The herbal composition GGEx18 from Laminaria japonica, Rheum palmatum, and Ephedra sinica reduces obesity via skeletal muscle AMPK and PPARα

Figures & data

Table 1.  Composition of GGEx18.

Scientific name	Common name	Family name	Part used	Content (%)
Laminaria japonica Aresch	Sea Tangle	Laminariaceae	Broad strips	40
Ephedra sinica Stapf	Chinese ephedra	Ephedraceae	Stem	40
Rheum palmatum L.	Chinese rhubarb	Polygonaceae	root	20

Table 2.  Sequences of primers used for the RT-PCR assays.

Genes	GeneBank	Primer sequences	Size (bp)
AMPKαl	NM00103367	Forward: 5′-agagggccgcaataaaagat-3′	177
		Reverse: 5′-tgftgtacaggcagetgagg-3′
AMPKα2	NM178143	Forward: 5′-cgccketagtcckccatcag-3′	219
		Reverse: 5′-atgtcacacgclitgetckg-3′ -
PPARα	NM011144	Forward: 5′-gcagetcgtacaggtcatca-3′	202
		Reverse: 5′-ckcitcatccccaagcgtag-3′
CPT-1	LOTT36	Forward: 5′-tatgtgaggatgetgettcc-3′	586
		Reverse: 5′-ctcggagagetaagettgtc-3′
VLCAD	AF017176	Forward: 5′-cgtcagaggtgtacittgatgg-3′	268
		Reverse: 5′-catggackcagtcacatackgc-3′
MCAD	NM007382	Forward: 5′-gacatitgaaagetgetagtg-3′	380
		Reverse: 5′-tcacgagetatgatcagcctctg-3′
ACOX	J02752	Forward: 5′-ackataifiggccaattittg-3′	196
		Reverse: 5′-tgtggcagtggificcaagcc-3′
UCP3	NM009364	Forward: 5′-ggagccatggcagtgaciagt-3′	180
		Reverse: 5′-tgtgatgftgggccaagtccc-3′
β-Actin	J00691	Forward: 5′-tggaatcckgtggcatccatgaaa-3′	350
		Reverse: 5′-taaaacgcagetcagtaacagtcc-3′

Figure 1.  GGEx18 inhibits high fat diet–induced body weight gain and total adipose tissue mass in C57BL/6J mice. Adult male C57BL/6 mice were fed a low-fat diet (normal), a high-fat diet (control), or the fat diet supplemented with 125, 250, and 500 mg/kg/day GGEx18 for 9 weeks. (A) Body weights at the end of the treatment period were statistically significant between normal and control groups (p < 0.05), and between the control group and the 250 mg/kg/day and 500 mg/kg/day GGEx18 groups (p < 0.05). (B) At the end of study, adipose tissue weights were measured. All values are expressed as the mean ± SD. #p < 0.05 compared with normal group, *p < 0.05 compared with control group. Normal, low fat diet-fed mice; control, high fat diet-fed mice; GGEx18, GGEx18-treated high fat diet-fed mice.

Figure 2.  GGEx18 inhibits high fat diet–induced increases in lipid accumulation in skeletal muscle of C57BL/6J mice. Adult male C57BL/6J mice were fed a low-fat diet (normal), a high-fat diet (control), or the high-fat diet supplemented with 125, 250, and 500 mg/kg/day for 9 weeks. (A) Histology showing transverse sections of skeletal muscle. (B) Higher magnitude of bracket area from (A). Shown are representative oil red O-stained sections (7-μm thick) of skeletal muscle. The number and intensity of stained fibers in the GGEx18 group were much less than that in the control group. Normal, low fat diet-fed mice; control, high fat diet-fed mice; GGEx18, GGEx18-treated high fat diet-fed mice.

Figure 3.  GGEx18 increases the mRNA expression levels of AMPK, PPARα, and PPARα target genes in skeletal muscle of C57BL/6J mice. (A) Adult male C57BL/6J mice were fed a low-fat diet (Normal), a high-fat diet (Control), or the high-fat diet supplemented with 250 mg/kg/day GGEx18 for 9 weeks. Total cellular RNA was extracted from skeletal muscle tissue, and mRNA levels were measured using RT-PCR. All values are expressed as the mean ± SD of relative density units using β-actin as a reference. *p < 0.05 compared with control group. (B) Representative RT-PCR bands from one of the three independent experiments are shown. Control, high fat diet–fed mice; GGEx18, GGEx18 (50 mg/kg/day)-treated high fat diet–fed mice.

Figure 4.  GGEx18 decreases triglyceride droplets in C2C12 cells. C2C12 cells were differentiated as described in the Materials and Methods section and triglycerides were stained with oil red O. (A) Non-differentiated cells (ND). Differentiated cells treated with (B) DMSO, (C) 10 μg/mL GGEx18, (D) 10 µM fenofibrate, and (E) 10 µM Wy14,643.

Figure 5.  Compound C increases GGEx18-induced decreases in triglyceride droplets in C2C12 cells. C2C12 cells were differentiated as described in the Materials and Methods section and triglycerides were stained with oil red O. Differentiated cells treated with (A) DMSO, (B) 10 μg/mL GGEx18, (C) 10 μg/mL GGEx18 plus 0.1 μM compound C, (D) GGEx18 plus 1 μM compound C, and (E) GGEx18 plus 10 μM compound C. Comp C, compound C.

Figure 6.  Compound C decreases GGEx18-induced increases in mRNA expression levels of AMPK, PPARα, and PPARα target genes in C2C12 cells. C2C12 cells were differentiated as described in the Materials and Methods section. C2C12 cells were treated with DMSO, 10 μg/mL GGEx18, and 10 μg/mL GGEx18 plus 1 µM compound C. Total cellular RNA was extracted from differentiated cells and mRNA levels were measured using transcription-polymerase chain reaction (RT-PCR). All values are expressed as the mean ± SD of relative density units using β-actin as a reference. #p < 0.05 compared with DMSO group, **p < 0.05 compared with GGEx18 group. (B) Representative RT-PCR bands from one of the three independent experiments are shown.