transcriptome of adipocyte. Results: (-)-Ephedrine and (+)-pseudoephedrine from E. intermedia, aloe-emodin and chrysophanol from R. palmatum and shikonin from L. erythrorhizon were identified as phytochemical components of DF. DF caused dose-dependent inhibition of fat accumulation in 3T3-L1 adipocytes. It also significantly reduced adipose tissue mass and adipocyte size in high-fat diet-induced obese mice. DF was found to down-regulate the expressions of the lipogenic transcription factors such as sterol regulatory element binding protein 1C (SREBP1C), peroxisome proliferator activated receptor gamma (PPARγ), and CCAAT/enhancer binding protein alpha (C/EBPα). Among the three preparations of DF, the 70% ethanol extract was the most effective. RNA-Seq analyses showed that DF treatment decreased the expression levels of up-regulators and increased those of down-regulators of

3

lipogenic transcription factors. Conclusions: DF preparations, among which 70% ethanol extract was the most effective, reduced fat accumulation in 3T3-L1 adipocytes and high-fat diet-induced obese mice through the down-regulation of lipogenic transcription factors SREBP1C, PPARγ and C/EBPα.

KEY WORDS: Polyherbal composition; Obesity; Fat; SREBP1C; PPARγ; C/EBPα

4

1. Introduction

Gangjihwan (DF) is a polyherbal composition consisting of Ephedra intermedia, Rheum palmatum and Lithospermum erythrorhizon. E.intermedia has been used traditionally to treat the common cold, rhinitis, asthma, allergies, and rheumatoid arthritis (Bielory, 2004). The ephedra alkaloids, such as (-)-ephedrine, (+)-pseudoephedrine, (-)-Nmethylephedrine,

(+)-N-methylpseudoephedrine,

(-)-norephedrine

and

(+)-

norpseudoephedrine, are reported to have thermogenic properties increasing metabolism and body heat (Abourashed et al., 2003). R. palmatum has been used to treat constipation, jaundice, gastro-intestinal hemorrhage, and ulcers through its function of clearing heat, purging fire, cooling blood, promoting blood circulation, and removing blood stagnation (Zhang et al., 2015). R. palmatum is reported to contain anthraquinone derivatives such as emodin, aloe-emodin, chrysphanol and physcion as well as dianthrones such as sennosides A, B, C and D, which function as natural laxatives (Kon et al., 2014). L. erythrorhizon has been used to treat hematemesis, hematuria, constipation, burns, eczema, and urinary tract infection through its function of improving blood circulation, reducing fever and removing toxins (Kim et al., 2015). Naphthoquinone derivatives including shikonin, acetylshikonin, and isobutylshikonin are reported to its major constituents (Chen et al., 2002). Previous studies have reported the anti-obesity effects of the polyherbal composition similar to DF. It has been reported that Gyeongshingangjeehwan 18 (GGEx18), a polyherbal composition composed of E. sinica, R. palmatum, and Laminaria japonica,

5

reduced obesity, insulin resistance and hepatic steatosis (Oh et al., 2015; Shin et al., 2012; Shin and Yoon, 2012). DF is a polyherbal composition similar to GGEx 18 modified by replacing L. japonica with L. erythrorhizon. E. intermedia has been used to control body weight, but adverse effects, such as heart palpitations and insomnia, have been reported (Shekelle et al., 2003). Recently, it was reported that R. palmatum showed protective effects against obesity and metabolic disorders (Sheng et al., 2012), and L. erythrorhizon suppressed fat accumulation in high-fat diet-induced obese mice (Gwon et al., 2012), suggesting that DF, a polyherbal composition of these three herbs, may have an efficient anti-obesity effect. DF has been used in Korean traditional medical clinics located in Busan area for more than 1,000 cases of obesity patients. The clinical outcome was 3 to 8 kg weight loss per month and variable among patients. The average treatment period was 3 month, and exact treatment period depends on weight loss outcome of each patient. Patients were treated with 6.4 g/day of DF, 3.2 g between breakfast and lunch and 3.2 g between lunch and dinner. DF has been clinically treated as powdered raw herb mixture of E. intermedia, R. palmatum and L. erythrorhizon, with a ratio of 4:4:2 (Table 1). E. intermedia is the chief herb which treats key symptoms of obesity. R. palmatum is the deputy herb which reinforces anti-obesity effect of E.intermedia and relieves its constipation-inducing side effect. L. erythrorhizon is the assistant herb which harmonizes the effects of E. intermedia and R. palmatum. 3T3-L1 cell, originally derived from mouse embryos, is widely used as an in vitro model of fat accumulation due to its ability to differentiate into adipocyte (Green and Meuth, 1974), and high-fat diet-fed mouse is a well-known in vivo model of obesity and metabolic syndrome (Fraulob et al., 2010). Studies using these in vitro and in vivo models

6

of obesity have identified major genes involved in fat accumulation. To accumulate fat in adipocytes, many enzymes involved in lipid synthesis are up-regulated by the lipogenic transcription factors such as sterol regulatory element binding protein 1C (SREBP1C), peroxisome proliferator activated receptor gamma (PPARγ), and CCAAT/enhancer binding protein α (C/EBPα) (Rosen and MacDougald, 2006; Rosen et al., 2000). In this study, we standardized the preparation method and chemical constituents of DF, investigated its effect on the inhibition of fat accumulation, and elucidated its mechanism of action in 3T3-L1 adipocytes and high-fat diet-induced obese mice.

7

2. Materials and methods

2.1. Reagents 3T3-L1 cells were obtained from the American Type Culture Collection (Manassas, VA, USA), and all cell culture reagents were purchased from Life Technologies, Inc. (Gibco, Grand Island, NY, USA). Seven week-old C57BL/6N male mice were obtained from Samtako Bio Korea, Inc. (Osan, Korea). Experimental food for the low-fat diet (fat calories 10%) and high-fat diet (fat calories 45%) were purchased from Research Diets (New Brunswick, NJ, USA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.2. Preparation and standardization of DF Herbal ingredients were purchased from Hwalim Pharmaceuticals (Busan, Korea). The herbal ingredients of DF were identified by one of the authors, Professor Soon Shik Shin, and voucher specimens were stored in the Department of Formula Sciences, College of Korean Medicine, Dong-eui University (Busan, Korea). Voucher specimen number is DFOS-20150804-004-H for E. intermedia, DFOS-20150804-009-H for R. palmatum and DFOS-20150804-005-H for L. erythrorhizon. The herbal composition of DF is shown in Table 1. A water extract of DF (DF-FW), a 30 % ethanol extract of DF (DF-GA30) and a 70 % ethanol extract of DF (DF-GA70) were prepared by extracting the herbal ingredients using a Soxhlet extractor. Extractions were performed for four hours at 65 ℃ followed by freeze-drying. For HPLC analyses, three DF preparations were dissolved to a concentration

8

of 20 mg/ml in 100% HPLC-grade methanol and filtered with 0.45 μm PTFE membrane filter (Adventec, Inc., Dublin, CA, USA). The chemical standards (-)-ephedrine and (+)pseudoephedrine (Sigma-Aldrich) were dissolved to a stock concentration of 1 mg/ml with 100% HPLC-grade methanol. The stock solutions were diluted to concentrations ranging from 0.125 to 1 mg/ml for experimental analyses. HPLC was performed on an Agilent 1260 Infinity system (Agilent Technologies, Waldbronn, Germany), which consisted of a 1260 quaternary pump, an auto-sampler and a multiple wavelength detector connected to a Hector-M C18 column (250 mm  4.6 mm i.d., RStech, Daejeon, Korea). Samples were eluted with mixtures of HPLC-grade acetonitrile (solvent A) and water buffered with 25 mM sodium dodecyl sulfate (solvent B). The gradient elution condition was 40% solvent A (0-25 min), 40-60% solvent A (25-35 min), 60% solvent A (35-40 min), 60-80% solvent A (40-50 min) and 80% solvent A (50-60 min) with a flow rate of 1.0 ml/min. Following the gradient elution, a final wash was performed with 40% solvent A for 10 minutes to prepare the column for the next analysis. Eluents for all chromatograms were followed by measuring absorbance at 215 nm.

Table 1. The herbal composition of DF Family Pharmaceutical name

Scientific name

Content Part used

name Ephedra intermedia Schrenk

Ephedrac

& C.A.Mey.

eae

Ephedrae Herba

(%)

Stem

9

40

Arnebiae/Lithospermi

Lithospermum erythrorhizon

Borragin Root

Radix

Siebold & Zucc

Rhei Radix et

aceae Polygona

Root/

ceae

Rhizome

20

Rheum palmatum L. Rhizoma

40

2.3. Adipocyte differentiation of 3T3-L1 cells 3T3-L1 cells were cultured to confluency in Dulbecco’s modified Eagle medium (DMEM) that was supplemented with 10% calf serum, 100 μg/mL streptomycin and 100 units/mL penicillin. After reaching confluency (day 0), the culture medium was changed to DMEM that contained 10 % fetal bovine serum, 1 g/mL insulin, 0.25 M dexamethasone and 0.5 mM 3-isobutyl-1-methylxanthine, and cells were cultured for two days. After two days (day 2), the medium was changed to DMEM containing 10% fetal bovine serum and 1 g/mL insulin; medium was also changed on day 4. Cells were harvested on day 7. DF-FW, DF-GA30 or DF-GA70 were dissolved in dimethylsulfoxide (DMSO) and added into the medium at day 0, day 2 and day 4. Control cells were treated with an equal amount of DMSO diluted in medium. Intracellular fat droplets were stained with oil red O dye and imaged under a light microscope at magnitude of x400 on day 7. Oil red O dye from the stained intracellular fat droplets was extracted with 60 % isopropanol and quantified by measuring optical density at 510 nm as previously described (Gustafson and Smith, 2006). Cell viability was determined using a CellCountEZ™ Cell Survival Assay Kit (Rockland Immunochemicals, Limerick, PA, USA), which is based on the ability of viable mammalian cells to convert hydroxyethyl disulfide into mercaptoethanol (Li et al., 2012).

10

2.4. High-fat diet-induced obesity experiments in mice The animal experiments were approved by the Ethical Committee of Animal Experimentation in Dong-eui University (approval number of R2015-009). After a oneweek adaptation period, mice were randomly divided into five groups. Each group was composed of 7 mice. The normal group was fed a low-fat diet (10% fat calories), while a control group and three experimental groups were fed a high-fat diet (45% fat calories). For the experimental groups, DF-FW, DF-GA30 or DF-GA70, which were suspended in distilled water, were orally administered at a dose of 250 mg/kg once a day for 8 weeks. The normal and control groups were orally administered an equal volume of distilled water. During the experimental period, the temperature was maintained at 21 ± 2°C, and the humidity was 55 ± 5%. The number of ventilations was 15 to 17 times per hour. The illumination was 150-300 lux with 12-hour light/dark cycles. The experimental diet and water were fed ad libitum. After 8 weeks of administration, mice were sacrificed by cervical dislocation and the weights of inguinal adipose tissue, mesenteric adipose tissue and retroperitoneal adipose tissue were measured. Samples of inguinal adipose tissues were fixed with 10% formaldehyde followed by paraffin embedding. The paraffin blocks were sliced into five-m-thick sections, stained with hematoxylin-eosin and imaged. The sizes of adipocytes in tissue sections were measured using image analysis software (Infinity analyze, Lumenera Corp., Ottawa, ON, Canada).

2.5. Real-time qPCR analysis

11

Total RNA was extracted from 3T3-L1 adipocytes and adipose tissues using an RNeasy kit (Qiagen, Hilden, Germany). One µg of RNA was reverse-transcribed using a cDNA reverse transcription kit (Applied Biosystems, Inc., Foster City, CA, USA). Realtime qPCR was performed using a StepOne Real-time PCR System (Applied Biosystems, Inc., Foster City, CA, USA). Reactions included the TaqMan gene expression master mix, TaqMan probe, primers, and 1/10 of cDNA reverse transcription product in a 20 L total reaction volume. The reaction mixtures were preheated at 95C for 10 min to activate the enzyme followed by 40 cycles of melting at 95C for 15 s and annealing/extension at 60C for 1 min. Assay-on-demand gene expression products (Applied Biosystems, Inc.), which contain TaqMan probe and primers for each gene, were used to evaluate the mRNA levels of ACC1 (Mm01304257_m1), FAS (Mm01253292_m1), SCD1 (Mm00772290_m1) SREBP1C (Mm00550338_m1), PPARγ (Mm00440945_m1), C/EBPα (Mm01265914_s1) and 18S rRNA (Hs99999901_s1). The level of 18S rRNA was used as an endogenous control as previously described (Gustafson and Smith, 2006). Expression levels for transcripts of interest were first normalized to levels of 18S rRNA in each sample then presented as ratios of experimental samples to control sample; untreated adipocytes were used for in vitro experiments and control group mice were used for in vivo experiments. The quantifications were performed according to the comparative Ct method (Livak and Schmittgen, 2001).

2.6. Protein extraction and Western blotting 3T3-L1 adipocytes were harvested using a cell scraper and lysed with ice-cold

12

RIPA buffer containing 25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS and a protease inhibitor cocktail (Sigma-Aldrich). The cell lysates were centrifuged at 14,000 rpm for 20 min at 4°C to remove insoluble materials. The protein concentrations were determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). Fifty micrograms of proteins from each cell lysate were separated on a 10% SDS-polyacrylamide gel at 80V for 1.5 h, and electro-transferred to a nitrocellulose membrane at 150 mA for 1.5 h. The membranes were blocked for 2 h at room temperature with phosphate buffered saline containing 5% skim milk and 0.1% Tween 20. Primary antibodies were added at a 1:1,000 dilution and incubated overnight at 4 ℃. Blots were incubated with 1:1,000 dilution of a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Protein bands were visualized using a chemiluminescence kit (Pierce, Rockford, IL, USA). An anti-ß-actin antibody was used as an endogenous control to confirm that an equal amount of protein was loaded in each lane. Anti-PPARγ (#2430) primary antibody, and anti-mouse (#7076S) / anti-rabbit (#7074) secondary antibodies were purchased from Cell Signaling (Beverly, MA, USA). Anti-SREBP1C (557036) antibody was purchased from BD Science (Franklin Lakes, NJ, USA). Anti-C/EBPα (sc-61) and antiβ-actin (sc-47778) antibodies were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA).

2.7. RNA-Seq analyses Total RNA was isolated from untreated 3T3-L1 adipocytes and DF-GA70-treated 3T3-L1 adipocytes using an RNeasy kit (Qiagen, Hilden, Germany). For each RNA sample,

13

the library construction was performed using the SENSE 3’ mRNA-Seq Library Prep Kit (Lexogen, Inc., Vienna, Austria) according to the manufacturer’s instructions. Highthroughput sequencing was performed using NextSeq 500 (Illumina, Inc., San Diego, CA, USA). RNA-seq data were submitted to GEO of NCBI with an accession number of GSE94257. The whole transcriptomes were compared, and we identified differentially expressed genes (DEGs) that displayed a greater than 2-fold change in expression upon treatment with DF-GA70. Functional analyses and regulation network of DEGs were generated through the use of Ingenuity Pathway Analysis (Qiagen bioinformatics, Redwood City, CA, USA) (www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis).

2.8. Statistical analyses Data were expressed as the mean ± standard deviation (SD) of at least three independent experiments. Statistical significance was determined using the unpaired t –test. All analyses were performed using SPSS ver. 21 (SPSS, Inc., Chicago, IL, USA).

3. Results 3.1. Standardization of DF The yields of DF extracts in water (DF-FW), 30% ethanol (DF-GA30), and 70% ethanol (DF-GA70) were 22.7%, 26.3%, and 22.9%, respectively. Among the diverse phytochemicals present in DF, two major components from E. intermedia, (-)-ephedrine and (+)-pseudoephedrine, were the most prominent (Fig. 1). Their calibration curves were y = 12,072.2485x + 65.3639 (R² = 0.9997) for (-)-ephedrine and y = 16,094.8252x - 1.0821

14

(R² = 1.0000) for (+)-pseudoephedrine. As shown in Table 2, levels of (-)-ephedrine were 68 times greater than levels of (+)-pseudoephedrine in all DF preparations. These two ephedra alkaloids were extracted at higher concentrations by solvents with lower polarity. 35.90 mg/g of (-)-ephedrine and 4.42 mg/g of (+)-pseudoephedrine were detected in the 70% ethanol extract. 20.10 mg/g of (-)-ephedrine and 3.17 mg/g of (+)-pseudoephedrine were present in the 30% ethanol extract. 12.92 mg/g of (-)-ephedrine and 1.63 mg/g of (+)pseudoephedrine were present in the water extract. Detailed analysis of the HPLC profile of DF-GA70 identified other phytochemicals including aloe-emodin and chrysophanol from R. palmatum as well as shikonin from L. erythrorhizon (Fig. 2A). The peaks of each phytochemicals were identified by the elution time of standards (Fig. 2B).

Fig. 1

(A) DF-GA70

(B) DF-GA30 (C) DF-FW

Table 2. Contents of (-)-ephedrine and (+)-pseudoephedrine in three DF preparations.

15

(-)-ephedrine (mg/g)

(+)-pseudoephedrine (mg/g)

DF-GA70

35.90 ± 0.09

4.42 ± 0.02

DF-GA30

20.10 ± 0.05

3.17 ± 0.01

DF-FW

12.92 ± 0.02

1.63 ± 0.02

Fig. 2 (A) DF-GA70

(B) Standard phytochemicals, aloe-emodin (1) and chrysopanol (5)

from R. palmatum, (+)-pseudoephedrine (2) and (-)-ephedrine (3) from E. intermedia and shikonin (4) from L. erythrorhizon

16

3.2. DF inhibited fat accumulation in 3T3-L1 adipocytes When intracellular fat droplets in 3T3-L1 adipocytes were stained using oil red O dye, we observed dose-dependent inhibition of fat accumulation by DF-FW, DF-GA30 or DF-GA70 for concentrations ranging from 5-20 μg/ml (Fig. 3). To quantify intracellular fat content, we extracted and measured oil red O (Fig. 4A). DF-GA70 had the strongest inhibitory effect among the three preparations, especially at low concentrations of 5-10 μg/ml. We measured the viability of cells treated with the three DF preparations. Cell viability after each treatment was identical to that of untreated adipocytes, indicating that the inhibitory effects of DF preparations on intracellular fat accumulation were not due to their cytotoxicity (Fig. 4B).

Fig. 3

17

Fig. 4

18

3.3. DF inhibited fat accumulation in high-fat diet-induced obese mice Inguinal adipose tissue, mesenteric adipose tissue and retroperitoneal adipose tissue were significantly increased in control mice that were fed a high-fat diet compared with normal mice that were fed a low-fat diet. Significant reductions of adipose tissues were observed in mice that were fed high-fat diet in concert with 250 mg/kg/day of DF-FW, DF-GA30 or DFGA70 for 8 weeks (Fig. 5). Of the three DF preparations, DF-GA70 showed the strongest effects on adipose tissue reduction. Histological analyses of inguinal adipocyte tissues showed that adipocytes of control mice that were fed a high-fat diet were significantly enlarged when compared with normal mice that were fed a low-fat diet. This enlargement was due to an increase in intracellular fat accumulation (Fig. 6A). The size of adipocytes was significantly reduced upon oral administration of DF preparations. All DF preparations reduced adipocyte size, while DF-GA70 showed the strongest effect (Fig. 6B).

19

Fig. 5 (A) inguinal adipose tissue, (B) mesenteric adipose tissue, (C) retroperitoneal adipose tissue

Fig. 6

20

3.4. DF down-regulated the expression of Lipid-synthesizing enzymes We used real-time qPCR to measure the expression levels of lipid-synthesizing enzymes such as acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FAS), and stearoyl-CoA desaturase 1 (SCD1). RNA was extracted from 3T3-L1 adipocytes treated with DF-FW, DF-GA30 or DF-GA70 for 7 days and from inguinal adipose tissues of mice orally treated with 250 mg/kg/day of each DF preparation for 8 weeks.

Fig. 7

In 3T3-L1 adipocytes, the expression levels of lipid-synthesizing enzymes, ACC1, FAS and

21

SCD1, were markedly up-regulated when compared with levels observed in preadipocytes. Expression levels of ACC1, FAS and SCD1 showed a significant, dose-dependent decrease when treated with DF preparations. Of the three DF preparations, DF-GA70 showed the strongest effect (Fig. 7A-C). In the high-fat diet-induced obese mice experiments, administrations of all three DF preparations significantly reduced expression levels of ACC1 in adipose tissue. Expression levels of FAS and SCD1 in adipose tissue were significantly reduced by the administrations of DF-GA30 and DF-70A (Fig. 7D-F).

3.5. DF down-regulated the expression of lipogenic transcription factors SREBP1C, PPARγ, and C/EBPα are lipogenic transcription factors that regulate the expression of many genes involved in lipid synthesis and fat accumulation (Rosen and MacDougald, 2006; Rosen et al., 2000). Using real-time qPCR, we observed that SREBP1C, PPARγ and C/EBPα all showed increased expression in 3T3-L1 adipocytes when compared with preadipocytes. Treatment with the DF preparations caused a significant decrease in the expression of these lipogenic transcription factors (Fig. 8A-C). All three preparations of DF caused a reduction in the expression of each lipogenic transcription factor when applied at concentrations ranging from 5 to 20 μg/ml. Of the three DF preparations, DF-GA70 showed the strongest effect. When administered to high-fat diet-induced obese mice at a dose of 250 mg/Kg/day for 8 weeks, all three DF preparations caused a significant decrease in the expression levels of SREBP1C. Administration of DFGA30 and DF-GA70 caused a significant down-regulation of PPARγ and C/EBPα (Fig. 8D-F).

22

Fig. 8

To elucidate the in vivo dose effects of DF, high fat diet-fed mice were orally treated for 8 weeks with 0, 150, 300, 600 mg/kg/day of DF-GA70, which has the strongest effects among three preparations of DF. The results showed that DF-GA70 induced dosedependent reductions of fat tissue as well as the expression levels of lipid-synthesizing enzymes and lipogenic transcription factors (Fig. 9).

23

Fig. 9

To elucidate the effects of DF on the protein levels of lipogenic transcription factors, proteins were extracted from 3T3-L1 adipocytes treated with DF-70GA, which has the strongest effects among three preparations of DF, and were analyzed by Western blotting. When DF-GA70 was treated for 7 days at doses ranging from 2 to 30 g/ml, we observed a dose-dependent decrease in the protein levels of PPARγ, C/EBPα and SREBP1C (Fig. 10A). The SREBP1C protein is synthesized as a 125-kDa precursor that undergoes proteolytic processing to generate a 68-kDa mature SREBP1C (Eberle et al., 2004; Jeon and Osborne, 2012; Wang et al., 1994). Data showed that both the precursor and

24

proteolyzed mature forms of SREBPIC were down-regulated by DF-GA70 treatment. When cells were treated with DF-GA70 for 2, 4 and 7 days, the protein levels of the lipogenic transcription factors were down-regulated when compared with untreated cells at the same differentiation day (Fig. 10B).

Fig. 10

3.6. DF decreased the up-regulators and increased the down-regulators of lipogenic transcription factors. To elucidate the mechanism for the down-regulations of lipogenic transcription factors by DF-GA70 treatment, we performed RNA-Seq analyses on 3T3-L1 adipocytes that were untreated or treated with 20 g/ml of DF-GA70 for 7 days. Scatter plot of RNASeq data of untreated adipocytes vs. DF-GA70-treated adipocytes is shown in Fig. 11. Up-

25

regulated genes by DF-GA70 treatment are colored red, and down-regulated genes are colored green. The whole transcriptomes were compared, and we identified differentially expressed genes (DEGs) with fold changes greater than 2 upon treatment with DF-GA70. The mRNA expression levels of the 24,421 genes were quantified, and 1,113 DEGs were found.

Fig. 11.

Ingenuity pathway analysis was used to find, among DEGs, up-regulators and down-regulator of SREBP1C, PPAR and C/EBP. We found 2 up-regulators and 5 down-

26

regulators of SREBP1C among DEGs. Their descriptions, expression levels and fold changes were shown in Table 3 and diagramed in Fig. 12A. Also found were 8 DEGs including 7 up-regulators and 1 down-regulators of PPAR (Table 4 and Fig. 12B), and 6 DEGs including 4 up-regulators and 2 down-regulators of C/EBP (Table 5 and Fig. 12C). RNA-Seq results were summarized in Fig. 12D. Some genes such as Ppara, Thra and Lep regulate multiple lipogenic transcription factors. Among 9 up-regulators of lipogenic transcription factors, 8 were decreased while only one of them was increased by DF-GA70. Among 6 down-regulators of lipogenic transcription factors, 4 were increased while only 2 of them were decreased by DF-GA70. As a result, most up-regulators were decreased and more down-regulators were increased, which resulted in the down-regulation of lipogenic transcription factors as shown in Fig. 8, 9 and 10.

Table 3. DF-induced fold changes in the up- or down-regulators of SREBP1C.

Function of DEGs

Fold

Expression Level

Change

(Read Count)

Gene Gene Reference

Symbol

DF-treated /

DF-

un-

untreated

treated

treated

Description (Alias) (Seo et al.,

Down-regulator

0.462

45

96

Clu

clusterin 2013) (Soukas et al.,

Down-regulator

4.612

70

15

Lep

leptin 2000)

Up-regulator

0.273

85

310

oxysterol binding

(Yan et al.,

protein

2007)

Osbp

27

peroxisome (Yoshikawa et Down-regulator

2.953

202

69

Ppara

proliferator activated al., 2003) receptor alpha receptor (TNFRSF)(Wang et al.,

Ripk2 Down-regulator

2.325

70

30

interacting serine2013)

(CARD3)

threonine kinase 2 (Yoon et al., Down-regulator

0.441

300

680

Sik1

salt inducible kinase 1 2009)

Up-regulator

0.341

132

387

thyroid hormone

(Araki et al.,

receptor alpha

2009)

Thra

Table 4. DF-induced fold changes in the up- or down-regulators of PPAR.

Function of DEGs

Fold

Expression Level

Change

(Read Count)

DF-treated / untreated

Gene Gene Reference

Symbol DF70treated

un-

Description (Alias)

treated

1-acylglycerol-3phosphate O-

Up-regulator

2.216

8247

3721

acyltransferase 2

(Gale et al.,

(lysophosphatidic

2006)

Agpat2

acid acyltransferase, beta) CCAAT/enhancer Up-regulator

0.394

8298

21074

(Xi et al., 2016)

Cebpb binding protein

28

(C/EBP), beta (Braga et al., Up-regulator

0.299

225

752

Fst

follistatin 2014) (Zhou et al.,

Down-regulator

4.612

70

15

Lep

leptin 2014)

Up-regulator

0.329

111

Ncoa2

nuclear receptor

(Hartig et al.,

(SRC-2)

coactivator 2

2011)

337

peroxisome (Goto et al., Up-regulator

2.953

202

69

Ppara

proliferator activated 2011) receptor alpha

Up-regulator

Up-regulator

0.341

0.255

132

58

thyroid hormone

(Ying et al.,

receptor alpha

2007)

Medag

mesenteric estrogen

(Zhang et al.,

(MEDA-4)

dependent adipose-4

2012)

387

Thra

226

Table 5. DF-induced fold changes in the up- or down-regulators of C/EBP.

Function of DEGs

Fold

Expression Level

Change

(Read Count)

DF-treated / untreated

Gene Gene Reference

Symbol DF70treated

un-

Description (Alias)

treated

CCAAT/enhancer (Abdou et al., Up-regulator

0.394

8298

21074

Cebpb

binding protein 2013) (C/EBP), beta

Up-regulator

Down-regulator

0.477

2.953

343

202

719

colony stimulating

(Chen et al.,

factor 1 (macrophage)

2013)

peroxisome

(Gervois et al.,

Csf1

69

Ppara

29

proliferator activated

2004)

receptor alpha

Up-regulator

Up-regulator

Down-regulator

0.258

0.341

2.530

26

132

418

99

runt related

(Guo et al.,

transcription factor 1

2012)

thyroid hormone

(Fozzatti et al.,

receptor alpha

2013)

tribbles homolog 1

(Satoh et al.,

(Drosophila)

2013)

Runx1

387

Thra

165

Trib1

Fig. 12

30

4. Discussion In this study, we standardized the preparation of DF by extracting its phytochemical constituents in water, 30% ethanol or 70% ethanol followed by HPLC profile analyses. Among the diverse phytochemicals of DF, (-)-ephedrine and (+)pseudoephedrine from E. intermedia were the most prominent. The highest concentrations of (-)-ephedrine and (+)-pseudoephedrine were detected in the 70% ethanol extract of DF (Fig. 1). Other phytochemicals of DF such as aloe-emodin and chrysophanol from R. palmatum, and shikonin from L. erythrorhizon were also identified (Fig. 2). We found that,

31

while all three preparations of DF reduced fat accumulation both in vitro (Fig. 3 and 4) and in vivo (Fig. 5 and 6), the 70% ethanol extract of DF was the most effective. DF treatment caused down-regulations of lipid-synthesizing enzymes ACC1, FAS and SCD1 both in vitro and in vivo (Fig. 7). ACC1 is a rate-limiting enzyme in the biogenesis of long chain fatty acids and catalyzes the conversion of acetyl-CoA to malonylCoA which is a precursor of fatty acid synthesis and an inhibitor of fatty acid oxidation (Fullerton et al., 2013). FAS is a key enzyme of lipid biosynthesis, and phytochemicals which inhibit FAS causes a reduction in fat accumulation (Liang et al., 2013). SCD1 catalyzes the conversion of saturated fatty acids, palmitate (16:0) and stearate (18:0), into monounsaturated fatty acids, palmitoleate (16:1) and oleate (18:1), respectively. The products of these conversion reactions represent the dominant constituents of triacylglycerols that accumulate as fat in adipocytes, and it was reported that the inhibition of SCD1 suppressed fat accumulation in adipocytes (Ralston and Mutch, 2015). The lipidsynthesizing enzymes ACC1, FAS and SCD1 are transcribed by lipogenic transcription factors SREBP1C, PPARγ and C/EBPα : The transcription of ACC1 is induced upon binding of SREBP1C to its promoter (Magana et al., 1997; Shimomura et al., 1999). SREBP1C binds to a regulatory element in the promoter of the FAS gene to induce its transcription (Shimomura et al., 1999). SCD1 mRNA expression is also induced by the binding of SREBP1C to its promoter (Paton and Ntambi, 2009). PPARγ has been reported to regulate the expression of FAS (Jin et al., 2012) and SCD1 (Ikeda et al., 2015). Transcription of ACC1 is known to be upregulated by C/EBPα (Tae et al., 1994). Results of this study presented that DF caused a significant and dose-dependent down-regulation of lipogenic transcription factors SREBP1C, PPARγ and C/EBPα, both in vitro and in vivo

32

(Fig. 8, 9 and 10). DF-GA70 contains diverse phytochemicals including (-)-ephedrine and (+)pseudoephedrine from E. intermedia, aloe-emodin and chrysophanol from R. palmatum, and shikonin from L. erythrorhizon with other unidentified phytochemicals (Fig. 2). It is logical to suggest that multiple phytochemical components of DF may affect multiple target genes rather than affecting a few genes. To find multiple genes affected by DF treatment, whole transcriptomes were compared in 3T3-L1 adipocytes untreated vs. treated with DFGA70 using RNA-Seq analyses. Among 9 up-regulators of SREBP1C, PPAR and C/EBP, 8 up-regulators were decreased by DF-GA70 treatment, while only 1 up-regulator was increased (Fig. 12D). Among 6 down-regulators of SREBP1C, PPAR and C/EBP, 4 down-regulators were increased, while 2 down-regulators were decreased. The results showed that majority of up-regulators of lipogenic transcription factors were decreased, and majority of down-regulators of them were increased, which resulted in the down-regulation of SREBP1C, PPAR and C/EBP. Up-regulators of lipogenic transcription factors such as CCAAT/enhancer binding protein  (Cebpb), thyroid hormone receptor alpha (Thra), and runt related transcription factor 1 (Runx1) were decreased by DF-GA70. Cebpb is an adipogenic transcription factor which induce PPARγ and C/EBPα by activating their promoters (Abdou et al., 2013). Thra is known to upregulates SREBP1C (Araki et al., 2009), PPARγ (Ying et al., 2007) and C/EBPα (Fozzatti et al., 2013). Runx1 is an upregulator of C/EBPα which binds in its promoter to induce mRNA expression (Guo et al., 2012). On the contrary, down-regulators of lipogenic transcription factors such as leptin (Lep) and tribbles homolog 1 (Trib1) were increased by DF-GA70. Lep is a down-regulator

33

of SREBP1 and PPARγ, and causes reduction in their expressions in mouse (Tobe et al., 2001). Trib1 is a down-regulator of C/EBPα; expression of Trib1 abolished C/EBPα levels while knockdown of Trib1 caused an increase in its levels (Ishizuka et al., 2014). Peroxisome proliferator activated receptor α (Ppara), a down-regulator of SREBP1C (Yoshikawa et al., 2003) and C/EBPα (Gervois et al., 2004), was increased, which resulted in their down-regulations. Ppara is known to up-regulate PPARγ (Goto et al., 2011), but its up-regulating effect was canceled by the increases of many down-regulators of PPARγ including Thra, Cebpb, Fst, Ncoa2 and Medag. These results suggest that the diverse phytochemicals of a polyherbal composition DF reduce fat accumulation by decreasing the majority of up-regulators and increasing down-regulators of the lipogenic transcriptional factors: SREBP1C, PPARγ and C/EBPα. RNA-Seq analyses were further described in the Supplementary Data.

5. Conclusion DF reduced fat accumulation in 3T3-L1 adipocytes and high-fat diet-induced obese mice. Among three preparations of DF, the 70% ethanol extract was the most effective. DF, which contains diverse phytochemical components, reduced the expressions of the lipidsynthesizing enzymes by decreasing the levels of up-regulators and increasing the levels of down-regulators of the lipogenic transcriptional factors such as SREBP1C, PPARγ and C/EBPα.

Acknowledgements This research was supported by the Korean Health Technology R&D Project

34

(HI15C0075) of the Ministry of Health & Welfare, Republic of Korea.

Conflict of interest The authors declare no conflict of interest.

Author contribution Design the experiments : Yoosik Yoon, Soon Shik Shin, and Heejung Yang Performed the experiments : Jewoong Jang, Yoonju Jung, Seyeon Chae, and Michung Yoon Analyzed the data : Soo Hyun Cho, and Michung Yoon Wrote the manuscript : Yoosik Yoon, Soon Shik Shin, and Heejung Yang

35

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Figure Legends

Fig. 1. HPLC chromatograms of DF preparations. The analytical conditions are described in the material and methods. (A) DF-GA70, DF 70% ethanol extract, (B) DF-GA30, DF 30% ethanol extract, (C) DFFW, DF water extract

Fig. 2. HPLC chromatograms of DF-GA70 and standard phytochemicals. (A) DF-GA70

(B) Standard phytochemicals, aloe-emodin (1) and chrysopanol (5) from R.

palmatum, (+)-pseudoephedrine (2) and (-)-ephedrine (3) from E. intermedia and shikonin (4) from L. erythrorhizon

Fig. 3. Effects of DF preparations on fat droplet formation in 3T3-L1 adipocytes. Fat droplets in preadipocytes, untreated adipocytes and adipocytes treated for 7 days with 5-20 g/ml of DF-FW, DF-GA30 or DF-GA70 were stained with oil red O dye and examined under a light microscope at 400X magnification with the scale bar = 100 m.

Fig. 4. Effects of DF preparations on intracellular fat accumulation and cell viability in 3T3-L1 adipocytes. (A) The inhibitory effect of DF on intracellular fat accumulation was determined by oil red O dye staining. Oil red O dye was extracted using isopropanol and was quantified by measuring optical density at 510 nm. *p<0.05, **p<0.01, ***p<0.001 compared with

44

untreated adipocytes. ###p<0.001 compared with preadipocytes. (B) Cell viability was determined by the ability of mammalian cells to convert hydroxyethyl disulfide into mercaptoethanol.

Fig. 5. Effects of DF preparations on fat accumulation in high-fat diet-induced obese mice. The normal group was fed a low fat diet, while other groups were fed a high-fat diet. Among the groups that were fed a high-fat diet, a control group was administered water while the remaining groups were administered 250 mg/kg/day of DF-FW, DF-GA30 or DFGA70. After 8 weeks of oral administration, adipose tissues were dissected and the weights of (A) inguinal adipose tissue, (B) mesenteric adipose tissue, and (C) retroperitoneal adipose tissue were measured. *p<0.05, **p<0.01, ***p<0.001 compared with the control group. ###p<0.001 compared with the normal group.

Fig. 6. Effects of DF preparations on the size of adipocytes in adipose tissue. Tissue sections were prepared from the inguinal adipose tissues of low fat diet-fed normal mice, high fat diet-fed control mice and high fat diet-fed mice orally administered with 250 mg/kg/day of DF-FW, DF-GA30 or DF-GA70 for 8 weeks. Samples of adipose tissues were fixed and paraffin-embedded. (A) Paraffin sections were stained with hematoxylineosin and imaged (magnification, 100×). (B) Cell sizes were measured using image analysis software. **p<0.01 compared with the control group. #p<0.05 compared with the normal group.

45

Fig. 7. Effects of DF preparations on the expression levels of lipid-synthesizing enzymes. (A-C) RNAs were extracted from 3T3-L1 adipocytes that were treated with 0, 5, 10, 15 or 20 μg/ml of DF-FW, DF-GA30 or DF-GA70 for 7 days. Messenger RNA levels of (A) ACC1, (B) FAS and (C) SCD1 were measured by real-time qPCR. *p<0.05, **p<0.01, ***p<0.001

compared

with

untreated

adipocytes.

###p<0.001

compared

with

preadipocytes. (D-F) RNAs were extracted from adipose tissues harvested from low fat diet-fed normal mice, high fat diet-fed control mice, and high fat diet-fed mice orally administered with 250 mg/kg/day of DF-FW, DF-GA30 or DF-GA70 for 8 weeks. Messenger RNA levels of (D) ACC1, (E) FAS and (F) SCD1 were measured by real-time qPCR. *p<0.05, ***p<0.001 compared with the control group. ###p<0.001 compared with the normal group.

Fig. 8. Effects of DF preparations on the expression levels of lipogenic transcription factors. (A-C) RNAs were extracted from 3T3-L1 adipocytes that were treated with 0, 5, 10, 15 or 20 μg/ml of DF-FW, DF-GA30 or DF-GA70 for 7 days. Messenger RNA levels of (A) SREBP1C, (B) PPAR and (C) C/EBP were measured by real-time qPCR. *p<0.05, **p<0.01, ***p<0.001 compared with untreated adipocytes. ###p<0.001 compared with preadipocytes. (D-F) RNAs were extracted from adipose tissues harvested from low-fat diet-fed normal mice, high-fat diet-fed control mice, and high-fat diet-fed mice orally administered with 250 mg/kg/day of DF-FW, DF-GA30 or DF-GA70 for 8 weeks. Messenger RNA level (D) SREBP1C, (E) PPAR and (F) C/EBP were measured by real-time qPCR. *p<0.05,

46

**p<0.01, ***p<0.001 compared with the control group. ###p<0.001 compared with the normal group.

Fig. 9. In vivo effects of DF-GA70 dose on high fat diet-fed mice. Mice were divided into 4 groups and fed a high-fat diet. A control group was administered 0 mg/kg/day while the remaining groups were administered 150, 300 or 600 mg/kg/day of DF-GA70 for 8 weeks. (A) Mesenteric adipose tissues were dissected and the weighed. (BF) RNAs were extracted from adipose tissues and mRNA levels of FAS (B), SCD1 (C), SREBP1C (D), PPAR (E) and C/EBP (F) were measured by real-time qPCR. *p<0.05 compared with the control group.

Fig. 10. Effects of DF-GA70 on the protein levels of lipogenic transcription factors. (A) Proteins were extracted from 3T3-L1 preadipocytes (lane 1), untreated adipocytes (lane 2) or adipocytes that were treated with 2, 4, 6, 10, 20 or 30 μg/ml of DF-GA70 for 7 days (lanes 3 to 8). Protein levels of PPAR, C/EBP and SREBP1C were analyzed by Western blotting. (B) Proteins were extracted from 3T3-L1 preadipocytes (lane 1), untreated adipocytes (lane 2) or adipocytes cultured without (-) or with (+) DF-GA70 for 2, 4 or 7 days (lanes 3 to 8). Protein levels of PPAR, C/EBP and SREBP1C were analyzed by Western blotting.

Fig 11. Scatter plot of RNA-Seq data of untreated adipocyte vs DF-GA70-treated adipocyte. Up-regulated genes are colored red, and down-regulated genes are colored green. Up and

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down 2 fold threshold line was shown as thin diagonal red and green line, respectively.

Fig. 12. Diagrams of the up-regulators and down-regulators of lipogenic transcription factors found among DEGs. DF-GA70-induced changes in the up- or down-regulators of SREBP1C (A), PPAR (B) and C/EBP (C) were shown along with their combination (D). Up-regulators were marked as red arrows, and down-regulators were marked as green dotted-line arrows. DF-GA70-induced fold changes were marked as color around the gene name: red for increased expressions and green for decreased expressions. More intense color shows greater fold change. Total RNA was isolated from untreated 3T3-L1 adipocytes and those treated with 20 μg/ml of DF-GA70 for 7 days. High-throughput sequencing was performed using NextSeq 500. Functional analyses and networks were generated through the use of Ingenuity Pathway Analysis (www.qiagenbioinformatics.com/products/ ingenuity-pathway-analysis).

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Graphical abstract

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