Skip to main content
NCBI home page
RSC Medicinal Chemistry logoLink to RSC Medicinal Chemistry
. 2020 Jun 5;11(7):814–822. doi: 10.1039/d0md00125b

In vitro anti-diabetic assessment of guavanoic acid functionalized gold nanoparticles in regulating glucose transport using L6 rat skeletal muscle cells

K Govindaraju a,✉, K S Uma Suganya a,b
PMCID: PMC7649838  PMID: 33479677

graphic file with name d0md00125b-ga.jpgGlucose uptake patterns of guavanoic acid and guavanoic acid functionalized gold nanoparticles in the presence of genistein (IRTK inhibitor) and wortmannin (PI3K inhibitor).

Abstract

Guavanoic acid functionalized gold nanoparticles exhibit anti-diabetic potential by improving insulin dependent glucose uptake in L6 rat skeletal muscle cells. The mode of action of the gold nanoparticles was established from the glucose uptake assay in the presence and absence of genistein and wortmannin. The anti-diabetic efficacy of guavanoic acid functionalized gold nanoparticles was put forth by in vitro assays like for PTP 1B, α-amylase and α-glucosidase enzyme activities. Studies on cytotoxicity revealed 50% inhibition of cells at 265 ± 0.01 μg mL–1. In the LDH enzyme release assay on differentiated L6 myoblasts treated with different concentrations (1–100 μg mL–1) of guavanoic acid functionalized gold nanoparticles, a viability of 75% at 100 μg mL–1 was observed.

Introduction

Type-2 diabetes mellitus is a chronic metabolic disorder which incites several difficulties in signaling. In accordance with FDA reports, approximately ten diabetic patients die on average every minute. The morbidity rate of this dangerous disease has been estimated to show an increase from 382 million in 2013 to 592 million by 2035. Almost 80% of the population in low and middle-income countries suffer from diabetes mellitus.1 The main pathophysiological state of type 2 diabetes is insulin resistance (decrease in cellular response to insulin), this includes destruction of the insulin signaling pathway, which leads to failure of insulin stimulated uptake of glucose in targeted tissues.2 Several factors involved in the activation/phosphorylation trigger the translocation of GLUT4 from the intracellular pool to the plasma membrane.3,4 Though a series of anti-diabetic drugs are available in the market, type 2 diabetes is still a huge burden in developed and developing countries.

Cellular delivery involving the transfer of drugs and various biomolecules to the cell membrane/cells has attracted increasing attention because of its importance in medicine and drug delivery. Nanocarriers have provided a new platform for delivery of a particular therapeutic agent to specific sites. Gold nanoparticles are one of the potent candidates for delivery of various payloads (proteins, DNA/RNA, bioactive molecules, etc.,) into their specific targets.5 Further exploitation of their unique physical and chemical properties will ease the transportation and delivery of the payloads. Recently, research towards functionalization of nanoparticles with novel bioactive molecules has shown greater potential to improve human health.6 Several pharmaceutical companies have received FDA approval for the use and development of nano-based drugs in the past few years.7 Medicinal plant Psidium guajava is a virtuous source containing health promoting secondary metabolites like flavonoids, tannins, glycosides, and terpenoids. Among which, triterpenoid 20β-acetoxy-2α,3β-dihydroxyurs-12-en-28-oic acid (guavanoic acid) possesses various pharmaceutical properties as reported elsewhere.8 The present study aims to investigate the anti-diabetic potential of guavanoic acid functionalized gold nanoparticles using an in vitro model.

Results and discussion

Theranostic nanomedicine is a promising therapeutic paradigm; it takes advantage of the high ability of nanoplatforms to deliver cargo and loads for various therapeutic applications.9 The resulting nanosystems, disease diagnosis and targeted drug delivery systems or therapeutic response monitoring are estimated to play a significant role in the emerging era of personalized medicine and many research attempts have been devoted towards that goal. Inorganic nanoparticles particularly, gold nanoparticles, possess versatile properties suitable for cellular targeted drug delivery, including wide bio-availability, functionality, bio-compatibility, controlled release of loaded drugs, etc.

Characterization of guavanoic acid functionalized gold nanoparticles

The high resolution transmission electron microscopy (HR-TEM) images revealed that the formed guavanoic acid functionalized gold nanoparticles were spherical in shape with an average size of 12 nm (Fig. 1).

Fig. 1. HR-TEM image of guavanoic acid functionalized gold nanoparticles.

Fig. 1

Open in a new tab

Studies on cell morphology

Studies on the different cellular morphologies of L6 myotubes were conducted (normal L6 myotube cells, early differentiation, 50% differentiated cells, differentiated cells) using a phase contrast microscope. The morphology assessments of the cells revealed structural changes in the shape of the cell as shown in Fig. 2a–d.

Fig. 2. Morphology images of L6 myotube cells (10×): a) L6 myotubes – normal cells; b) L6 myotubes – early differentiation; c) L6 myotubes – 50% differentiated cells; d) L6 myotubes – differentiated cells.

Fig. 2

Open in a new tab

Studies on cell viability

The MTT assay was performed to assess the cell viability of L6 myotubes treated with guavanoic acid functionalized gold nanoparticles. From the assay, it was determined that the L6 myotube cells treated with various concentrations of gold nanoparticles ranging from 5–320 μg mL–1 for 48 h resulted in an IC50 value of 265 ± 0.01 μg mL–1 (Fig. 3). The cytotoxicity of gold nanoparticles is well documented and depends upon their size and shape and the presence of functionalized surface ligands.10–12 The cytotoxic effect of gymnemic acid synthesized gold nanoparticles on 3T3-L1 adipocyte cells exposed to a higher concentration of 1000 μM showed 56.67% cell viability.13 The present study has revealed the less cytotoxic nature of guavanoic acid functionalized gold nanoparticles.

Fig. 3. Cell viability of L6 myotubes treated with guavanoic acid functionalized gold nanoparticles.

Fig. 3

Open in a new tab

Glucose uptake measurements in L6 myotubes

To study the effect of guavanoic acid and guavanoic acid functionalized gold nanoparticles on glucose transport, L6 myotubes were treated with different concentrations of guavanoic acid and guavanoic acid functionalized gold nanoparticles at two different time points (12 and 24 h), respectively. Different time and dose dependent increases in glucose uptake were observed upon treatment with guavanoic acid and guavanoic acid functionalized gold nanoparticles. At 24 h, maximum glucose uptake was achieved with a maximum activity at 10 μg mL–1 and 50 ng mL–1 for guavanoic acid and guavanoic acid functionalized gold nanoparticles, respectively (Fig. 4a and b). To confirm the enhancement of glucose transport, guavanoic acid and guavanoic acid functionalized gold nanoparticles were treated with an insulin receptor and P13K and were further checked using an inhibition assay. Pre-treatment with genistein (a specific inhibitor for the tyrosine kinase-insulin receptor) followed by incubation with guavanoic acid/guavanoic acid functionalized gold nanoparticles resulted in glucose uptake values of 22.5% and 74.5%, respectively (Fig. 5a). Pre-treatment with wortmannin (P13K inhibitor)14 and incubation with guavanoic acid/guavanoic acid functionalized gold nanoparticles resulted in glucose uptake values up to 49.56% and 73.69%, respectively (Fig. 5b). Skeletal muscle tissue is the key insulin target in maintaining glucose homeostasis (stimulation of glucose uptake mediated by GLUT4 translocation).15 L6 myotubes are used as a model system for studying the glucose uptake and insulin resistance16 and hence used for the glucose uptake assay in the present study. Transporter of GLUT4 to the surface of the cell occurs through the insulin regulated pathway and involving the insulin receptor (IR) protein tyrosine kinase activity followed by the tyrosine phosphorylation of the IR substrate proteins. Further, the activation of a complex network of downstream molecules14,17 is also possible for the insulin-independent glucose uptake and the translocation of GLUT4 to the plasma membrane is mediated by the activation of 5′ adenosine monophosphate-activated protein kinase (AMPK).17 Research reports have shown that the AMPK and its signaling pathway are possible molecular targets in the development of new drugs for the treatment of diabetes (type 2) and obesity.18–21 In the present study, we have demonstrated the anti-diabetic effect of guavanoic acid/guavanoic acid functionalized gold nanoparticles in vitro (Fig. 4). Further, the glucose uptake activation was strongly inhibited by genistein upon treatment with guavanoic acid/guavanoic acid functionalized gold nanoparticles, suggesting that the glucose transport takes place by involvement of a dependent insulin receptor. Several reports highlighted that P13K plays a major role in insulin signaling pathway by regulating the insulin mediated glucose transport.14 Pretreatment of wortmannin (P13K inhibitor) resulted in a decrease in the glucose uptake activity of the guavanoic acid and guavanoic acid functionalized gold nanoparticles, suggesting the involvement of P13K in enhancing glucose transport.

Fig. 4. Comparative analysis of a) guavanoic acid and b) guavanoic acid functionalized gold nanoparticles on 2-deoxy-d-3[H]-glucose uptake activities. Dose response analysis at 24 h. The results were expressed as percentage glucose uptake with respect to the vehicle control. The positive control rosiglitazone (50 μM) showed 129.5% uptake. The values are mean ± SE (n = 3 duplicates). (*), p < 0.05 compared with the control.

Fig. 4

Open in a new tab

Fig. 5. Glucose uptake patterns of guavanoic acid and guavanoic acid functionalized gold nanoparticles in the presence of a) genistein (IRTK inhibitor) and b) wortmannin (PI3K inhibitor). L6 myotubes were treated with genistein (50 μM) and wortmannin (100 nM) 30 min prior to the incubation with guavanoic acid (10 μg mL–1), guavanoic acid functionalized gold nanoparticles (50 ng mL–1) and insulin (100 nM) followed by the 2-deoxy-d-[1-3H]-glucose uptake assay. The results were expressed as percentage glucose uptake with respect to the vehicle control. The values are mean ± SE (n = 3 duplicates). (*), p < 0.05 compared with the control.

Fig. 5

Open in a new tab

PTP 1B inhibitory activity and cytotoxicity by LDH release

An in vitro anti-diabetic study by a dose and time dependent PTP 1B inhibitory assay was conducted using guavanoic acid functionalized gold nanoparticles. As shown in Fig. 6a guavanoic acid functionalized gold nanoparticles exhibit a significant inhibitory effect with an IC50 of 650 ± 0.02 ng mL–1 similar to the positive control RK-682 (IC50 – 5 μM) and it is 100% inhibited at the dose of 25 μg mL–1. Time dependent PTP 1B inhibitory action was monitored by treating with IC50 (650 ng mL–1) and 1 μg mL–1 concentrations and 100% PTP 1B inhibition was achieved at 60 and 40 min, respectively (Fig. 6b). Insulin mediated glucose transport is negatively regulated by protein tyrosine phosphatases (PTPs); among the PTP family, PTP 1B is a key modulator of insulin signal transduction by acting at downstream signaling molecules (IRS1 and P13K)22 and has high expression under insulin-resistant conditions which are related to obesity.23 Cytotoxicity assessment using the LDH enzyme revealed that the differentiated L6 myoblasts treated with different concentrations (1–100 μg mL–1) of guavanoic acid functionalized gold nanoparticles had a viability from 95% to 75% (Fig. 7) for the whole spectrum of the study.

Fig. 6. a) Dose–response and b) time-dependent bar diagrams for guavanoic acid functionalized gold nanoparticle inhibition of protein tyrosine phosphatase (PTP) 1B.

Fig. 6

Open in a new tab

Fig. 7. Cytotoxic effect on L6 myotubes treated with guavanoic acid functionalized gold nanoparticles by LDH release measurements.

Fig. 7

Open in a new tab

Studies on the inhibition of α-amylase and α-glucosidase enzymes

Fig. 8 shows the % inhibition (ng mL–1) of α-amylase and α-glucosidase treated with different concentrations of guavanoic acid functionalized gold nanoparticles. α-Amylase and α-glucosidase inhibitory activities have IC50 values of 829 ± 0.02 ng mL–1 and 975 ± 0.01 ng mL–1, respectively. The IC50 value of standard drug acarbose against α-amylase is 655 ± 0.01 mg mL–1 and for α-glucosidase is 705 ± 0.01 mg mL–1. Inhibition of α-amylase and α-glucosidase activities is reported as suitable for controlling postprandial hyperglycaemia in type 2 diabetes.24,25 The mechanism of the activity on carbohydrate binding regions of α-amylase and α-glucosidase enzymes, endoglucanases that catalyze the hydrolysis of internal α-1,4-glucosidic linkages in starch related polysaccharides, has also been targeted for the suppression of postprandial hyperglycemia. The enzymes are responsible for hydrolyzing dietary starch into maltose which is then broken down to glucose prior to absorption.26 In the present study, guavanoic acid functionalized gold nanoparticles show 100% inhibition in the concentration of 10 μg and are shown to be promising candidates to control release of glucose by inhibiting the breakdown of starch by metabolic enzymes and could be developed as potent anti-diabetic nanomaterials.

Fig. 8. Inhibition of alpha amylase & alpha glucosidase enzymes (%) by guavanoic acid functionalized gold nanoparticles and reference alpha amylase glucosidase inhibitor (acarbose) (values are expressed as mean ± SD, n = 3) (*), p < 0.05 compared with the control.

Fig. 8

Open in a new tab

Surface topography by AFM

AFM studies were carried out for the undifferentiated and differentiated L6 myotubes and differentiated L6 myotubes treated with guavanoic acid functionalized gold nanoparticles. The undifferentiated L6 myotube cells show a spherical-like structure and an intact membrane architecture with a smooth surface (Fig. 9i), whereas the differentiated L6 myotube cells show an elongated rod-like structure with a smooth surface (Fig. 9ii). The guavanoic acid functionalized gold nanoparticle treated differentiated L6 myotube cells show a rod-like morphology with a smooth surface without any changes in morphological features which indicates that guavanoic acid functionalized gold nanoparticles did not cause any membrane damage (Fig. 9iii). Noteworthily, guavanoic acid functionalized gold nanoparticles exhibit potent activity compared to guavanoic acid in all the experimental assays, indicating the significant contribution of the metallic gold. Being less toxic and having potent glucose uptake activity suggest that bio-functionalized gold nanoparticles could be developed further to treat diabetic mellitus.

Fig. 9. (i) Atomic force microscopy (AFM) image of undifferentiated L6 myotubes (A) and 3D topography of undifferentiated L6 myotubes (B); (ii) atomic force microscopy (AFM) image of differentiated L6 myotubes (A) and 3D topography of differentiated L6 myotubes (B) revealing spindle shaped cells; (iii) AFM image of guavanoic acid functionalized gold nanoparticle treated L6 myotubes (A) and 3D topography of treated L6 myotubes (B).

Fig. 9

Open in a new tab

Conclusions

In the present study, the potent in vitro anti-diabetic activity of guavanoic acid functionalized gold nanoparticles was studied using L6 rat skeletal muscle cell lines. The guavanoic acid functionalized gold nanoparticles were found to improve insulin-dependent glucose uptake activity. The modes of action were confirmed by the glucose uptake assay in the presence and absence of genistein and wortmannin. Further, the in vitro inhibitory effects of PTP 1B and carbohydrate metabolizing enzymes such as α-amylase and α-glucosidase enzymes were studied. A cytotoxicity assay was carried out and showed an IC50 of 265 ± 0.01 μg mL–1. AFM studies of the undifferentiated and differentiated L6 myotubes and differentiated L6 myotube cells treated with guavanoic acid functionalized gold nanoparticles showed an intact membrane architecture with a smooth surface. Thus, the reported guavanoic acid functionalized gold nanoparticles have enormous potential for the development of future therapeutics to treat diabetic complications.

Experimental section

Preparation of guavanoic acid functionalized gold nanoparticles

The synthesis of guavanoic acid mediated gold nanoparticles was carried out using a previously established procedure.27 Briefly, 1 mL of guavanoic acid (5 mg) was added into 49 mL of 1 mM aqueous HAuCl4 solution. The reaction solution turned from pale yellow to a ruby red color which indicates the formation of guavanoic acid functionalized gold nanoparticles. The size and morphology of guavanoic acid functionalized gold nanoparticles were examined using HR-TEM. The samples were prepared on carbon-coated copper grids and the films on the grids were allowed to dry prior to measurements (JEOL 3010 model microscope) operated at an accelerating voltage of 120 keV.

L6 rat skeletal muscle cell culture and development of insulin resistance

L6 rat skeletal muscle cells were obtained from National Centre for Cell Science (NCCS), Pune. The cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and supplemented with antibiotic and antimycotic solution [penicillin (120 units per mL), gentamycin (160 μg mL–1), streptomycin (75 μg mL–1) and amphotericin B (3 μg mL–1)] in a CO2 environment using a CO2 incubator.2 For differentiation, the L6 cells were transferred to a differential medium (DMEM with 2% FBS) for 4–5 days post-confluence. The differentiation was observed by the multiple nucleation of cells; the differentiated cells were incubated with a high glucose medium (medium with 25 mM L–1 glucose) for 24 h. After that, the cells were allowed to acquire an insulin-resistant state for further experimentation.28

Cell viability assay

A cytotoxicity assay was carried out with the conventional MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay. Briefly, L6 cells were seeded at a density of 1 × 105 cells per well in a 96 well microtiter plate supplemented with DMEM, 10% FBS and antibiotic solution and grown for 72 h. The medium was replaced with varying concentrations of guavanoic acid functionalized gold nanoparticles (5, 10, 20, 40, 80, 160 and 320 μg mL–1). The treated cells were thoroughly washed with 1X PBS and were incubated with 10 μL MTT (from a stock solution of 5 mg mL–1 MTT) for 4 h. After incubation, the so-formed purple formazan crystals were solubilized in 100 μL DMSO and the optical density was measured at 570 nm using an ELISA reader (Bio-Tek, Winooski, VT, USA).29

2-Deoxy-d-[1-3H] glucose uptake measurement

The differentiated L6 myoblast cells grown in 24 well plates were subjected to the glucose uptake assay as reported earlier.2 The cells were treated with guavanoic acid functionalized gold nanoparticles for 24 h and glucose uptake data were corrected for non-specific uptake in the presence of 10 μM cytochalasin B. The assays were carried out in triplicate. The results have been expressed as the percentage of glucose uptake with respect to the guavanoic acid control. Rosiglitazone was used as the positive control. For inhibition studies (P13K and IRTK), L6 myotube cells were treated with genistein (50 μM) and wortmannin (100 nm)30,31 30 min prior to the incubation with guavanoic acid and guavanoic acid functionalized gold nanoparticles followed by the glucose uptake assay.

PTP 1B inhibition assay

Protein tyrosine phosphatase 1B (PTP 1B) inhibitory activity was measured as per manufacturer’s protocol using a calorimetric assay kit (Calbiochem, Merck Cat. No. 539736).

Cytotoxicity assessment by the lactate dehydrogenase (LDH) assay

The LDH release assay was carried out by following protocols reported previously.32 The assay quantitatively measures the LDH, a stable cytosolic enzyme released during cell lysis. The assay was carried out using 96 well cell culture plates with 2 × 104 cells/200 μL per well. The cells upon treatment with concentrations ranging from 1–10 μg mL–1 guavanoic acid functionalized gold nanoparticles in L6 myotubes were measured at a time point of 24 h. 0.05% Triton X-100 was used to induce maximal lysis. The plate was read at 490 nm using an ELISA reader (Bio-Tek, Winooski, VT, USA). LDH release (%) was calculated using the following formula:

graphic file with name d0md00125b-t1.jpg 1

α-Amylase and α-glucosidase enzyme inhibition assay

Acarbose (standard drug) was dissolved in sodium acetate buffer to obtain a stock solution of 1 mg mL–1. Various concentrations ranging from 100 ng–10 μg guavanoic acid functionalized gold nanoparticles, 1 mL of 1% w/v soluble starch solution, 1 mL of α-amylase enzyme and 2 mL of 0.1 M sodium phosphate buffer (pH 7.4) were added. Then this solution was incubated for 1 h at 37 °C. After incubation, 0.1 mL of iodine–iodide indicator was added and measured at 565 nm using a UV-visible spectrophotometer (Shimadzu 1800). 0.1 M sodium acetate buffer was used as a blank. The reaction without extract was used as a control.33 Inhibition of the enzyme activity was calculated by using the following formula:

graphic file with name d0md00125b-t2.jpg 2

For the α-glucosidase enzyme inhibition assay, various concentrations ranging from 100 ng–10 μg guavanoic acid functionalized gold nanoparticles, 0.1 mL of α-glucosidase enzyme (1 U mL–1), and 1 mL of 0.2 M Tris buffer (pH 8) were added. Then the mixture was incubated for 60 min at 35 °C. The reaction was terminated by heating for 2 min in a boiling water bath. The amount of liberated glucose is measured by the glucose oxidation method at 540 nm using a UV-visible spectrophotometer (Shimadzu 1800). Acarbose was used as a standard drug. Distilled water was used as a blank. The percentage inhibition was calculated using the formula34

graphic file with name d0md00125b-t3.jpg 3

AFM studies

AFM studies were carried out using an NTEGRA Prima-NTMDT, Ireland microscope with the samples drop cast on clean glass slides. AFM studies were performed using a Nanonics supersensor AFM probe glass tip (15 nm diameter), in scanning mode.

Statistical analysis

All data were expressed as mean ± SEM. Statistical significance between means of the independent groups was analyzed using one-way ANOVA and p < 0.05 was considered to be statistically significant.

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgments

The authors thank the Board of Research in Nuclear Sciences (BRNS), Government of India for the financial support. We also thank the management of Sathyabama Institute of Science and Technology, Chennai for its strong support in the research activities.

References

  1. IDF DIABETES ATLAS (2013) Sixth Edition, http://www.diabetes.org/diabetes-basics/statistics/?referrer=https://www.google.co.in/.
  2. Sujatha S., Anand S., Sangeetha K. N., Shilpa K., Lakshmi J., Balakrishnan A., Lakshmi B. S. Int. J. Diabetes Mellitus. 2010;2:101–109. [Google Scholar]
  3. Myers M. G., White M. F. Diabetes. 1993;42:643–650. doi: 10.2337/diab.42.5.643. [DOI] [PubMed] [Google Scholar]
  4. Ducluzeau P. H., Fletcher L. M., Welsh G. I., Tavare J. M. J. Cell Sci. 2002;115:2857–2866. doi: 10.1242/jcs.115.14.2857. [DOI] [PubMed] [Google Scholar]
  5. Ghosh P., Han G., De M., Kim C. K., Rotello V. M. Adv. Drug Delivery Rev. 2008;60:1307–1315. doi: 10.1016/j.addr.2008.03.016. [DOI] [PubMed] [Google Scholar]
  6. Watkins R., Wu L., Zhang C., Davis R. M., Xu B. Int. J. Nanomed. 2015;10:6055–6074. doi: 10.2147/IJN.S92162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Patra C. R., Bhattacharya R., Mukhopadhyay D., Mukherjee P. Adv. Drug Delivery Rev. 2010;62:346–361. doi: 10.1016/j.addr.2009.11.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ngbolua K. N., Lufuluabo L. G., Moke L. E., Bongo G. N., Liyongo C. I., Ashande C. M., Sapo B. S., Gbolo B. G., Mpiana P. T. Phytomedicine. 2018;5:7–13. [Google Scholar]
  9. Xie J., Lee S., Chen X. Adv. Drug Delivery Rev. 2010;62:1064–1079. doi: 10.1016/j.addr.2010.07.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jennings T. and Strouse G., in Bio-Applications of Nanoparticles, ed. C. W. C. Warren, Landes Bioscience and Springer Science+Business Media, Tallahassee, 2007, pp. 34–47. [Google Scholar]
  11. Chithrani B. D., Ghazani A. A., Chan W. C. W. Nano Lett. 2006;6:662–668. doi: 10.1021/nl052396o. [DOI] [PubMed] [Google Scholar]
  12. Khan J. A., Pillai B., Das T. K., Singh Y., Maiti S. ChemBioChem. 2007;8:1237–1240. doi: 10.1002/cbic.200700165. [DOI] [PubMed] [Google Scholar]
  13. Rajarajeshwari T., Shivashri C., Rajasekar P. RSC Adv. 2014;4:63285–63295. [Google Scholar]
  14. Khan A. H., Pessin J. E. Diabetologia. 2002;45:1475–1483. doi: 10.1007/s00125-002-0974-7. [DOI] [PubMed] [Google Scholar]
  15. Klip A., Ishiki M. Endocrinology. 2005;146:5071–5078. doi: 10.1210/en.2005-0850. [DOI] [PubMed] [Google Scholar]
  16. Klip A., Guma A., Ramlal T., Bilan P. J., Lam L., Leiter L. A. Endocrinology. 1992;130:2535–2544. doi: 10.1210/endo.130.5.1572281. [DOI] [PubMed] [Google Scholar]
  17. Virkamaki A., Ueki K., Kahn C. R. J. Clin. Invest. 1999;103:931–943. doi: 10.1172/JCI6609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Goodyear L. J., Kahn B. B. Annu. Rev. Med. 1998;49:235–261. doi: 10.1146/annurev.med.49.1.235. [DOI] [PubMed] [Google Scholar]
  19. Kenner K. A., Anyanwu E., Olefsky J. M., Kusari J. J. Biol. Chem. 1996;271:19810–19816. doi: 10.1074/jbc.271.33.19810. [DOI] [PubMed] [Google Scholar]
  20. Gum R. J., Gaede L. L., Koterski S. L., Heindel M., Clampit J. E., Zinker B. A., Trevillyan J. M., Ulrich R. G., Jirousek M. R., Rondinone C. M. Diabetes. 2003;52:21–28. doi: 10.2337/diabetes.52.1.21. [DOI] [PubMed] [Google Scholar]
  21. Nichols A. J., Mashal R. D., Balkan B. Drug Dev. Res. 2006;67:559–566. [Google Scholar]
  22. Gum R. J., Gaede L. L., Koterski S. L., Heindel M., Clampit J. E., Zinker B. A., Trevillyan J. M., Ulrich R. G., Jirousek M. R., Rondinone C. M. Diabetes. 2003;52:21–28. doi: 10.2337/diabetes.52.1.21. [DOI] [PubMed] [Google Scholar]
  23. Calera M. R., Vallega G., Pilch P. F. J. Biol. Chem. 2000;275:6308–6312. doi: 10.1074/jbc.275.9.6308. [DOI] [PubMed] [Google Scholar]
  24. Inzucchi S. E. JAMA, J. Am. Med. Assoc. 2002;287:360–372. doi: 10.1001/jama.287.3.360. [DOI] [PubMed] [Google Scholar]
  25. Puls W., Keup U. Diabetologia. 1973;9:97–101. doi: 10.1007/BF01230687. [DOI] [PubMed] [Google Scholar]
  26. Vishnu Kiran M., Murugesan S. J. Chem. Pharm. Res. 2013;5:1001–1008. [Google Scholar]
  27. Khaleel Basha S., Govindaraju K., Manikandan R., Seog Ahn J., Bae E. Y., Singaravelu G. Colloids Surf., B. 2010;75:405–409. doi: 10.1016/j.colsurfb.2009.09.008. [DOI] [PubMed] [Google Scholar]
  28. Huang C., Somwar R., Patel N., Niu W., Torok D., Klip A. Diabetes. 2002;51:2090–2098. doi: 10.2337/diabetes.51.7.2090. [DOI] [PubMed] [Google Scholar]
  29. Uma Suganya K. S., Govindaraju K., Ganesh Kumar V., Prabhu D., Arulvasu C., Stalin Dhas T., Karthick V., Changmai N. Appl. Surf. Sci. 2016;371:415–424. [Google Scholar]
  30. Merlijn B., Peter J. A., Maassen J. A. Biochem. Biophys. Res. Commun. 2005;326:511–514. doi: 10.1016/j.bbrc.2004.11.055. [DOI] [PubMed] [Google Scholar]
  31. Cheng Z., Pang T., Gu M., Gao A. H., Xie C. M., Li J. Y., Nan F. J., Li J. Biochim. Biophys. Acta, Gen. Subj. 2006;1760:1682–1689. doi: 10.1016/j.bbagen.2006.09.007. [DOI] [PubMed] [Google Scholar]
  32. Sangeetha K. N., Shilpa K., Jyothi Kumari P., Lakshmi B. S. Phytomedicine. 2013;20:213–220. doi: 10.1016/j.phymed.2012.10.011. [DOI] [PubMed] [Google Scholar]
  33. Malik C. P. and Singh M. B., Plant enzymology and histoenzymology, Kalyani publishers, New Delhi, 1980, p. 278. [Google Scholar]
  34. Krishnaveni S., Balasubramanian T., Sadasivam S. Food Chem. 1984;15:229–232. [Google Scholar]

Articles from RSC Medicinal Chemistry are provided here courtesy of Royal Society of Chemistry

RESOURCES