Biological pH Range Research Articles

A water-soluble colorimetric chemosensor for sequential probing of Cu2+ and S2− and its practical applications to test strips, reversible test, and water samples

A water-soluble colorimetric chemosensor NHOP ((E)-1-(2-(2-(2-hydroxy-5-nitrobenzylidene)hydrazineyl)-2-oxoethyl)pyridin-1-ium) chloride) was developed for the sequential probing of Cu2+ and S2−. NHOP underwent a color change from pale yellow to colorless in the presence of Cu2+ in pure water. The binding ratio between NHOP and Cu2+ was confirmed to be 1:1 by the Job plot and ESI-MS (electrospray ionization mass spectrometry). The detection limit of NHOP for Cu2+ was calculated as 0.15 μM, which was far below the EPA (Environmental Protection Agency) standard (20 μM). The NHOP-coated test strip was able to easily monitor Cu2+ in real-time. Meanwhile, the NHOP-Cu2+ complex reverted from colorless to pale yellow in the presence of S2− through the demetallation. The stoichiometric ratio between NHOP-Cu2+ and S2− was determined to be 1:1 by analyzing the Job plot and ESI-MS. The detection limit of NHOP-Cu2+ for S2− was calculated as 0.29 μM, which was very below the WHO (World Health Organization) guideline (14.7 μM). NHOP successfully achieved the quantification for Cu2+ and S2− in water samples. NHOP could work as a sequential probe for Cu2+ and S2− at the biological pH range (7.0–8.4). Moreover, NHOP could successively probe Cu2+ and S2− at least three cycles because of its reversible property. The detection mechanisms of NHOP for Cu2+ and NHOP-Cu2+ for S2− were demonstrated with Job plot, ESI-MS, and DFT (density functional theory) calculations. Therefore, NHOP could work as an efficient sequential probe for Cu2+ and S2− in environmental systems.

An efficient copper(II) salamo-based complex fluorescence chemosensor for detecting sulfide ions: Structure exploration and practical application

A novel salamo-based fluorescent chemical sensor l-Cu2+ was designed and prepared via the reaction of H2L and Cu(OAc)2·H2O. The whole research has found that Cu(II) atom in the complex crystal are located in the N2O2 cavity of H2L, forming a four-coordination system. Hirshfeld surface analysis confirmed the presence of hydrogen bonding and π···π stacking interactions. As a probe molecule, l-Cu2+can selectively and sensitively detect S2- without interference from other ions, and can be effectively used within the biological pH range. In addition, DFT theoretical calculations and molecular electrostatic potential analysis (ESP) were used to investigate the electron cloud density and positive and negative electrostatic potentials inside the probe, as well as the mechanism for identifying sulfur ions. Finally, the probe l-Cu2+has shown good practical application effects and can recognize S2− in three different water samples. It can also be used as a qualitative detection strip. l-Cu2+complex possesses excellent application prospect in bacteriostasis.

An AIE-Active probe for detection and bioimaging of pH values based on lactone hydrolysis reaction.

Cellular pH homeostasis is essential for many physiological and pathological processes. pH monitoring is helpful for the diagnosis, treatment and prevention of disorders and diseases. Herein, we developed a ratiometric fluorescent pH probe (TCC) based on a coumarin derivative containing a highly active lactone ring. TCC exhibited a typical AIE effect and emitted blue fluorescence under weak acidic condition. When under weak basic condition, the active lactone moiety underwent a hydrolysis reaction to afford a water-soluble product, which gave red-shifted emission. The emission color change from blue through cyan and then to yellow within pH 6.5-9.0 which is approximate to the biological pH range. And the fluorescence color change along with pH value is reversible. Furthermore, TCC was successfully utilized in the detection of the intracellular pH change of live HeLa cells, which indicated that TCC had practical potential in biomedical research.

Poly(maleic anhydride) Shell Modified with Negatively and Positively Charged Groups to Control Zeta Potential and Hydrodynamic Size of Encapsulated Quantum Dots at Variable pH

AbstractWe prepared water‐soluble quantum dots (QDs) with tunable ξ‐potential varied from −38 to +20 mV and controlled sensitivity to pH level. Poly(maleic anhydride‐alt‐1‐tetradecene) (PMAT) was modified with bifunctional molecules containing sulfate, sulfonate, phosphate, phosphonate, and quaternary ammonium in different proportions. The sulfonate shell of QDs leads to a stable negative ξ‐potential in the biological pH range from 4.5 to 9.5, while modification with quaternary ammonium groups gives QDs with positive and pH‐independent charge. If nearly half of carboxyl groups of PMAT were modified with quaternary ammonium groups, ξ‐potential begins pH‐dependent with the isoelectric point at pH≈5.5. To obtain pH‐independent, neutrally charged QDs, the rest of carboxyl groups can be converted into sulfonate groups at a 1 : 1 ratio; sulfonate‐quaternary ammonium QDs show high colloidal stability in solutions with high ionic strength. In addition, encapsulated QDs show an unusual correlation between their hydrodynamic size and ζ‐potential: when ζ‐potential approaches zero, the hydrodynamic size markedly increases due to a decrease in the electrostatic repulsion component of the QDs diffusion coefficient.

One-/Two-Photon Excited Cell Membrane Imaging and Tracking by a Photoactive Nanocage.

Cell membrane imaging by predesigned molecular and supramolecular photoluminescence probes is of great importance in understanding the nano-biointeractions for potential applications in cellular tracking, drug delivery, cancer diagnosis, and treatment. Herein, we report an effective strategy for cell membrane imaging in both living cell and tissue levels on the basis of a multifunctional nanocage (MOC-16) integrating one-/two-photon active phosphorescence, high charges, balanced hydrophobicity/lipophilicity, and proton sensitivity attributes. The intrinsic optical characters, including strong one-/two-photon excitation and pH-dependent red emission, make MOC-16 powerful optical probes for membrane imaging in living cell and tissue levels under both visible and near-infrared irradiations. Meanwhile, the highly positive charges of +28 endow MOC-16 with adequate water solubility and deprotonation ability while still maintaining its hydrophobicity, thus enabling balanced hydrophobic-lipophilic interactions at the nano-biointerface to facilitate a pH-dependent membrane absorption within the biological pH range of 5.3-7.4. However, the low-charged RuL3 metalloligand or polyethylene glycol (PEG)-modified MOC-16PEG with less hydrophobicity cannot offer enough nano-biointeractions for cell membrane tracking. These findings advance the fundamental understanding of nano-biointerface interactions of MOCs with cell membranes and provide further guidance in their biological applications.

Development of Mebeverine HCl Prolonged release Mini-tablets by thermoplastic granulation technique

Mebeverine HCl is a BCS class-I drug and thus it possesses high solubility in aqueous media across the biological pH range. The marketed reference product is a multi-unit particulate system (MUPS) containing prolonged release pellets filled in hard gelatin capsule. In conventional manufacturing process, a huge quantity of solvents (aqueous and/or organic) is used to manufacture such dosage form. Additionally, it demands more processing time and efforts. Therefore, a prolonged release capsules dosage form of Mebeverine HCl was formulated using thermoplastic (melt) granulation technique without usage of any solvent. Prolonged release minitablets sized 2 mm in diameter were developed as per quality by design principles. A 23 full-factorial design of experiment was applied to optimize levels of drug release controlling ingredients which includes a hydrophobic meltable binder (hydrogenated castor oil) cum matrixing agent, a hydrophilic meltable binder (polyethylene glycol) which may act as pore former also, and a release controlling polymer (ethyl cellulose). The optimized formulation was found stable. Dissolution profiles of the optimized formulation were found similar to the marketed reference product in different media across the physiological pH range. In conclusion, the explored solvent less process was capable to manufacture the MUPS dosage form of Mebeverine HCl prolonged-release capsules, which is stable and pharmaceutically equivalent with the reference product. The developed process is more beneficial to small and medium scale industry, as it does not require any special and costly equipment, significantly decreases manufacturing cost and increases productivity compared to conventional process, which is mentioned in literature.

Red-Near-Infrared Fluorescent Probe for Time-Resolved in Vivo Alkaline Phosphatase Detection with the Assistance of a Photoresponsive Nanocontainer.

The monitoring of alkaline phosphatase (ALP) activity in different tissues is significant for disease diagnosis and therapy. However, the time-resolved in vivo sensing of ALP activity remained unresolved. Herein, a novel red-near-infrared fluorescent ALP probe (Cl2-BDCM-ALP) based on a dichloro-substituted dicyanomethylene-4H-chromene derivative was designed and synthesized with high fluorescence efficiency and stability under biological pH range. By using Cl2-BDCM-ALP, ALP activity under an acidic microenvironment such as a tumor site can be sensitively imaged, which cannot be achieved by some previously reported ALP probes. By further loading the Cl2-BDCM-ALP into a near-infrared (NIR) light-responsive nanocontainer, time-resolved long-term imaging of ALP activity was facilely achieved with noninvasive NIR light remote control. Time-resolved variation of ALP activity of the drug-induced acute liver injury mice was successfully monitored in vivo for the first time. This strategy holds great promise in the in situ ALP detection under a broad pH range with temporal resolution.

Kilogram-scale synthesis of carbon quantum dots for hydrogen evolution, sensing and bioimaging

The development of large-scale synthetic methods for high quality carbon quantum dots (CQDs) is fundamental to their applications. However, the macroscopic preparation and scale up synthetic of CQDs is still in its infancy. Here, we report a facile, green, kilogram-scale synthesis of high quality fluorescent CQDs derived from poplar leaves via a one-step hydrothermal method. Notably, the throughput of CQDs can reach a level up to as high as 1.4975 kg in one pot. The structure and properties of the as-prepared CQDs were assessed through TEM, XRD, XPS and various spectroscopic methods. The obtained high quality CQDs with a photoluminescent quantum yield of 10.64% showed remarkable stability in aqueous media, rich functional groups, high photostability, consistent photoluminescence within biological pH range and low cytotoxicity. On account of these good properties, we demonstrated the multifunctional application to electrocatalytic water splitting, Fe3+ sensing and bioimaging. It showed remarkable electrocatalytic activity, Fe3+ sensitivity and good biocompatibility. This study provides a green, facile, inexpensive and large-scale method for producing high quality CQDs, which provides application value for large-scale production of CQDs.

PH-Sensitive Visible or Shortwave Infrared Quantum Dot Nanoprobes Using Conformation-Switchable Copolymeric Ligands

Intracellular and extracellular pH are key parameters in many physiological processes and diseases. For example, the extracellular pH of the tumor micro-environment is slightly more acidic than in healthy tissue. In vivo mapping of the extracellular pH within the tumor would therefore improve our understanding of the tumor physiology. Fluorescent semiconductor quantum dots (QDs) represent interesting probes for in vivo imaging, in particular in the shortwave infrared (SWIR) range. Here, pH-sensitive QD nanoprobes are developed using a conformation-switchable surface chemistry. The central fluorescent QD is coated with a copolymer ligand and conjugated to gold nanoparticle quenchers. As the pH decreases from physiological (7.5) to slightly acidic (5.5-6), the copolymer reversibly shrinks, which increases the energy transfer between the QD and the gold quenchers and modulates the QD fluorescence signal. This enables the design of ratiometric QD probes for biological pH range emitting in the visible or SWIR range. In addition, these probes can be easily encapsulated and remain functional within ghost erythrocyte membranes, which facilitate their in vivo application.

A nanomolar detection of mercury(II) ion by a chemodosimetric rhodamine-based sensor in an aqueous medium: Potential applications in real water samples and as paper strips

A new promising rhodamine based colorimetric and fluorometric chemosensor, RDV has been designed and synthesized for specific detection of Hg2+ ion. It acts as highly selective "turn-on" fluorescent chemosensor for Hg2+ ion without interference from other competitive metal ions in aqueous acetonitrile medium. The drastic color change with addition of Hg2+, from colorless to pink, indicates RDV can acts as "naked-eye" indicator for Hg2+. The Hg2+ promoted selective hydrolysis of appended vinyl ether group in RDV followed by Hg2+ chelated complex formation with concomitant opening of spirolactam ring is the plausible sensing mechanism. The detailed absorption, fluorescence, 1H NMR, 13C NMR and mass spectrometry confirms the proposed sensing mechanism. The limit of detection (LOD) of Hg2+ by RDV is 136 nM indicating the high sensitivity towards Hg2+. The RDV shows consistent spectroscopic response in biological pH range 4-10. In addition to explore practical applicability of RDV, its paper strips have been made and used to detect Hg2+ in pure water solution up to 10 ppm level. Furthermore, the potential application of RDV for the sensing of Hg2+ in real water samples (tap water and drinking waters from different sources) were also monitored and demonstrated.

Polyethylene glycol-modified cystamine for fluorescent sensing

Tedious polymeric modifications, laborious syntheses, low water solubility, high cytotoxicity and low quantum yields are still the major concerns related to fluorescent materials used for biosensing applications. Here we report one-pot facile synthesis of polyethylene glycol (PEG)-modified cystamine-based multifunctional fluorescent dyes named as CPEG-168, CPEG-1000 and CPEG-2000, which can be used for biosensing and detection of both cations and anions in pure aqueous medium. Rapid response toward thiols could be achieved due to the presence of disulfide functionality and reversible redox property between thiol and disulfide. A cyclic structure is formed by (NH–H) intramolecular hydrogen bonding to produce electron dense region for strong fluorescence emission. The presence of –NH group made them sensitive toward Cu2+ ions, while excellent sensitivity toward pH and F− ions was provided by intramolecular hydrogen bonding between –NH groups. The increase in chain length of PEGs enhanced the fluorescence emission intensities due to the formation of more rigid structure, and hence, the desired quantum yields can be tuned by changing the chain length of PEG. The aforementioned properties along with high brightness in aqueous solution, at biological pH and temperature range, make these fluorescent dyes potential candidates for biosensing applications and detection of cations and anions.

2-(2′-Hydroxyphenyl)-benzothiazole (HBT)-terpyridine conjugate: A highly specific ICT based fluorescent probe for Zn2+ ions and its application in confocal cell imaging

Abstract A new fluorescent sensor based on benzothiazole-terpyridine conjugate is reported. It functions as a fluorescent sensor that is highly selective for the Zn2+ ions via internal charge transfer (ICT) mechanism in DMSO: H2O medium. The 42 nm blue-shifted emission upon addition of Zn2+ to the sensor is attributed to the capture of Zn2+ by the terpyridine moiety that inhibits ICT from the terpyridine to the benzthiazole moiety. The sensing is supported by the Job’s plot, ESI–MS, 1H NMR titration experiments. Importantly, this probe shows high selectivity over other biologically relevant metal ions. Also, anions do not show any effect. In addition, the detection limit is found to be up to 35 nM which is acceptable within the EPA (US) limit. The probe is also able to function within a broad biological pH range of 6–8.

Rhodamine – Cyclohexane diamine based “turn-on” fluorescence chemosensor for Cr3+: Photophysical & confocal cell imaging studies

A new rhodamine based fluorescent turn on sensor L has been reported as a highly selective and sensitive chemosensor for the Cr3+ ion in a CH3CN-H2O solution. In presence of Cr3+ ion, the strong fluorescence enhancement is attributed due to the interaction of Cr3+ with L, leading to opening of the spirolactam ring of rhodamine B to the amide form. The Cr3+ sensing mechanism is also supported by the Job’s plot, ESI-MS, 1H NMR titration experiments. Importantly, this probe shows high selectivity over other biologically relevant metal ions. Also, anions do not show any effect. In addition, the detection limit is found to be up to 7.5 nM which is acceptable within the EPA (US) limit. The probe is also able to function within a broad biological pH range of 4–8. Furthermore, the probe L shows cellular penetrability for its potential use in confocal cell imaging studies.

“Turn off-on” phosphorescent sensor for biothiols based on a Ru-Cu ensemble

Glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) are three major biothiols, which play key roles in various biological systems. Accordingly, the development of phosphorescent probe of biothiols has attracted great attention in clinical applications. Here we report a ruthenium polypyridyl complex (1), which shows large phosphorescence quenching with 2.0 equivalents of Cu2+ ions. The non-phosphorescent 1−Cu ensemble sensor is formed and further applied to detect biothiols in living cancer cells. Among the various amino acids, GSH, Cys and Hcy induce distinct turn-on phosphorescence with high selectivity and insensitivity to biological relevant pH range and other proteins. As we know, this is the first report that Ru−Cu ensemble is used for probing endogenous thiols in the living cells.

A biotinylated piperazine-rhodol derivative: a 'turn-on' probe for nitroreductase triggered hypoxia imaging.

We developed a nitroreductase responsive theranostic probe 1; it comprises biotinylated rhodol in conjunction with p-nitrobenzyl functionality. The probe 1 showed a remarkable fluorescence 'turn-on' signal in the presence of nitroreductase under physiological conditions. The probe is considerably stable within a wide biological pH range (6-8) and also is very sensitive toward a reducing micro-environment e.g. liver microsome. Further, it enables providing cellular and in vivo nematode images in a reducing microenvironment.

Water soluble cationic porphyrin showing pH-dependent optical responses to G-quadruplexes: Applications in pH-sensing and DNA logic gate

A water soluble cationic porphyrin derivative 5,10,15,20-tetra-{4-[3-(1-methyl-1-piperidinyl) propoxy]phenyl}porphyrin (TMPipPrOPP), which showed attractive pH-dependent optical responses to G-quadruplexes, was synthesized and characterized. Under molecular crowding conditions, a stable TMPipPrOPP/G-quadruplex complex was formed in acidic pH range. Alkalization of the system to neutral and basic pH caused destruction of the complex due to reduced electrostatic interactions. The formation and destruction of the TMPipPrOPP/G-quadruplex complex was reversibly adjusted by pH, accompanied by repeated on-off switching of the colorimetric and fluorescent responses of TMPipPrOPP to G-quadruplexes. In comparison, no optical responses were observed for single-stranded, duplex and i-motif DNAs in the tested pH range (5.0–8.5). Besides highlighting the importance of designing novel drugs targeting G-quadruplexes in specific genome regions, this work may also provide a useful tool for G-quadruplex-based biosensing and nanomachine construction. One example is the design of colorimetric and fluorescent sensors for ratiometric pH-sensing. The proposed sensing platform can be used not only for highly sensitive pH-sensing in a narrow biological pH range, but also for pH detection in a broad range. Another example is the construction of DNA-based dual-output all-optical molecular logic gates that can perform basic AND, NAND, OR, NOR, INHIBIT and IMPLICATION logic operations.

Extraction and stability of bovine serum albumin (BSA) using cholinium-based Good's buffers ionic liquids

Good's buffers ionic liquids (GB-ILs), composed of cholinium-based cations and Good's buffers anions, display self-buffering characteristics in the biological pH range, and their polarity and hydrophobicity can be easily tuned by a proper manipulation of their ions chemical structures. In this work, the extraction ability for bovine serum albumin (BSA) of aqueous biphasic systems (ABS) formed by polypropylene glycol 400 (PPG 400) and several GB-ILs was evaluated. ABS formed by PPG 400 and cholinium chloride ([Ch]Cl), GBs, and sucrose were also investigated for comparison purposes. It is shown that BSA preferentially migrates for the GB-IL-rich phase, with extraction efficiencies of 100%, achieved in a single-step. Dynamic light scattering, and circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopies were employed to evaluate the effect of the investigated cholinium-based GB-ILs on the BSA stability, and compared with results obtained for the respective GBs precursors, [Ch]Cl and sucrose, a well-known protein stabilizer. Molecular docking studies were also carried out to investigate on the binding sites of GB-IL ions to BSA. The experimental results confirm that BSA has a higher stability in GB-ILs than in any of the other compounds investigated.

Reflection-mode micro-spherical fiber-optic probes for in vitro real-time and single-cell level pH sensing

pH sensing at the single-cell level without negatively affecting living cells is very important but still a remaining issue in the biomedical studies. A 70μm reflection-mode fiber-optic micro-pH sensor was designed and fabricated by dip-coating thin layer of organically modified aerogel onto a tapered spherical probe head. A pH sensitive fluorescent dye 2′,7′-Bis (2-carbonylethyl)-5(6)-carboxyfluorescein (BCECF) was employed and covalently bonded within the aerogel networks. By tuning the alkoxide mixing ratio and adjusting hexamethyldisilazane (HMDS) priming procedure, the sensor can be optimized to have high stability and pH sensing ability. The in vitro real-time sensing capability was then demonstrated in a simple spectroscopic way, and showed linear measurement responses with a pH resolution up to an average of 0.049 pH unit within a narrow, but biological meaningful pH range of 6.12–7.81. Its novel characterizations of high spatial resolution, reflection mode operation, fast response and high stability, great linear response within biological meaningful pH range and high pH resolutions, make this pH probe a very cost-effective tool for chemical/biological sensing, especially within the single cell level research field.

Abstract 5519: In vivo fluorescence lifetime imaging differentiates the bound versus unbound status of a cell surface receptor targeted ligand.

Abstract In vivo fluorescence lifetime imaging allows for the isolation of probe-specific fluorescence lifetime from surrounding tissue auto-fluorescence. Fluorescence lifetime is not affected by agent concentration or depth, or image acquisition parameters. However, fluorescence lifetime can be affected by the in vivo tissue microenvironment, e.g. pH or hypoxia. In this work, fluorescence lifetime is used to characterize the presence and fate of a high-affinity peptidyl delta opioid receptor (δOR) ligand conjugated to the near-infrared fluorescent IRDye800CW (Li-Cor). HCT116 colorectal cancer cells engineered to over-express the δOR and parental non-expressing HCT116 cells were bilaterally xenografted subcutaneously into the flanks of athymic nude mice. At a series of time-points following tail vein injection of the ligand, in vivo fluorescence images images were acquired using the Optix MX3 scanner (Advanced Research Technologies). For 24 h, the ligand fluoresence lifetime was significantly different (p<0.05) in the δOR positive tumors relative to the negative tumors. At 48 hours, the fluorescence lifetime in the negative tumor decreased and was no longer different. We hypothesize that the early difference in fluorescence lifetime is attributed to the bound state of the ligand on receptor positive tumors relative to the unbound state in negative tumors. Over time, unbound ligand clears from the negative tumor leaving only a low concentration of ligand retained by non-specific interactions. In vitro studies demonstrated that the fluoresecence lifetime of this ligand is not altered within the biological pH range. However, the tumor microenvironment is complex and multiple factors may be influencing the observed differences. Future plans include studies of 3D cell cultures using varied microenvironmental conditions and studies on δOR endogenous cell lines. Citation Format: Yoli JeuneSmith, Allison S. Cohen, Narges K. Tafreshi, Josef Vagner, Robert Gillies, David Morse. In vivo fluorescence lifetime imaging differentiates the bound versus unbound status of a cell surface receptor targeted ligand. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5519. doi:10.1158/1538-7445.AM2013-5519

Synthesis, characterization and anti-diabetic effect of bis[(1- R-imidazolinyl)phenolato]oxovanadium(IV) complexes

The five-coordinate oxovanadium(IV) complexes; [VO(pimin) 2] ( 1a), [VO(Etpimin) 2] ( 2) and [VO(EtOHpimin) 2] ( 3), were prepared by reacting the ligands; 2-(2′-hydroxyphenyl)-1 H-imidazoline (piminH), 2-(2′-hydroxyphenyl)-1-ethylimidazoline (EtpiminH) and 2-(2′-hydroxyphenyl)-1-ethanolimidazoline (EtOHpiminH), with VOSO 4. The complexes were characterized by elemental analysis, IR, UV–Vis and cyclic voltammetry. All complexes show V O stretching vibrations between 932 and 987 cm −1. The presence of three d–d transition occurring between 400 and 625 nm and the irreversible oxidation (V IV → V V) between 400 and 490 mV confirm the d 1 electronic configuration of the complexes. The solid state structures of [VO(pimin) 2] ( 1a) and its autoxidation hydrolysis product [VO 2(pimin)(piminH′)] ( 1b) were determined by single crystal X-ray diffraction. The geometry of [VO(pimin) 2] was found to be intermediate between trigonal bipyramidal and square pyramidal and sits on a crystallographic twofold axis, while the geometry of [VO 2(pimin)(piminH′)] was distorted trigonal bipyramidal. Potentiometric titrations were used to determine the protonation and stability constants for the ligands and oxovanadium(IV) complexes, respectively. The species existing over a biological pH range were also investigated. The in vitro studies indicated that the oxovanadium(IV) complexes were effective in enhancing glucose uptake in the 3T3-L1 adipocytes, C2C12 muscle cells and Chang liver cell lines. In these cell lines, the anti-hyperglycemic effect was equivalent to or surpassed the effect of metformin.