Regulation of peroxiredoxin-3 gene expression under basal and hyperglycemic conditions: Key roles for transcription factors Sp1, CREB and NF-κB.

Peroxiredoxin-3 (Prx-3), a thioredoxin-dependent peroxidase located exclusively in the mitochondrial matrix, catalyses peroxides/peroxinitrites. Altered levels of Prx-3 is associated with diabetic cardiomyopathy (DCM). However, molecular mechanisms of Prx-3 gene regulation remain partially understood. We undertook a systemic analysis of the Prx-3 gene to identify the key motifs and transcriptional regulatory molecules. Transfection of promoter-reporter constructs in the cultured cells identified -191/+20 bp domain as the core promoter region. Stringent in silico analysis of this core promoter revealed putative binding sites for specificity protein 1 (Sp1), cAMP response element-binding protein (CREB) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Interestingly, while co-transfection of the -191/+20 bp construct with Sp1/CREB plasmid diminished Prx3 promoter-reporter activity, mRNA and protein levels, co-transfection with NF-κB expression plasmid augmented the same. Consistently, inhibition of Sp1/CREB/NF-κB expression reversed the promoter-reporter activity, mRNA and protein levels of Prx-3, thereby confirming their regulatory effects. ChIP assays provided evidence for interactions of Sp1/CREB/NF-κB with the Prx-3 promoter. H9c2 cells treated with high glucose as well as streptozotocin (STZ)-treated diabetic rats showed time-dependent reduction in promoter activity, endogenous transcript and protein levels of Prx-3. Augmentation of Sp1/CREB protein levels and their strong binding with Prx-3 promoter are responsible for diminished Prx-3 levels under hyperglycemia. The activation/increase in the NF-κB expression under hyperglycemia was not sufficient to restore the reduction of endogenous Prx-3 levels owing to its weak binding affinity. Taken together, this study elucidates the previously unknown roles of Sp1/CREB/NF-κB in regulating Prx-3 gene expression under hyperglycemic condition.

Protein extracts from Momordica charantia var. charantia and M. charantia var. muricata show anti-lipidemic and antioxidant properties in experimental type 2 diabetic rats.

Plant-derived compounds are used to manage dyslipidemia and oxidative stress in type 2 diabetic condition. In this study, anti-lipidemic and antioxidant properties of the protein extracts from "Charantia" (PEC) and "Muricata" (PEM) varieties of Momordica charantia were analyzed by quantifying lipids, hepatic, renal, and oxidative stress markers, and histopathological examination of liver and kidney tissues. Protein extracts were orally administered at 10 (PEC10, PEM10) or 20 mg/kg body weight (PEC20, PEM20). Levels of cholesterol, low-density lipoprotein, and triglycerides decreased but high-density lipoprotein increased significantly in treated rats as compared to untreated diabetic rats (p < .01), and attained normal physiological range in both doses. Levels of superoxide dismutase, catalase, glutathione peroxidase, and reduced glutathione increased but thiobarbituric acid reactive substances decreased significantly in treated rats as compared to untreated diabetic rats (p < .01), and attained normal physiological range in PEM20 only. Histopathological examinations supported a protective role for the protein extracts against oxidative stress. PRACTICAL APPLICATIONS: Momordica charantia, a well-known medicinal plant is traditionally used for treating diabetes in India as well as other countries. The whole plant was shown to have medicinal importance. Anti-diabetic potential of this plant was scientifically established largely using organic extracts and water extract mainly from the fruits of this plant. However, protein extracts from the seeds and fruit pulp of this plant were also proven to have anti-diabetic activity. The present study illustrates the anti-lipidemic and antioxidant effect of protein extracts from the fruit pulp of two varieties of M. charantia (Charantia and Muricata) in Streptozotocin-induced type 2 diabetic rats. This study provides experimental evidence in support of its use in the management of type 2 diabetes-related complications.

Inhibition of New Delhi Metallo-ß-Lactamase 1 (NDM-1) Producing Escherichia coli IR-6 by Selected Plant Extracts and Their Synergistic Actions with Antibiotics.

Improper use of antibiotics has led to a great concern in the development of pathogenic microbial resistance. New Delhi metallo-ß-lactamase 1 (NDM-1) producing bacteria are resistant to most of the ß-lactam antibiotics, and so far, no new compounds have been clinically tested against these bacteria. In this study, ethanol extracts from the leaves of 240 medicinal plant species were screened for antibacterial activity against an NDM-1 Escherichia coli strain. The extracts that showed antibacterial activity were then tested for minimum inhibitory concentrations (MICs) and zones of inhibition. The extract from Combretum albidum G. Don, Hibiscus acetosella Welw. ex Hiern, Hibiscus cannabinus L., Hibiscus furcatus Willd., Punica granatum L., and Tamarindus indica L. showed bactericidal activity between 5 and 15 mg/ml and the MIC was between 2.56 and 5.12 mg/ml. All six plant extracts inhibited activity of the NDM-1 enzyme in vitro, and the IC50 value ranged between 0.50 and 1.2 ng/µl. Disruption of bacterial cell wall integrity by the plant extracts was clearly visible with scanning electron microscopy. Increases in membrane permeability caused 79.4-89.7% bacterial cell deaths as investigated by fluorescence-activated cell sorting. All the plant extracts showed synergistic effects when combined with colistin [fractional inhibitory concentration (ΣFIC) = 0.125-0.375], meropenem (ΣFIC = 0.09-0.313), and tetracycline (ΣFIC = 0.125-0.313). Thus, the plant extracts can be fractionated for the identification of active compounds, which could be used as new antibacterial compounds for the development of drugs against NDM-1 E. coli in addition to their use in combination therapy.

Identification of species adulteration in traded medicinal plant raw drugs using DNA barcoding.

Plants are the major source of therapeutic ingredients in complementary and alternative medicine (CAM). However, species adulteration in traded medicinal plant raw drugs threatens the reliability and safety of CAM. Since morphological features of medicinal plants are often not intact in the raw drugs, DNA barcoding was employed for species identification. Adulteration in 112 traded raw drugs was tested after creating a reference DNA barcode library consisting of 1452 rbcL and matK barcodes from 521 medicinal plant species. Species resolution of this library was 74.4%, 90.2%, and 93.0% for rbcL, matK, and rbcL + matK, respectively. DNA barcoding revealed adulteration in about 20% of the raw drugs, and at least 6% of them were derived from plants with completely different medicinal or toxic properties. Raw drugs in the form of dried roots, powders, and whole plants were found to be more prone to adulteration than rhizomes, fruits, and seeds. Morphological resemblance, co-occurrence, mislabeling, confusing vernacular names, and unauthorized or fraudulent substitutions might have contributed to species adulteration in the raw drugs. Therefore, this library can be routinely used to authenticate traded raw drugs for the benefit of all stakeholders: traders, consumers, and regulatory agencies.

Multi-locus DNA barcoding identifies matK as a suitable marker for species identification in Hibiscus L.

The genus Hibiscus L. includes several taxa of medicinal value and species used for the extraction of natural dyes. These applications require the use of authentic plant materials. DNA barcoding is a molecular method for species identification, which helps in reliable authentication by using one or more DNA barcode marker. In this study, we have collected 44 accessions, representing 16 species of Hibiscus, distributed in the southern peninsular India, to evaluate the discriminatory power of the two core barcodes rbcLa and matK together with the suggested additional regions trnH-psbA and ITS2. No intraspecies divergence was observed among the accessions studied. Interspecies divergence was 0%-9.6% with individual markers, which increased to 0%-12.5% and 0.8%-20.3% when using two- and three-marker combinations, respectively. Differentiation of all the species of Hibiscus was possible with the matK DNA barcode marker. Also, in two-marker combinations, only those combinations with matK differentiated all the species. Though all the three-marker combinations showed 100% species differentiation, species resolution was consistently better when the matK marker formed part of the combination. These results clearly showed that matK is more suitable when compared to rbcLa, trnH-psbA, and ITS2 for species identification in Hibiscus.

In vitro and in vivo α-amylase and α-glucosidase inhibiting activities of the protein extracts from two varieties of bitter gourd (Momordica charantia L.).

BACKGROUND: α-amylase and α-glucosidase digest the carbohydrates and increase the postprandial glucose level in diabetic patients. Inhibiting the activity of these two enzymes can control postprandial hyperglycemia, and reduce the risk of developing diabetes. Bitter gourd or balsam pear is one of the important medicinal plants used for controlling postprandial hyperglycemia in diabetes patients. However, there is limited information available on the presence of α-amylase and α-glucosidase inhibiting compounds. In the current study, the protein extracts from the fruits of M. charantia var. charantia (MCC) and M. charantia var. muricata (MCM) were tested for α-amylase and α-glucosidase inhibiting activities in vitro, and glucose lowering activity after oral administration in vivo. RESULTS: The protein extract from both MCC and MCM inhibited the activity of α-amylase and α-glucosidase through competitive inhibition, which was on par with Acarbose as indicated by in vitro percentage of inhibition (66 to 69 %) and IC50 (0.26 to 0.29 mg/ml). Both the protein extracts significantly reduced peak blood glucose and area under the curve in Streptozotocin-induced diabetic rats, which were orally challenged with starch and sucrose. CONCLUSIONS: Protein extracts from the fruits of the two varieties of bitter gourd inhibited α-amylase and α-glucosidase in vitro and lowered the blood glucose level in vivo on par with Acarbose when orally administrated to Streptozotocin-induced diabetic rats. Further studies on mechanism of action and methods of safe and biologically active delivery will help to develop an anti-diabetic oral protein drug from these plants.