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ORIGINAL ARTICLE
Year : 2010  |  Volume : 1  |  Issue : 1  |  Page : 24-26 Table of Contents     

Negative regulation of glucose uptake by Costus pictus in L6 myotube cell line


1 Department of Pharmaceutical Biotechnology, J.S.S. College of Pharmacy, Ootacamund - 643 001, Tamil Nadu, India
2 The Himalaya Drug Company, Makali, Bangalore - 560 123, India
3 Department of Pharmaceutical Science, L. M. College of Science & Technology (Pharmacy Wing), Jodhpur - 342 003, Rajasthan, India

Date of Web Publication20-Sep-2010

Correspondence Address:
Anil Pareek
Department of Pharmaceutical Science, L. M. College of Science & Technology, Jodhpur-342 003, Rajasthan
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0976-9234.68872

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   Abstract 

The leaf of Costus pictus D. Don is considered as an antidiabetic in folklore medicine and is known to reduce the blood sugar, similar to insulin. The aim of this study is to investigate the effect of ethanolic extract of C. pictus leaf on glucose uptake by L6 myotube cell line (skeletal muscle cells) involved in glucose utilization. Ethanolic extract of C. pictus leaf was analyzed to study GLUT4 translocation and glucose uptake activity, but it has no direct peripheral action at 300 μg/ml dose comparable with insulin and metformin. The glucose uptake was not enhanced by the extract of C. pictus leaf.

Keywords: Costus pictus , diabetes, GLUT4 glucose uptake, insulin, L6 cell line


How to cite this article:
Pareek A, Suthar M, Godavarthi A, Goyal M, Bansal V. Negative regulation of glucose uptake by Costus pictus in L6 myotube cell line. J Pharm Negative Results 2010;1:24-6

How to cite this URL:
Pareek A, Suthar M, Godavarthi A, Goyal M, Bansal V. Negative regulation of glucose uptake by Costus pictus in L6 myotube cell line. J Pharm Negative Results [serial online] 2010 [cited 2019 Nov 22];1:24-6. Available from: http://www.pnrjournal.com/text.asp?2010/1/1/24/68872


   Introduction Top


Diabetes is a chronic disorder of the metabolism of carbohydrates, proteins and fat, and caused due to absolute or relative degree of insulin resistance. [1] It has became an epidemic with a worldwide incidence of 5%, the number likely to increase from 135 million in 1995 to 300 million in 2025. [2] There are more than 30 million people with diabetes mellitus in India and the incidence is increasing. [3] Insulin and various types of hypoglycemic agents such as biguanids and sulfonylureas, old and new, are available for the treatment of diabetes. However, none of these medications is ideal due to toxic side effects and, in some cases, diminution of response after prolonged use. [4] So, with increasing incidence of diabetes mellitus in rural population throughout the world and due to adverse effects of synthetic medicine, there is a clear need for development of indigenous, inexpensive botanical source of antidiabetic crude or purified drug. [5] Many plants reported useful for the treatment of diabetes mellitus in ayurveda system of medicine have been tested on experimental animals. [6] Costus pictus D. Don (Zingiberaceae) syn. Costus mexicanus (DC.) Greene, commonly known as spiral ginger, stepladder or insulin plant, originated in Mexico. In India, it is grown in gardens as an ornamental plant, especially in Kerala, at every household. The species is similar to Costus speciosus (Koenig) Sm., which is commonly known as "Channakkoova" in Kerala, and leaves of both these species are being used by many people in Kerala for diabetes mellitus. [7],[8] Powder of the leaves of the medicinal plant C. pictus, known to posses therapeutic effect when administered to streptozotocin-induced diabetic rats, is found to reduce blood glucose level by 21% after 15 days of administration. [9] The methanol extract of C. pictus leaf is used to lower blood glucose level in alloxan-induced diabetic rats. [10] The antihyperglycemic and insulin secretory activity of an aqueous extract of C. pictus leaf was investigated in streptozotocin-induced diabetic rats. [11] Antioxidant [12],[13] and diuretic activity [14] have been evaluated. The antidiabetic property of C. pictus might be due to one or many mechanisms, viz., promotion of insulin, decrease in absorption of intestinal glucose, inhibition of gluconeogenesis, promotion of peripheral utilization of glucose.

Although antidiabetic activity of C. pictus extract has been reported, the exact mechanism of this effect is yet to be elucidated. The L6 cell line is the best characterized cellular model origin to study glucose uptake and GLUT4 translocation. [15],[16] Therefore, we evaluated the effect of ethanolic extract of C. pictus leaf on glucose uptake through glucose transport in L6 cell lines.


   Materials and Methods Top


Plant material

The leaves of C. pictus were collected from Centre of Indian Medical Heritage (CIMH), AVP campus, Kanjikode, Palakkad. Kerala, identified and authenticated by comparing with voucher specimen by Dr. Suresh Baburaj, Director, Survey of Medicinal Plants and Collection Unit, Ootacamund. The voucher specimen was deposited at our department for future reference.

Chemicals and cells

Fetal bovine serum was purchased from Invitrogen (Carlsbad, CA, USA). Dulbecco's modified Eagle's medium (DMEM) and other culture products were purchased from GIBCO BRL (San Diego, CA, USA). Trypsin, versene, glucose in PBS solution (TPVG) solution, bovine serum albumin (BSA), insulin, metformin and glucose kit were obtained from Randox Laboratory Ltd. (Ardmore, United Kingdom); dibasic sodium hydrogen phosphate, sodium bicarbonate, magnesium chloride, calcium chloride, potassium chloride and sodium chloride were from Ranbaxy Laboratories Ltd. (Mohali, India) and SD Fine Chem. (Mumbai, India). All chemicals and solvents used were of analytical grade. The L6 cell line (RAT) was obtained from National Centre for Cell Sciences (Pune, India).

Preparation of plant extract

The leaves of the C. pictus were washed thoroughly with tap water, shade-dried, cut into small pieces and crushed to moderately coarse powder. Extraction was done using 95% ethanol in a soxhlet apparatus for 6 hours. The extract was concentrated using rotary evaporator at 40-45C under reduced pressure.

Cytotoxicity assay

The extract was separately dissolved in 1 ml of dimethyl sulfoxide (DMSO) and the volume was made up to 10 ml with maintenance medium to obtain a stock solution of 1 mg/ml concentration, sterilized by filtration and further dilutions were made from the stock. The cytotoxicity assays were carried out using 0.1 ml of cell suspension containing 10,000 cells seeded in each well of a 96-well microtitre plate (Tarsons India Pvt. Ltd., Kolkata, India). Fresh medium containing different concentrations of the test sample was added after 24 hours of seeding. Control wells were incubated without test sample and with maintenance medium (2% serum). The microtitre plates were incubated at 37C in a humidified incubator with 5% CO 2 for a period of 72 hours. The morphology of the cells was inspected under the microscope for detectable alterations, i.e., loss of monolayer, granulation and vacuolization in the cytoplasm. The cytopathogenic effect (CPE) was scored. The 50% cytotoxic concentration (CTC 50 ) was determined by the standard Microculture Tetrazolium MTT. [17],[18],[19]

Preparation of cell culture

Monolayers of L6 cells were maintained at subconfluent conditions in growth media (DMEM with 4.5 g/l glucose, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% fetal bovine serum). Cells were maintained in a humidified 37C incubator with ambient oxygen and 5% CO 2 . Cells were maintained in continuous passage by trypsinization of subconfluent cultures using TPVG solution.

Glucose uptake by l6 cell line

Glucose uptake activity was estimated by the methods described by Pareek et al.[15] Cells were cultured on 6-well plates and incubated for 48 hours at 37C in a CO 2 incubator. When semi-confluent monolayer was formed, the culture was renewed with serum free DMEM containing 0.2% BSA and incubated for 18 hours at 37C in the CO 2 incubator. After 18 hours, the medium was discarded and cells were washed with krebs ringer phosphate (KRP) buffer once. The cells are treated with insulin, standard drug and plant extract, and glucose (1 M) was added and incubated for half an hour. The supernatant was collected for glucose estimation and glucose uptake was terminated by washing the cells three times with 1 ml ice-cold KRP buffer. Cells were subsequently lysed by freezing and thawing three times. Cell lysate was collected for glucose estimation. Glucose uptake was calculated as the difference between the initial and final glucose content in the incubated medium by Glucose oxidase-Peroxidase (GOD-POD) method. Six groups containing five wells of plate (n = 5) each were taken as given in [Table 1].
Table 1 : Effect of C. pictus on glucose uptake in L6 cell line

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Statistical analysis

Data were presented as mean ± standard error and analyzed using one way analysis of variance (ANOVA) with Dunnett test.


   Results Top


The extract of C. pictus was evaluated for its cytotoxic activity by 3-(4, 5 dimethyl thiazole-2 yl) - 2, 5-diphenyl tetrazolium bromide (MTT) assay. C. pictus extract showed CTC 50 value of 342.5 μg/ml in L6 cell line. In vitro study on glucose utilization in L6 cell line was studied and results are given in [Table 1] and [Figure 1].
Figure 1 : Effect of C. pictus on glucose uptake in L6 cell line

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   Discussion Top


Glucose uptake was not significantly increased by C. pictus extract and the extract was found to be less effective than insulin. The glucose uptake was significantly more in all the groups tested except in the C. pictus group, when compared to the control group. Results were compared with those of insulin (injectable antidiabetic) and metformin (oral antidiabetic), which were used as a standard antidiabetic drugs. Insulin (1 IU/ml) and metformin (100 μg/ml) enhanced the glucose uptake by 148.79 ± 4.64% and 71.50 ± 6.25%, respectively, over control. The extract was also tested with insulin to confirm any synergistic effect, but results indicated that the extract did not have any synergistic effect with insulin. The extract and insulin enhanced the glucose uptake in L6 cells by 149.76 ± 5.15% over control, when used together. In India, C. pictus is grown in garden, especially in Kerala, where the fresh raw leaves are eaten by diabetic people. It is turning out be a munching dietary supplement for diabetes, as it is thought to lower raised blood sugar level considerably.

In conclusion, we found that the extract of C. pictus leaves does not significantly stimulate glucose uptake by L6 skeletal muscle cells. It appears that C. pictus has no direct peripheral action. Further studies with estimation of insulin and insulin receptor may give more insight into the mechanism of the antidiabetic activity of C. pictus D. Don.


   Acknowledgments Top


The authors wish to thank Dr. P. Vijayan, Head of Department of Pharmaceutical Biotechnology, J.S.S. college of pharmacy, Rocklands, Ootacamund, for his valuable support and guidance to complete this work.

 
   References Top

1.Jarald E, Joshi SB, Jain DC. Diabetes Vs herbal medicines. IJPT 2008;7:97-100.  Back to cited text no. 1      
2.Turben H. Genetics of type 2 diabetes. Curr Sci 2002;83:1477-82.  Back to cited text no. 2      
3.Shankar P, Sundarka MK. Management of type 2 diabetes: Evidence based approach. J Indian Acad Clin Med 2001;2:244-50.  Back to cited text no. 3      
4.Dixit VP, Joshi S. Antidiabetic effect of alfala and injection in chicks: A biochemical evaluation. Indian J Physiol Pharmacol 1985;29:47-50.  Back to cited text no. 4      
5.Venkatesh S, Reddy GD, Reddy BM, Ramesh M, Apparao AV. Antihyperglycemic activity of Carulluma asttenuate. Fitoterapaia 2003;74:272-7.  Back to cited text no. 5      
6.Grover JK, Yadav S, Vats V. Medicinal plants of India with antidiabetic potential. J Ethanopharmacol 2002;81:81-100.  Back to cited text no. 6      
7.Merina B. Insulin plant in gardens. Nat Prod Radiance 2004;3:349-50.  Back to cited text no. 7      
8.Merina B. Toxicity studies of the herb Costus pictus D.Don. Available from: http://www.pharmainfo.net [last cited on 2005].  Back to cited text no. 8      
9.Devi VD, Urooj A. Hypoglycemic potential of Morus Indico L. and Costus igneus. Nak.: A preliminary study. Indian J Exp Biol 2008;46:614-6.  Back to cited text no. 9      
10.Jothivel N, Ponnusamy SP, Appachi M, Singaravel S, Rasilingan D, Deivasigamani K, et al. Anti-diabetic activity of methanol leaf extract of Costus pictus D.Don in alloxan-induced diabetic rats. JHS 2007;53:655-63.   Back to cited text no. 10      
11.Gireesh G, Thomas SK, Joseph B, Paulose CS. Antihyperglycemic and insulin secretory activity of Costus pictus leaf extract in streptozotocin induced diabetic rats and in vitro pancreatic islet culture. J Ethnopharmacol 2009;123:470-4.  Back to cited text no. 11  [PUBMED]  [FULLTEXT]  
12.Dhanabal SP, Kumar A, Chandrasekar R, John S, Joseph S, James M, et al. Hypoglycemic and antioxidant activities of Costus mexicanus (Costaceae). Aryavaidyan. 2007;21:53-8.  Back to cited text no. 12      
13.Jayasri MA, Mathew L, Radha A. A report on the antioxidant activity of leaves and rhizomes of Costus pictus D. Don. IJIB 2009;5:20-6.  Back to cited text no. 13      
14.Camargo ME, Najera RC, Torres RS, Aldete ME. Evaluation of the diuretic effect of the Costus pictus D. Don in rats. Proc West Pharmacol Soc 2006;49:72-4.  Back to cited text no. 14      
15.Gupta RN, Pareek A, Rathore GS, Suthar M. Study of glucose uptake activity of Helicteres isora Linn. Fruits in L-6 cell lines. Int J Diabetes Dev Ctries 2009;29:170-3.  Back to cited text no. 15      
16.Patel MB, Mishra SH. Cell lines in diabetes. Phcog Rev 2008;2:188-205.  Back to cited text no. 16    Medknow Journal  
17.Denirot F, Lang R. Rapid colorimetric assay for cell growth and survival: Modification to tetrazolium dye, procedure giving improved sensitivity and reliability. J Immunol Methods 1986;89:271-7.  Back to cited text no. 17      
18.Freshney RI. Culture of animal cells: A manual of basic technique. In: Liss AR, editor. 4 th ed. New York. A John Wiley and Sons; 2003.   Back to cited text no. 18      
19.Edmondson JM, Linda SA, Martinez AO. A rapid and simple MTT-based spectrophotometric assay for determining drug sensitivity in monolayer culture. J Tissue Cult Methods 1988;11:15-7.  Back to cited text no. 19      


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1]


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