Effects of a multi-herbal extract on type 2 diabetes
© Yeo et al; licensee BioMed Central Ltd. 2011
Received: 14 October 2010
Accepted: 4 March 2011
Published: 4 March 2011
An aqueous extract of multi-hypoglycemic herbs of Panax ginseng C.A.Meyer, Pueraria lobata, Dioscorea batatas Decaisne, Rehmannia glutinosa, Amomum cadamomum Linné, Poncirus fructus and Evodia officinalis was investigated for its anti-diabetic effects in cell and animal models.
Activities of PPARγ agonist, anti-inflammation, AMPK activator and anti-ER stress were measured in cell models and in db/db mice (a genetic animal model for type 2 diabetes).
While the extract stimulated PPARγ-dependent luciferase activity and activated AMPK in C2C12 cells, it inhibited TNF-α-stimulated IKKβ/NFkB signaling and attenuated ER stress in HepG2 cells. The db/db mice treated with the extract showed reduced fasting blood glucose and HbA1c levels, improved postprandial glucose levels, enhanced insulin sensitivity and significantly decreased plasma free fatty acid, triglyceride and total cholesterol.
The aqueous extract of these seven hypoglycemic herbs demonstrated many therapeutic effects for the treatment of type 2 diabetes in cell and animal models.
Caused by complex interactions of multiple factors, diabetes mellitus type 2 (type 2 diabetes) is characterized by decreased secretion of insulin by the pancreas and resistance to the action of insulin in various tissues (eg muscle, liver, adipose), leading to impaired glucose uptake . Management of type 2 diabetes usually begins with change of diet and exercise  and most patients ultimately require pharmacotherapy, such as oral anti-diabetic drug (OAD) . OADs include sulfonylurea, non-sulfonylurea secretagogues, biguanides (eg metformin), thiazolidinediones (eg TZD or glitazone) and glucosidase inhibitors and glucagon-like peptide-1 (GLP-1) inhibitor. All OADs, however, have adverse effects, eg weight gain with sulfonylurea, non-sulfonylurea secretagogues or TZD, edema and anemia with TZD .
A variety of medicinal herbal products including herbs used in Chinese medicine have beneficial effects on diabetes  and used as non-prescription treatment for diabetes ; many of these herbs have been formulated into multi-herbal preparation for enhanced effects . While traditional formulae are often prescribed, their efficacy has yet to be investigated; recently, anti-diabetic multi-herbal formulae were studied and reported [6, 7].
The present study reports a new anti-diabetic formula consisting of seven herbs, namely hypoglycaemic cadidates including Panax ginseng C.A.Meyer, Pueraria lobata, Dioscorea batatas Decaisne, Rehmannia glutinosa , Amomum cadamomum Linné , Poncirus fructus  and Evodia officinalis  which are available in South Korea. This formula's anti-diabetic molecular mechanisms and anti-hyperglycemic effects are demonstrated in cell models and db/db mice respectively.
The dried herbs of Panax ginseng C.A. Meyer (Aralia family), Pueraria lobata (Pea family), Dioscorea batatas DECAISNE (Dioscoreaceae), Rehmannia glutinosa (Phrymaceae), Amomum cadamomum Linné (Zingiberaceae), Poncirus fructus(Rhamnaceae)) and Evodia officinalis DODE(Rutaceae) were purchased from Kwangmyungdang Natural Pharmaceutical (Korea) and identified morphologically, histologically and authenticated by Professor Su-In Cho (School of Korean Medicine, Pusan National University, Korea) according to standard protocol in National Standard of Traditional Medicinal Materials of The Korean Pharmacopeia . Voucher specimens of all seven species were deposited in Pusan National University, Korea.
Powders of the herbs were mixed in equal amount (200 g each) and extracted in hot-water. The extract was freeze dried to powder and melt by dimethylsulfoxide (DMSO) when used. Macelignan, an active compound of Myristica fragrans Houtt (Myristicaceae), was prepared for positive control .
Cell lines of human embryonic kidney (HEK) 293 (CRL-1573), 3T3-L1 pre-adipocytes (CL-173), HepG2 hepatocytes (HB-8065) and C2C12 skeletal myoblast cells (CRL-1772) were obtained from the American Type Culture Collection (ATCC, USA). HEK293 and HepG2 were cultured in Dulbecco's modified Eagle's medium (DMEM) containing glucose (Invitrogen, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco BRL, USA). The 3T3-L1 pre-adipocytes were differentiated as described previously . C2C12 skeletal myoblast cells were grown in DMEM supplemented with 2% horse serum to induce differentiation into myotubes.
The PPAR ligand-binding activity was measured with a GAL4/PPAR chimera assay and PPRE-tk-Luc reporter assay as described previously . HEK293 cells were transfected with pFA-PPARγ and pFR-Luc (UAS-Gal4-luciferase) and treated with the extract, rosiglitazone (Alexis Biochemicals, USA) or macelignan at doses ranging from 2 to 10 μmol/L for 24 hours. For PPRE-tk-Luc reporter assay, HepG2 (2 × 105 cells/well) were transfected with PPRE-tk-Luc and incubated with the extract, rosiglitazone or macelignan for 24 hours. The luciferase activities were then determined with a luciferase assay system kit (Promega, USA).
To determine the anti-inflammatory activities and anti-endoplasmic recticulum (ER) stress, we transfected HepG2 cells (2 × 105 cells/well) with NFkB-Luc reporter or ERSE-Luc reporter using a Cignal™ Reporter Assay kit (SABiosciences, USA). The cells were then incubated with the extract, rosiglitazone or macelignan for 24 hours. The luciferase activities were determined with a Dual-Glo Luciferase assay system kit (Promega, USA).
We performed Real-time RT-PCR to determine the expression of adipose fatty acid-binding protein (aP2), acyl-CoA synthetase (ACS) and carnitine palmitoyltransferase-1 (CPT-1). The total RNA was extracted with TRIzol reagent (Invitrogen, USA) and subjected to reverse transcription with M-MLV Reverse Transcriptase (Promega, USA). The total RNA was then amplified (with gene-specific primers) and quantified with a fluorescence thermocycler (iQ™5, Multicolor Real-Time PCR System, Bio-Rad, USA).
Western blot analysis
Total proteins were extracted with PRO-PREP reagent (iNtRON Biotechnology, Korea) and immuno-blotted with the antibodies of p-AMPK, IkBα, GRP78 or p-elf2α (Santa Cruz Biotechnology, USA) . The immune complexes were identified with an enhanced chemiluminescence detection system (Amersham Biosciences, Sweden) according to the manufacturer's instructions and in conjunction with a Fluorochem gel image analyzer (MF-Chem:BIS 3.2, Alpha Innotech, USA).
Twenty-eight (28) male C57BL/KsJ-db/db mice aged 8 weeks were purchased from Jackson Laboratory (USA) and individually housed in polycarbonate cages under a 12-hour light-dark cycle at 21-23°C and 40-60% humidity. After a 2-week adaptation period, the body weight and fasting blood glucose level of the 10-week-old mice were measured. Then, the mice were equally divided into four groups (n = 7): (1) diabetic control, (2) rosiglitazone, (3) macelignan and (4) treatment (with the extract). All groups were fed a standard AIN-76 semi-synthetic diet (American Institute of Nutrition) and three experimental groups (rosigltiazone, macelignan and treatment) were orally administered with rosiglitazone (10 mg/kg body weight), macelignan (15 mg/kg body weight) or the extract (150 mg/kg body weight) for three weeks. After starved for 12 hours, the mice were anesthetized with ether and their blood samples were collected from the inferior vena cava for the measurement of the blood and plasma biomarkers such as HbA1c and insulin. All animal handlings during the experiments were in accordance with the Pusan National University guidelines for the care and use of laboratory animals.
Fasting blood glucose, blood HbA1c and plasma biomarker analyses
During the experiments, the fasting blood glucose concentration was monitored by a Glucometer (GlucoDr, Allmedicus, Korea) with venous blood drawn from the mouse tail vein after a 12-hour fast. Moreover, the blood glycosylated hemoglobin (HbA1c) collected from sacrificed mice was measured with a MicroMat™ II Hemoglobin A1c Test (Bio-Rad Laboratories, USA). All blood samples obtained were centrifuged at 1000 × g for 15 min at 4°C for biochemical analysis. The plasma insulin, glucagon and C-peptide levels were measured with the enzyme-linked immunosorbent assay (ELISA) kits (ALPCO Diagnostics, USA).
Furthermore, the plasma lipids such as total cholesterol and triglyceride were determined with commercial kits (Sigma-Aldrich, USA) while the plasma free fatty acid (FFA) concentration was determined with an ACS (acyl-CoA synthetase)-ACOD(ascorbate oxidase) method (Wako Pure Chemical Industries, Japan).
Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT)
On the third week of treatment, an intraperitoneal glucose and insulin tolerance test (IPGTT and IPITT) were performed on all db/db mice after a 12-hour overnight fast. To determine the glucose and insulin tolerance, we injected the mice intraperitoneally with glucose (0.5 g/kg body weight) or insulin (2 unit/kg body weight). The glucose concentrations of blood drawn from the tail vein were determined immediately upon collection at 30, 60 and 120 min after glucose injection or at 30, 60 and 120 min after insulin injection.
All statistical tests were two-sided, and the level of significance was set at 0.05. All data are presented as mean ± standard deviation (SD) for all groups. Statistical analyses were performed with the SPSS, version 18(SPSSInc., Chicago, IL, USA). One-way ANOVA(analysis of variance) with post-hoc test by Duncan's multiple-range test was used to examine differences among groups. The data were analyzed by Student's t-test for two group comparison.
Effect on PPARγ agonist
Effect on AMPK activation
Effect on inflammatory processes
Effect on attenuation of ER stress
Effects on body weight change and fasting blood glucose in db/db mice
Effects on postprandial glucose and insulin sensitivity in db/db mice
Effects on plasma lipids in db/db mice
Effects of the extract on the plasma lipid profiles in db/d b mice
2.28 ± 0.21a
0.94 ± 0.05c
1.70 ± 0.21b
1.75 ± 0.11b
296.2 ± 59.5a
109.4 ± 29.2c
259.0 ± 54.9ab
217.9 ± 34.9b
Total cholesterol (mg/dL)
146.1 ± 15.0b
181.9 ± 5.84a
110.0 ± 22.4c
119.4 ± 3.41c
Effects on glycosylated hemoglobin level and plasma biomarkers in db/db mice
Effects of the extract on concentrations of blood and plasma biomarkers in db/db mice
10.7 ± 0.46a
1.48 ± 0.89b
0.37 ± 0.07a
3.12 ± 0.73b
4.68 ± 1.11b
7.40 ± 0.88c
3.43 ± 1.05a
0.32 ± 0.02a
4.76 ± 1.09a
9.67 ± 3.05ab
10.8 ± 0.25a
1.52 ± 0.12b
0.23 ± 0.05b
4.14 ± 0.35ab
6.74 ± 1.31b
9.3 ± 0.80b
3.15 ± 1.43a
0.21 ± 0.02b
4.79 ± 0.44a
14.2 ± 7.55a
In this study, we tested a formulation of seven medicinal herbs including Panax ginseng C.A.Meyer for the anti-diabetic effects in cells and in vivo. We found that the extract from the seven herbs functioned as PPARγ agonists and an AMPK activators, as well as inhibitors of inflammation and ER stress. PPARγ can improve insulin sensitivity and glucose tolerance by regulating lipid storage, glucose homeostasis and adipokine regulation . The TZD group, especially rosiglitazone and troglitazone, are agonists of PPARγ . The extract significantly increased the PPARγ-dependent luciferase activity in vitro and stimulated the formation of lipid droplets and the expression of aP2 upon transient transfection of 3T3-L1 cells. Rb1, the most abundant ginsenoside in ginseng root, increases the expression of mRNA and protein of PPARγ and exerts anti-diabetic and insulin-sensitizing activities . 20(S)-protopanaxatriol (PPT), a ginsenoside metabolite, increases PPARγ-transactivation activity with an activity similar to troglitazone, and up-regulates the expression of PPARγ target genes such as aP2, LPL and PEPCK . Therefore, the activity of PPARγ against may be due to Panax ginseng. Further studies are required to confirm this speculation.
Activation of AMPK enhances insulin sensitivity through increased glucose uptake and lipid oxidation in skeletal muscle and inhibition of glucose and lipid synthesis in the liver . Metformin acts as an activator of AMPK in the liver and skeletal muscle . The present study demonstrated that the extract activated AMPK in C2C12 and induced increased expression of AMPK target genes. Ginsenoside Rh2 and Rg3, a red ginseng rich constituent, activates AMPK significantly in 3T3-L1 adipocytes and to contribute to antiobesity effects [22, 23]. Further studies are required to characterize which herb activates AMPK.
Inflammatory cytokines and IKK attenuate insulin signaling through serine phosphorylation of IRS-1 . High doses of salicylates, which block the IKKb activity, ameliorate hyperglycemia and insulin resistance in diabetes and obesity . Our results showed that the extract effectively suppressed NFkB-dependent luciferase activity in TNF-α-treated HepG2 cells and increased the IkB level, suggesting that the extract blocked the activation of the NF-κB pathways.
By activating c-Jun amino-terminal kinase (JNK), which induces insulin resistance in liver and skeletal muscle and inhibits beta cell function, ER stress induces the development of type 2 diabetes . Thus, agents that alleviate ER stress may act as potent anti-diabetic agents. Chemical or biological compounds such as macelignan , chromium-phenylalanine , PBA (phenyl butyric acid)  or TUDCA (tauroursodeoxycholic acid)  or molecular chaperon have been shown to inhibit ER stress and enhance insulin sensitivity, thereby normalizing hyperglycemia. The present study found that the extract alleviated ER stress and efficiently suppressed ERSE-dependent transactivation in thapsigargin-treated HepG2 and expression of ER stress marker proteins. In future studies, we will determine the optimal combination ratio for this formulation and isolate its active fractions.
The aqueous extract of these seven hypoglycemic herbs demonstrated anti-diabetic effects on type 2 diabetes.
aminoimidazole carboxamide ribonucleotide
AMP-activated protein kinase
adipose fatty acid-binding protein 2
ER stress response element
free fatty acids
elongation initiation factor
blood glycosylated hemoglobin
high density lipoprotein-cholesterol
human embryonic kidney293
intraperitoneal glucose tolerance test
intraperitoneal insulin tolerance test
c-Jun N-terminal kinases
oral antidiabetic drug
phenyl butyric acid
peroxisome proliferator-activated receptor
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (331-2008-1-E-00036) and Pusan National University (Program Post-Doc 2009).
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