A novel phenolic formulation for treating hepatic and peripheral insulin resistance by regulating GLUT4-mediated glucose uptake.

Chronic insulin resistance suppresses muscle and liver response to insulin, which is partially due to impaired vesicle trafficking. We report here that a formula consisting of resveratrol, ferulic acid and epigallocatechin-3-O-gallate is more effective in ameliorating muscle and hepatic insulin resistance than the anti-diabetic drugs, metformin and AICAR. The formula enhanced glucose transporter-4 (GLUT4) translocation to the plasma membrane in the insulin-resistant muscle cells by regulating both insulin-independent (calcium and AMPK) and insulin-dependent (PI3K) signaling molecules. Particularly, it regulated the subcellular location of GLUT4 through endosomes to increase glucose uptake under insulin-resistant condition. Meanwhile, this phytochemicals combination increased glycogen synthesis and decreased glucose production in the insulin-resistant liver cells. On the other hand, this formula also showed anti-diabetic potential by the reduction of lipid content in the myotubes, hepatocytes, and adipocytes. This study demonstrated that the three phenolic compounds in the formula could work in distinct mechanisms and enhance both insulin-dependent and independent vesicles trafficking and glucose transport mechanisms to improve carbohydrate and lipid metabolism.

Tamarindus indica seed-shell nanoparticles­silver nanoparticles-Ceratonia silique bean gum composite for copper-micro mesh grid electrode fabrication and its application for glucose detection in artificial salivary samples.

This study used a new approach to fabricate a glucose detection system based on nano-engineered biomaterials. The fabrication steps included strategic synthesis, integration and stabilization of biological and metal nanoparticles in superabsorbent hydrogel gum matrix. The design of the high-performance electrochemical biosensor platform includes copper-micro mesh grid electrode modified with polymer phase comprising of silver nanoparticles surface coroneted with Ceratonia silique locust bean gum (LBG), Tamarindus indica seed-shell nanoparticles and glucose oxidase (GOx). Fundamental assessment of catalytic properties of the nanobiocomposite films on copper grid probe were performed by cyclic voltammetry, amperometry, differential pulse voltammetry. Probes showed good repeatability, reproducibility, selectivity, and long-term stability. The GOx was well-immobilized and stabilized by C. siliqua nano-matrix, with 85% and 98% activity retention when stored at different condiions for 6 month and 3 months, respectively. The fabricated grid-platform exhibited linear response in a wide range of glucose concentration, with detection limit of 1.0 nM (S/N = 3) and sensitivity 38.7 mA nM-1 cm-2. The bionanomaterial-based sensor was successfully applied for ultra-low glucose detection in artificial salivary samples. The designed sensor, perhaps with further modifications, has potential for the next generation of sensing platform in various biological fluids especially for non-invasive glucose detection for diabetic patients.

Amelioration of insulin resistance using the additive effect of ferulic acid and resveratrol on vesicle trafficking for skeletal muscle glucose metabolism.

Dysregulation of vesicle trafficking in muscle is one of the factors responsible for the pathogenesis of insulin resistance (IR). Ferulic acid (FER) and resveratrol (RSV) are known to have hypoglycemic property. In this study, differentiated L6 myotubes were induced with palmitate as a model of IR. Chemical ablation of muscle vesicles was used to investigate how FER and RSV influence glucose utilization. Results showed that both FER and RSV elicit glucose uptake and promote glycogen synthesis in insulin-resistant muscle cells. Mechanistic studies further showed that FER markedly enhances the transferrin receptor-containing endosomal compartment activities via phosphoinositide 3-kinase (PI3K)/atypical protein kinase C-dependent pathway, while RSV facilitates glucose transporter storage vesicles (GSV) trafficking via an exercise-like effect of conventional protein kinase C/5'-adenosine monophosphate-activated protein kinase (AMPK) modulation. Therefore, these two phenolic compounds promoted glucose transport through two separate routes, and they had an additive effect on the increase of glucose uptake in insulin-resistant muscle cells. These findings provide a basis for the understanding of the antidiabetic potential of RSV and FER combination.

Exploration of Chitinous Scaffold-Based Interfaces for Glucose Sensing Assemblies.

: The nanomaterial-integrated chitinous polymers have promoted the technological advancements in personal health care apparatus, particularly for enzyme-based devices like the glucometer. Chitin and chitosan, being natural biopolymers, have attracted great attention in the field of biocatalysts engineering. Their remarkable tunable properties have been explored for enhancing enzyme performance and biosensor advancements. Currently, incorporation of nanomaterials in chitin and chitosan-based biosensors are also widely exploited for enzyme stability and interference-free detection. Therefore, in this review, we focus on various innovative multi-faceted strategies used for the fabrication of biological assemblies using chitinous biomaterial interface. We aim to summarize the current development on chitin/chitosan and their nano-architecture scaffolds for interdisciplinary biosensor research, especially for analytes like glucose. This review article will be useful for understanding the overall multifunctional aspects and progress of chitin and chitosan-based polysaccharides in the food, biomedical, pharmaceutical, environmental, and other diverse applications.

4-Hydroxybenzoic acid serves as an endogenous ring precursor for antroquinonol biosynthesis in Antrodia cinnamomea.

Antrodia cinnamomea, an endemic fungus species of Taiwan, has long been used as a luxurious dietary supplement to enhance liver functions and as a remedy for various cancers. Antroquinonol (AQ), identified from the mycelium of A. cinnamomea, is currently in phase II clinical trials in the USA and Taiwan for the treatment of non-small-cell lung cancer. In the previous studies, we have demonstrated that AQ and 4-acetylantroquinonol B (4-AAQB) utilize orsellinic acid, via polyketide pathway, as the ring precursor, and their biosynthetic sequences are similar to those of coenzyme Q. In order to test 4-hydroxybenzoic acid (4-HBA), synthesized via shikimate pathway, is the ring precursor of AQ analogs, the strategy of metabolic labeling with stable isotopes was applied in this study. Here we have confirmed that 4-HBA serves as the ring precursor for AQ but not a precursor of 4-AAQB. Experimental results indicated that A. cinnamomea preferentially utilizes endogenous 4-HBA via shikimate pathway for AQ biosynthesis. Exogenous tyrosine and phenylalanine can be utilized for AQ biosynthesis when shikimate pathway is blocked by glyphosate. The benzoquinone ring of 4-AAQB is synthesized only via polyketide pathway, but that of AQ is synthesized via both polyketide pathway and shikimate pathway. The precursor-products relationships diagram of AQ and 4-AAQB in A. cinnamomea are proposed based on the experimental findings.

4-Acetylantroquinonol B from antrodia cinnamomea enhances immune function of dendritic cells against liver cancer stem cells.

The functions of 4-acetylantroquinonol B (4-AAQB), a ubiquinone derivative isolated from the mycelium of Antrodia cinnamomea, in immunotherapy for liver cancer were investigated. We found that 4-AAQB could inhibit liver cancer stem cell related manifestations and activate the antitumor ability of dendritic cells. Specifically, 4-AAQB can inhibit EpCAM, AFP and related pathways of HepG2 cells. It also significantly decreases the expression of ß-catenin, inhibits the tumorigenicity and decreases the secretion of immune escape related cytokines. Moreover, 4-AAQB can stimulate the proliferation of immune cells and promote the endocytosis of immature dendritic cells. When co-cultured immature dendritic cells with EpCAM+ HepG2 cells, 4-AAQB enhanced the expression of MHC class I and II on the surface of liver cancer stem cells and dendritic cells, increased the expression of costimulatory molecules CD80 of dendritic cells and cytokines related to immune activation. In conclusion, 4-AAQB from Antrodia cinnamomea can enhance immune function of dendritic cells against liver cancer stem cells, and may have the potential to be used for liver cancer prevention and immunotherapy.

Electrically nanowired-enzymes for probe modification and sensor fabrication.

Enzymes are highly specific and selective due to their precise, intricate three-dimensional catalytic- structure. Electron transfer in enzymes normally occurs through an active-metal centers or tunneling events that are highly insulated by the surrounding globular protein structure. In case of electrochemically active enzymes/proteins, the distance between the redox-active cofactor and the electrode surface plays key role during direct communication. Therefore, the long electron-tunneling distance can be overcome by introducing mobile redox mediators such as nanostructures specially nanowires which can diffuse into and out of the enzyme active site, ferrying reducing or oxidizing equivalents with them. Therefore, nanowire-conjugated enzymes have gained great interest in the development of biosensor devices and other electrocatalytic-biological applications. Herein we present a comprehensive review about the electrochemical enzyme-based sensor using nanowires. Over the past decade, nanowires were investigated as a versatile platform for various applications including sensors and biosensors because of their high aspect ratio and a high surface-to-volume ratio. This review aimed to summarize some of the recent developments in the enzyme based sensor research that have been achieved with various metallic and non-metallic one-dimensional nanostructure i.e. nanowires. Due to low or no toxicity and biocompatibility, enzymes conjugated with nanowires are still highly specific, sensitive and biologically active. This review demonstrates the potential usability of nanowired-enzymes for the bioanalytical applications. The review includes various types of nanowires, mode of the enzyme integration or immobilization methodologies, probe modification, biosensor fabrication and real or spiked sample testing. Biosensor parameters such as linear range and sensitivity, selectivity and detection limit of reported sensors were also considered herein. We also introduce some of the new nanowire materials which have not yet been used for biosensing or biosensor application. The limitations, challenges and prospects for the use of nanowired-enzymes in electrochemical and other real-time sensing systems as well as fabrication technologies are also discussed in this review.

6-shogaol induces autophagic cell death then triggered apoptosis in colorectal adenocarcinoma HT-29 cells.

6-shogaol is a phytochemical of dietary ginger, we found that 6-shogaol could induced both autophagic and apoptotic death in human colon adenocarcinoma (HT-29) cells. Results of this study showed that 6-shogal induced cell cycle arrest, autophagy, and apoptosis in HT-29 cells in a time sequence. After 6h, 6-shogal induced apparent G2/M arrest, then the HT-29 cells formed numerous autophagosomes in each phase of the cell cycle. After 18h, increases in acidic vesicles and LAMP-1 (Lysosome-associated membrane proteins 1) showed that 6-shogaol had caused autophagic cell death. After 24h, cell shrinkage and Caspase-3/7 activities rising, suggesting that apoptotic cell death had increased. And after 48h, the result of TUNEL assay indicated the highest occurrence of apoptosis upon 6-shogaol treatment. It appeared that apoptosis is triggered by autophagy in 6-shogaol treated HT-29 cells, the damage of autophagic cell death initiated apoptosis program.

Biosynthesis of Antroquinonol and 4-Acetylantroquinonol B via a Polyketide Pathway Using Orsellinic Acid as a Ring Precursor in Antrodia cinnamomea.

Antroquinonol (AQ) and 4-acetylantroquinonol B (4-AAQB), isolated from the mycelium of Antrodia cinnamomea, have a similar chemical backbone to coenzyme Q (CoQ). Based on the postulation that biosynthesis of both AQ and 4-AAQB in A. cinnamomea starts from the polyketide pathway, we cultivated this fungus in a culture medium containing [U-13C]oleic acid, and then we analyzed the crude extracts of the mycelium using UHPLC-MS. We found that AQ and 4-AAQB follow similar biosynthetic sequences as CoQ. Obvious [13C2] fragments on the ring backbone were detected in the mass spectrum for [13C2]AQ, [13C2]4-AAQB, and their [13C2] intermediates found in this study. The orsellinic acid, formed from acetyl-CoA and malonyl-CoA via the polyketide pathway, was found to be a novel benzoquinone ring precursor for AQ and 4-AAQB. The identification of endogenously synthesized farnesylated intermediates allows us to postulate the routes of AQ and 4-AAQB biosynthesis in A. cinnamomea.

Curcumin Induces Apoptosis of Colorectal Cancer Stem Cells by Coupling with CD44 Marker.

This study investigated the effect of curcumin on colorectal cancer stem cells (CCSCs) and its possible mechanism. Comparison of the metabolic profiles of human adenomatous polyp (N = 61) and colorectal cancer (CRC) (N = 57) tissue found statistically significant differences (p < 0.05) in their composition of adenosine monophosphate (AMP), adenine, 5'-methythioadenosine, 3-hydroxybutyric acid, prostaglandin E2, threonine, and glutamine. Our cell culture model study found that curcumin treatment (50 µM for 48 h) did indeed increase apoptosis of CRC cells as well as of CCSCs, but at a significant level only in CD44(+) cells. Further metabolic profile studies of the CRC, CD44(+), and CD44(-) cells indicated that curcumin treatment increased glyceraldehyde and hydroxypropionic acid in CD44(-) cells but decreased glutamine content in both curcumin-treated CRC and CD44(+) cells. Based on our comparison of the metabolic profiles of human tissues and cancer cells, we suggest that curcumin might couple with CD44 and that curcumin-CD44(+) coupling at the cell membrane might have some blocking effect on the transport of glutamine into the cells, thus decreasing the glutamine content in the CD44(+) cells and inducing apoptosis.

Fenugreek hydrogel-agarose composite entrapped gold nanoparticles for acetylcholinesterase based biosensor for carbamates detection.

A biosensor was fabricated to detect pesticides in food samples. Acetylcholinesterase was immobilized in a novel fenugreek hydrogel-agarose matrix with gold nanoparticles. Transparent thin films with superior mechanical strength and stability were obtained with 2% fenugreek hydrogel and 2% agarose. Immobilization of acetylcholinesterase on the membrane resulted in high enzyme retention efficiency (92%) and a significantly prolonged shelf life of the enzyme (half-life, 55 days). Transmission electron microscopy revealed that, gold nanoparticles (10-20 nm in diameter) were uniformly dispersed in the fenugreek hydrogel-agarose-acetylcholinesterase membrane. This immobilized enzyme-gold nanoparticle dip-strip system detected various carbamates, including carbofuran, oxamyl, methomyl, and carbaryl, with limits of detection of 2, 21, 113, and 236 nM (S/N = 3), respectively. Furthermore, the fabricated biosensor exhibited good testing capabilities when used to detect carbamates added to various fruit and vegetable samples.

Curcumin-loaded polymeric nanoparticles for enhanced anti-colorectal cancer applications.

The purpose of the present study was to fabricate polymeric nanoparticles as drug carriers for encapsulated curcumin with enhanced anti-colorectal cancer applications. Nanoparticles were formulated from chitosan and gum arabic, natural polysaccharides, via an emulsification solvent diffusion method. The formation of curcumin nanoparticles was confirmed by Fourier transform infrared spectroscopy and differential scanning calorimeter. The results show that curcumin was entrapped in carriers with +48 mV, 136 nm size, and high encapsulation efficiency (95%). Based on an in vitro release study, we inferred that curcumin nanoparticles could tolerate hydrolysis due to gastric juice or small intestinal enzymes, and therefore, it should reach the colon largely intact. In addition, curcumin nanoparticles had higher anti-colorectal cancer properties than free curcumin due to greater cellular uptake. Therefore, we concluded that curcumin was successfully encapsulated in chitosan-gum arabic nanoparticles with superior anti-colorectal cancer activity.

Invertase-nanogold clusters decorated plant membranes for fluorescence-based sucrose sensor.

In the present study, invertase-mediated nanogold clusters were synthesized on onion membranes, and their application for sucrose biosensor fabrication was investigated. Transmission electron microscopy revealed free nanoparticles of various sizes (diameter ~5 to 50 nm) along with clusters of nanogold (~95 to 200 nm) on the surface of inner epidermal membranes of onions (Allium cepa L.). Most of the polydispersed nanoparticles were spherical, although some were square shaped, triangular, hexagonal or rod-shaped. Ultraviolet-visible spectrophotometric observations showed the characteristic peak for nanoparticles decorated invertase-onion membrane at approximately 301 nm. When excited at 320 nm in the presence of sucrose, the membranes exhibited a photoemission peak at 348 nm. The fluorescence lifetime of this nanogold modified onion membrane was 6.20 ns, compared to 2.47 ns for invertase-onion membrane without nanogold. Therefore, a sucrose detection scheme comprised of an invertase/nanogold decorated onion membrane was successfully developed. This fluorescent nanogold-embedded onion membrane drop-test sensor exhibited wide acidic to neutral working pH range (4.0-7.0) with a response time 30 seconds (<1 min). The fabricated quenching-based probe had a low detection limit (2x10(-9) M) with a linear dynamic range of 2.25x10(-9) to 4.25x10(-8) M for sensing sucrose. A microplate designed with an enzyme-nanomaterial-based sensor platform exhibited a high compliance, with acceptable percentage error for the detection of sucrose in green tea samples in comparison to a traditional method. With some further, modifications, this fabricated enzyme-nanogold onion membrane sensor probe could be used to estimate glucose concentrations for a variety of analytical samples. Graphical abstract Synthesis and characterization of invertase assisted nanogold clusters on onion membranes and their application for fluorescence-based sucrose sensor.

A well-refined in vitro model derived from human embryonic stem cell for screening phytochemicals with midbrain dopaminergic differentiation-boosting potential for improving Parkinson's disease.

Stimulation of endogenous neurogenesis is a potential approach to compensate for loss of dopaminergic neurons of substantia nigra compacta nigra (SNpc) in patients with Parkinson's disease (PD). This objective was to establish an in vitro model by differentiating pluripotent human embryonic stem cells (hESCs) into midbrain dopaminergic (mDA) neurons for screening phytochemicals with mDA neurogenesis-boosting potentials. Consequently, a five-stage differentiation process was developed. The derived cells expressed many mDA markers including tyrosine hydroxylase (TH), ß-III tubulin, and dopamine transporter (DAT). The voltage-gated ion channels and dopamine release were also examined for verifying neuron function, and the dopamine receptor agonists bromocriptine and 7-hydroxy-2-(dipropylamino)tetralin (7-OH-DPAT) were used to validate our model. Then, several potential phytochemicals including green tea catechins and ginsenosides were tested using the model. Finally, ginsenoside Rb1 was identified as the most potent phytochemical which is capable of upregulating neurotrophin expression and inducing mDA differentiation.

Concentration variation and molecular characteristics of soluble (1,3;1,6)-ß-D-glucans in submerged cultivation products of Ganoderma lucidum mycelium.

(1,3)-ß-D-Glucans with (1,6)-ß-D-glucosyl branches are bioactive polysaccharides in fruiting bodies and mycelia of Ganoderma lucidum, a mushroom used in traditional Chinese medicine. Submerged cultivation of mycelium is one of the more efficient means of generating polysaccharides from this fungus. Twelve mycelium samples examined in this study demonstrated the quantitative and qualitative molecular characteristics of soluble (1,3;1,6)-ß-D-glucans. It was observed that the concentration of soluble (1,3;1,6)-ß-D-glucan varied substantially from 1.3 to 79.9 mg/dL. (1,3;1,6)-ß-D-Glucans also preserved their molecular characteristics with degrees of branching (DB) of 0.21-0.36 and molecular masses of 10(5)-10(6) g/mol for those samples with substantial quantities of ß-D-glucan. Using the high aggregating tendency of these molecules, (1,3;1,6)-ß-D-glucans were successfully purified via fractional precipitation with 35% (v/v) ethanol. (1,3;1,6)-ß-D-Glucan was proposed as a putative bioactive marker for immunomodulation because it was the most abundant polysaccharide in G. lucidum mycelium products to stimulate macrophage RAW 264.7 cells to release TNF-α.

Skin permeation of D-limonene-based nanoemulsions as a transdermal carrier prepared by ultrasonic emulsification.

Nanoemulsions can be used for transporting pharmaceutical phytochemicals in skin-care products because of their stability and rapid permeation properties. However, droplet size may be a critical factor aiding permeation through skin and transdermal delivery efficiency. We prepared D-limonene nanoemulsions with various droplet sizes by ultrasonic emulsification using mixed surfactants of sorbitane trioleate and polyoxyethylene (20) oleyl ether under different hydrophilic-lipophilic balance (HLB) values. Droplet size decreased with increasing HLB value. With HLB 12, the droplet size was 23 nm, and the encapsulated ratio peaked at 92.3%. Transmission electron microscopy revealed spherical droplets and the gray parts were D-limonene precipitation incorporated in spherical droplets of the emulsion system. Franz diffusion cell was used to evaluate the permeation of D-limonene nanoemulsion through rat abdominal skin; the permeation rate depended on droplet size. The emulsion with the lowest droplet size (54 nm) achieved the maximum permeation rate. The concentration of D-limonene in the skin was 40.11 µL/cm(2) at the end of 360 min. Histopathology revealed no distinct voids or empty spaces in the epidermal region of permeated rat skin, so the D-limonene nanoemulsion may be a safe carrier for transdermal drug delivery.

Chitosan-guar gum-silver nanoparticles hybrid matrix with immobilized enzymes for fabrication of beta-glucan and glucose sensing photometric flow injection system.

Simple and fast photometric flow injection analysis system was developed for sensing of ß-1,3-glucan from medicinal mushroom Ganoderma lucidum during fermentation. For this purpose, the chitosan-guar gum-silver nanoparticle-beta glucanase (Ch-GG-AgNPs-ßG) beads and Ch-GG-AgNPs-GOD (glucose oxidase) beads were prepared. The bead packed mini-columns were then used to assemble a flow injection analysis (FIA) system for the detection of ß-(1→3)-d-glucan biomarker or glucose. This colorimetric flow system can detect glucose and glucan with detection limits as low as 50ngmL(-1) and 100ngmL(-1) (S/N=3), respectively. The analysis time of this FIA was approximately 40s, which is faster than the previously reported glucan sensors. The glucose and glucan calibration curves were obtained in the range of 0.25-1.25µgmL(-1) (R(2)=0.988) and 0.2-1.0µgmL(-1)(R(2)=0.979), respectively. The applicability of the nano-bio-composite FIA sensor system for spiked and real ß-(1→3)-d-glucan samples were tested, and the accuracy of the results were greater than 95%. Thus, the designed FIA provides a simple, interference free and rapid tool for monitoring glucose and ß-glucan content, which can be used for various food samples with a little modification.

Enhancement of 4-acetylantroquinonol B production by supplementation of its precursor during submerged fermentation of Antrodia cinnamomea.

The antiproliferation activity of the ethanol extract of A. cinnamomea mycelium on hepatocellular cancer cells HepG2 was found to be associated with aroma intensity of the broth during fermentation. We hypothesized that some of the volatile compounds are the precursors of the key bioactive component 4-acetylantroquinonol B of this fungus. The major volatile compounds of A. cinnamomea were identified by GC/MS, and they are oct-1-en-3-ol, linalool, methyl phenylacetate, nerolidol, γ-cadinene and 2,4,5-trimethoxybenzaldehyde (TMBA). TMBA and nerolidol were further selected and used as supplements during fermentation. It was found that both of them could increase the production of 4-acetylantroquinonol B and enhance the antiproliferation activity of the fungus. In addition, the TMBA was identified as the most promising supplement for increasing the bioactivity of A. cinnamomea during cultivation.

Antihepatoma and liver protective potentials of ganoderma lucidum ( ling zhi) fermented in a medium containing black soybean ( hei dòu) and astragalus membranaceus ( sheng huáng qí).

The antihepatoma activity and liver protective function of the fermentation products (5 L fermenator) of Ganoderma lucidum (GL; Ling Zhi) cultivated in a medium containing black soybean (BS; Hei Dòu) and Astragalus membranaceus (AM; Sheng Huáng Qí) at different fermentation temperatures were investigated in this study. Hep 3B cells pretreated with lovastatin were used to study the antihepatoma activity, and possible active components were analyzed by reverse-phase high-performance liquid chromatography. Carbon tetrachloride (CCl4)-induced primary rat hepatocyte injury was further used to evaluate the liver protective activity of the fermentation products. While all the GL broth filtrates do not inhibit the growth of Hep 3B cells, the ethanolic extract from GL-2 mycelia (GL-2-mE), cultivated in the medium containing BS (50 g/L) and AM (20 g/L) at 24°C for 11 days showed the best antihepatoma activity (IC50 26.6 µg/mL) than the other ethanolic extracts from GL mycelia, GL fruiting body, BS, and AM did. The antihepatoma activities were correlated with some unknown active components in these samples. Furthermore, GL-2-mE (100 µg/mL) without harmful effect on the growth of normal primary rat hepatocytes significantly maintained cell viability, reduced lactate dehydrogenase leakage, lowered lipid peroxidation, and increased glutathione peroxidase and glutathione S-transferase activities in the CCl4-induced damaged primary rat hepatocytes.

Anti-diabetic effect of a traditional Chinese medicine formula.

An anti-diabetic TCM formula consisting of Schizandra chinensis Baill. (SC), Coptis chinensis (CC), Psidium guajava L. leaves (PG) and Morus alba L. leaves (MA) was developed based on its α-glucosidase, DPP-4 and AGE inhibitory activities in vitro using response surface methodology (RSM). Then, the in vivo study was carried out to confirm the anti-diabetic function of the formula. RSM results showed that the optimum anti-diabetic TCM formula is the combination SC (3000 µg mL(-1)), CC (80 µg mL(-1)), PG (374.56 µg mL(-1)) and MA (480 µg mL(-1)). For the in vivo study, insulin resistant mice were induced by high-fat/high-sucrose (HF/HS) feeding for 6 weeks. Administration of the developed formula significantly decreased non-fasting blood glucose in the HF/HS diet mice. Moreover, the formula decreased blood glucose levels in the insulin tolerance test. These results indicated that the anti-diabetic mechanism of the formula might be due to decreased insulin resistance. The serum fructosamine level in the high dose group was significantly lower than the HF/HS and normal control groups, indicating that the formula could improve middle term glucose levels and reduce the risk of complications. The contents of berberine and 1-deoxynojirimycin in the formula were 4.7 ± 0.4 and 77.1 ± 1.1 µg mL(-1), respectively. These two compounds can be used as indicators for quality control during production.