Study of glucose uptake activity of Helicteres isora Linn . fruits in L6 cell lines

The effect of hot water extract of fruits of Helicteres isora on glucose uptake was studied in rodent skeletal muscle cells (L-6 cells) involved in glucose utilization. H. isora is an antidiabetic medicinal plant being used in Indian traditional medicine. Hot water extracts were analysed for glucose uptake activity and found to be signifi cantly active at 200 μg/ml dose comparable with insulin and metformin. Elevation of glucose uptake by H. isora in association with glucose transport supported the upregulation of glucose uptake. It was concluded that hot water extract of H. isora activate glucose uptake in L-6 cell line of mouse skeletal muscles.

The effect of hot water extract of fruits of Helicteres isora on glucose uptake was studied in rodent skeletal muscle cells (L-6 cells) involved in glucose utilization.H. isora is an antidiabetic medicinal plant being used in Indian traditional medicine.Hot water extracts were analysed for glucose uptake activity and found to be signifi cantly active at 200 µg/ml dose comparable with insulin and metformin.Elevation of glucose uptake by H. isora in association with glucose transport supported the upregulation of glucose uptake.It was concluded that hot water extract of H. isora activate glucose uptake in L-6 cell line of mouse skeletal muscles.

Introduction
It is well known that the incidence of diabetes mellitus is high all over the world, especially in Asia.Diff erent types of oral hypoglycaemic agents such as biguanides and sulfonylureas available along with insulin for the treatment of diabetes mellitus, [1] have side eff ects associated with their usage. [2,3]There is a growing interest in herbal remedies because of their effi cacy, minimal side eff ects and relatively low costs.Herbal drugs or their extracts are prescribed widely, even when their biological active compounds are unknown.Even the World Health Organization (WHO) approves the use of plant drugs for diff erent diseases, including diabetes mellitus. [4,5][8] Insulin increases glucose uptake in skeletal muscle by increasing functional glucose transport molecules in the plasma membrane.Glucose transport in skeletal muscle can also be stimulated by contractile activity.The maximal eff ects of insulin and contractile activity on glucose transport are additive. [9] skeletal muscle, both insulin and contractile activity stimulate translocation of glucose transporter GLUT-4 protein from an intracellular membrane pool to the plasma membrane. [9]Resistance to this stimulatory eff ect of insulin is a major pathological feature of diabetes. [10]relatively little-studied response to adrenoceptor activation is facilitation of glucose uptake.Adrenoceptors are classiÞ ed into three main subtypes: α 1 -, α 2 -, and ß-adrenoceptors.ß-Adrenoceptor stimulation increases glucose uptake in rodent skeletal muscle [11][12][13][14] and brown adipose tissue.This eff ect is mediated primarily by the ß 3 -adrenoceptor in brown adipose tissue, [15,16] it is also mediated by ß 2 -adrenoceptors in skeletal muscle cells. [14] cells represent a good model for glucose uptake because they have been used extensively to elucidate the mechanisms of glucose uptake in muscle, have an intact insulin signaling pathway, and express the insulinsensitive GLUT4.
Helicteres isora L. is a large shrub of the family Sterculiaceae, which occurs, often gregariously, throughout India, from Jamuna eastwards to Bihar and Bengal and southwards in central, western and southern India and Andaman islands.It is one of the best known shrub mentioned in the articles of the Hindu Materia Medica and used as one of the Jamu medicines. [17]It is one of the Jamu raw materials used in traditional folk medicine in Indonesia; it is called "Buah Kayu Ules or Ulet-Ulet" on Java island and is used for the treatment of gastrospasm and as an anthelmintic for tapeworm in Indonesia, and as antispasmodic, antipyretic, antidiarrhoic and antidysenteric in Saudi Arabia [18] and as a tonic compound aft er childbirth in the Malay Islands. [19]mbers of this plant are used as anthelmintic and anticolic, while fruits are used as anti-colic, anticonvulsant, and in abdominalgia. [20]In addition, Hatt ori and coworkers reported an inhibitory activity of the water extract of fruits of H. isora against reverse transcriptase from avian myeloblastosis virus (AMVRT) [21] and antihuman immunodeficiency virus-type-1 (anti-HIV-1) activity. [22]Traditionally, the root juice is claimed to be useful in diabetes, emphysema, and a favourite cure for snakebite. [23,24]From the roots, betulic acid, daucosterol, sitosterols, isorin [25] can be isolated.Cucurbitacin B and isocucurbitacin B were isolated and reported to possess cytotoxic activity. [26]though the hypoglycemic eff ect of H. isora extract has been reported, the exact mechanism of this eff ect has yet to be elucidated.Therefore, we evaluated the eff ect of hot water extract of H. isora fruits on glucose uptake through glucose transports in skeletal muscle cells (L-6 cell line).

Plant material
The fruits of H. isora (L.) were collected from the local market of Ootacamund, in June 2006 and authenticated by Director, Survey of medicinal plants and collection unit, Ootacamund, Tamilnadu, India.

Chemicals
Fetal bovine serum was purchased from Invitrogen (Carlsbad, CA, USA).Dulbecco's modiÞ ed Eagle's medium (DMEM) and other culture products were purchased from GIBCO BRL (San Diego, CA, USA).TPVG solution, Bovine serum albumin (BSA), Insulin, Metformin, Glucose kit was obtained from Randox, Dibasic sodium hydrogen phosphate, sodium bicarbonate, magnesium chloride, calcium chloride, potassium chloride, sodium chloride were from Ranbaxy Laboratories Ltd., Mohali and SD Fine Chem., Mumbai, India.All chemicals and solvents used were of analytical grade.

Plant extract
The collected fruits were shade dried, coarsely powdered and extracted with hot water by maceration process.The extract was Þ ltered and concentrated in vacuum and kept in a vacuum desiccator for complete removal of solvent.Hot water extract of fruits of H. isora was obtained with an yield of 1.8%.

In vitro glucose uptake activity Preparation of cell culture
Monolayer of L-6 cells was maintained at sub conß uent conditions in growth media containing DMEM with 4.5 g/l glucose, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine serum.Cells were maintained in a humidiÞ ed 37°C incubator with ambient oxygen and 5% CO 2 .Cells were maintained in continuous passage by trypsinization of sub conß uent cultures using TPVG solution.
Glucose uptake assay Cells were cultured on 6 well plates and incubated for 48 h at 37°C in a CO 2 incubator.When semi conß uent monolayer was formed, the culture was renewed with serum free DMEM containing 0.2% BSA and incubated for 18 h at 37°C in the CO 2 incubator.Aft er 18 h, the media was discarded and cells were washed with KRP buff er once.The cells were treated with Insulin, standard drug and plant extract and added glucose (1M) and incubated for half an hour.The supernatant was collected for glucose estimation and glucose uptake was terminated by washing the cells thrice with 1 ml ice-cold KRP buff er.Cells were subsequently lysed by freezing and thawing thrice.Cell lysate was collected for glucose estimation.
Glucose uptake was calculated as the diff erence between the initial and Þ nal glucose content in the incubated medium by GOD-POD method as follows: Mix 10 µl of sample and 1 ml of reagent, incubate for 25 min at 15-25°C or 10 min at 37°C.Measure the absorbance of the standard (A standard) and the sample (A sample ) against the reagent blank within 60 min, the time interval from sample addition to read time must be exactly the same for standard/control and sample.Six groups containing Þ ve wells of plate (n = 5) each were taken as given in Table 1.

Results and Discussion
Glucose utilization in L-6 cell lines was studied in vitro.The results are given in Table 2 and Figure 1.
The given results show that hot water extract of fruit of H. isora enhance the glucose uptake by 28.99 % over control at 200 µg/ml dose.Results were compared with insulin (injectable anti diabetic) and metformin (oral anti diabetic), which were used as the standard anti diabetic drugs.Insulin (1IU/ml) and metformin (100 µg/ml) enhance the glucose uptake by 148.79 % and 71.50% over control.Extract was also tested with insulin to conÞ rm any synergistic eff ect, but results indicate that extract does not have any synergistic eff ect with insulin.Extract and insulin enhance the glucose uptake in L-6 cells by 149.28% over control when used in combination.
Skeletal muscle is the primary site responsible for postprandial glucose use.Furthermore, it is the most abundant tissue in the whole body, and thus, proper function of skeletal tissue is important to maintain normal blood glucose level. [27]Defects in insulin stimulated skeletal muscle glucose uptake are common pathological states in non-insulin-dependent diabetes mellitus (NIDDM). [28]GLUT4 is the major glucose transporter expressed in insulin responsive tissue such as skeletal muscle and adipose tissue, where they respond to an acute insulin challenge by translocating GLUT4 rapidly from an intracellular membrane storage site to the plasma membrane. [29]e results obtained in the present study clearly demonstrate that the H. isora hot water extract enhances glucose uptake under in vitro conditions.This may be due to its eff ect on the number of receptors located in the skeletal muscle cell line.Group 3 800 µl of KRP buffer, 100 µl of metformin (1mg/ml) and 100 µl of glucose solution (1M).

Figure 1 :[
Figure 1: Effect of hot water extract of fruits of Helicteres isora on glucose uptake in L-6 cell line