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ORIGINAL ARTICLE
Year : 2019  |  Volume : 10  |  Issue : 1  |  Page : 25-31  

Hibiscus sabdariffa linn fruits methanolic extract and fractions mediated glucose uptake stimulation and glucose transporter 4 regulation


1 Department of Biotechnology and Medical Engineering, Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, Johor Bahru, Johor, Malaysia
2 Medical Technology Division, Malaysian Nuclear Agency, Selangor, Malaysia
3 Department of Biosciences and Health Sciences, Faculty of Bioscience and Bioengineering, Universiti Teknologi Malaysia, Skudai, Malaysia

Date of Web Publication22-Aug-2019

Correspondence Address:
Siti Pauliena Mohd Bohari
Department of Bioscience, Faculty of Science, Universiti Teknologi Malaysia, Johor Bahru 81310, Johor
Malaysia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jpnr.JPNR_14_18

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   Abstract 


Objective: As such, this study reports the effects of Hibiscus sabdariffa Linn fruit methanolic extract and fractions (ethyl acetate, n-hexane, and butanol) on glucose uptake stimulation, as well as regulation of glucose transporter-4 (GLUT4) in L6 myotubes and 3T3F442A adipocytes cells. Materials and Methods: The stimulating effects of methanolic extract and fractions of H. sabdariffa Linn fruit on glucose uptake in 3T3F442A adipocytes through in vitro assay assessment are reported. The GLUT4 translocation of L6 myotubes membrane was also investigated. Results: The outcomes of this study revealed that methanolic extract and fractions were unable to induce glucose uptake in 3T3F442A adipocytes. In addition, the methanolic extract and fractions were incapable of enhancing both the distribution and the regulation of GLUT4 in L6 myotubes, in relation to the control. Conclusion: The systematic empirical works performed in this study conclude that the H. sabdariffa Linn derivatives are unsuitable potentials for the progression of new anti-diabetic drugs.

Keywords: 3T3F442A adipocytes, diabetes, glucose uptake, Hibiscus sabdariffa Linn


How to cite this article:
Shadhan RM, Bohari SP, Adam Z, Jamaluddin H. Hibiscus sabdariffa linn fruits methanolic extract and fractions mediated glucose uptake stimulation and glucose transporter 4 regulation. J Pharm Negative Results 2019;10:25-31

How to cite this URL:
Shadhan RM, Bohari SP, Adam Z, Jamaluddin H. Hibiscus sabdariffa linn fruits methanolic extract and fractions mediated glucose uptake stimulation and glucose transporter 4 regulation. J Pharm Negative Results [serial online] 2019 [cited 2019 Nov 20];10:25-31. Available from: http://www.pnrjournal.com/text.asp?2019/10/1/25/265143




   Introduction Top


The high prevalence of the type 2 diabetes mellitus is at an alarming rate as this particular type of diabetes is accounted for >90% of those diagnosed with diabetes across the globe, along with the escalating morbidity rate that results in a huge economic burden.[1] This Type 2 diabetes has been characterized by impotent insulin sensitivity and insulin secretion defects.[2] Low insulin sensitivity in peripheral cells can lead to chronic hyperglycemia and acute glycemic fluctuations,[3] which often cause microvascular and macrovascular complications.[4] Nevertheless, the type 2 diabetes treatment can be achieved by normalizing glycemia through nonpharmacological therapies, such as diet change, regular exercise, and weight loss. This can also be attained through pharmacological therapies using oral anti-diabetic drugs, for example, biguanides, thiazolidinediones (TZDs), and sulfonylureas (SUs).[5] In general, oral anti-diabetic drugs are introduced when nonpharmacological therapies fail to control glycemia.[3] Despite the abundant oral anti-diabetic drugs, the inefficacy of these drugs in treating Type 2 diabetes mellitus has made this type of diabetes dominant across nations.[6] The low demand for these drugs could be due to their undesirable side effects. For instance, treatment of SUs has been linked with weight gain and hypoglycemia. Metformin, TZDs, and acarbose treatment cause gastrointestinal problems. Meanwhile, TZDs therapy is often associated with peripheral edema.[3] Such negative drawbacks involving these oral anti-diabetic drugs have prompted a further search to develop alternative therapeutic agents in treating type 2 diabetes mellitus, which are not only effective but also safe with minimal side effects.

The mechanism of glucose uptake into peripheral cells (liver, muscle, and adipocytes) has been reckoned to be responsible for minimizing hyperglycemia. This uptake, which is mediated by insulin, refers to a type of anabolic hormone for regulating metabolisms of lipids, proteins, and carbohydrates.[7] Such insulin-mediated glucose uptake into peripheral cells can be enhanced with insulin sensitizers, such as biguanides and TZDs.[8] In addition, medicinal plants, for example, Lagerstroemia speciosa,[9] Amomi semen,[10] and Cortex Phellodendri,[11] have been proven to possess the capability of enhancing the uptake of insulin-mediated glucose into adipocytes cells. Furthermore, Aegle marmelos and Syzygium cumini plants have revealed to augment glucose uptake into L6 myotubes.[12] These medicinal plants, therefore, have the potential of boosting glucose uptake, either by mimicking or by sensitizing insulin action.[13]

As such, this study had been carried out to examine the effects of methanolic extract and fractions of Hibiscus sabdariffa Linn fruit on: (1) the glucose uptake in 3T3F442A adipocytes, as well as (2) the regulation of glucose transporter-4 (GLUT4) in L6 myotubes.


   Materials and Methods Top


Reagents

All chemicals, including trypsin, 2 Deoxy-D-[3H] glucose, penicillin/streptomycin, Dulbecco's Modified Eagle Medium (DMEM), and fetal bovine serum (FBS) had been retrieved from Invitrogen Corporation, USA, whereas 3-(4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide) (MTT) had been obtained from Sigma Chemical Co., USA.

Plant material and extraction procedure

The whole fruits of H. sabdariffa Linn were bought from a local herbal shop (Johor Bahru, Malaysia). Initially, the purchased fresh fruits were washed under running tap water before they were air-dried for a week. Next, the dried fruits were finely powdered by using a grinder. In adhering to the procedures specified by Chumsri et al.,[14] the powdered sample was soaked in methanol and water solution at a ratio of 4:1 for 96 h to attain the first batch of crude methanolic extract. After that, the extract was collected and placed in a rotary evaporator (EYELA N-1000, EYELA, Tokyo, Japan) before drying the gummy extract with a freeze dryer (Beta 2–4 LD plus LT, Martin Christ, Germany). With the same protocol adhered, four batches of crude extracts had been retrieved and pooled.[5] These extracts were further subjected to successive solvent-solvent partitioning from the least polar solvent (n-hexane) to the most polar solvents (ethyl acetate and butanol). Besides, the H. sabdariffa Linn plant has been officially documented by the Forest Research Institute Malaysia with a database card number bearing 280615-14.

Cell culture

The cells of 3T3F442A fibroblasts and L6 myoblast had been obtained from the European Collection of Cell Cultures (ECACC, England). These cells had been cultured in DMEM which was supplemented with 10% of FBS and 1% of penicillin/streptomycin, in a humidified incubator at 37°C that contained CO2 (5%) and air (95%).

3-(4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide) assay

The amount of viable cells found within the sample was determined by using MTT assay. The 3T3F442A fibroblasts and L6 myoblast cells were trypsinized and seeded into a 96-well plate with the respective seeding densities of 2 × 105 and 2.5 × 105 cells/well.[15] Following a 24-h incubation period, all cells had been treated with varied H. sabdariffa Linn fruit methanolic extract and fractions (7.81‒1000 μg/mL) concentrations, and then, were incubated for another 72 h. After that, phosphate buffer saline (5 mg/mL) was used to dissolve MTT, which was later filtered with a 0.22 μm Sartorius syringe filter (Sartorius Stedim Malaysia Sdn Bhd, KL, Malaysia) for sterilization purpose. Next, MTT solution (20 μL) had been included in every well, where the plate was later incubated again for 4 h at 37°C. On the 4-h incubation process, medium from every well had been carefully drained. The next step was to add 1 NHCl-isopropanol buffer (225 μL) into all the emptied wells to solubilize the purple crystals.[15] Finally, the rate of absorbance had been read at 560 nm by using a PromegaGloMax microplate reader (Promega, Wisconsin, USA).[16]

Examination of glucose uptake activity in 3T3F442A adipocytes displayed by Hibiscus sabdariffa Linn extracts and fractions

The 3T3F442A fibroblasts had been kept at 37°C and humidified with CO2 (5%) in DMEM medium, which was further supplemented with 1% (v/v) of antibiotic solution (50,000 IU/L penicillin-streptomycin), and 10% (v/v) of FBS. The cells that resembled the fibroblast had been distinguished to identify adipocytes spontaneously once confluency was attained. Next, 5 μg/mL of insulin was supplemented to the medium of complete culture. In fact, the differentiation magnitude had been verified by the viscous media found in every well; indicating the secretion of free fatty acids generated by the cells. After that, these confluent cells had been seeded in a plate with 12 wells with 2 × 105 cells/well concentration, which was kept at 37°C for overnight and humidified with CO2(5%) for attachment to occur before further analyses. After being left for an overnight, serum-free DMEM was used to wash the cells three times, and the cells were re-incubated for 2 h with the serum-free DMEM. Later, the cells were re-washed for three times after the period of starvation using Krebs-Ringer Bicarbonate Buffer (KRB). Next, varied concentrations of H. sabdariffa Linn fruit methanolic extract and fractions had been applied to pre-incubate with the adipocytes cells at 37°C for 30 min. In this study, the positive control was represented by rosiglitazone maleate. In the attempt of initiating the reaction of glucose uptake, 2-deoxy-[3H]-glucose (500 μL) (1 μCi/mL) diluted in glucose (0.1 mM) had been included into every well, except blank, which was later incubated at 37°C for 1 h. Following the incubation, ice-cold KRB buffer had been used to wash the cells three times, which was later solubilized by adding 0.1% of Safety Data Sheet that had been dissolved in pH 7.4 phosphate buffer. After transferring each well content into scintillation vials, 15 mL of scintillation cocktail (Ultima GoldTM LLT) had been included. The liquid scintillation counter (PerkinElmer, USA) was employed to measure the radioactivity of cells.

Detection of cell surface glucose transporter-4

The GLUT4 within the contents of L6 myoblasts had been measured by using ELISA (Uscn Life Science Inc., United States), which refers to a commercially available kit that is comprised sandwiched enzyme immunoassay meant to detect and measure the presence of GLUT4 in human cell lysates and tissue homogenates, to name a few, in a quantitative and in vitro manner. This kit also provides a microplate that has an antibody pre-coat specifically for GLUT4. Meanwhile, the concentration of GLUT4 within the samples was identified by extrapolating the standard curve obtained from the determined GLUT4 concentration. Next, 100 μL of each blank, standard, and sample dilution had been included into determined wells for incubation at 37°C for 1 h. Later, after each well content was drained without washing, re-incubation was performed after adding 100 μL of detection (reagent A) at 37°C for 1 h. Subsequently, the retrieved solution had been aspirated and swabbed thrice in a washing solution (350 μL). The solution was re-incubated at 37°C for another 30 min after adding detection buffer (reagent B) (100 μL). Following the aspiration and washing processes, each well was added with substrate solution (90 μL), and again, incubated at 37°C for 10 until 20 min. Finally, each well was added with stop solution (50 μL), and the absorbance rate was determined at a wavelength of 450 nm.

Statistical analyses

Each measurement had been performed thrice to calculate the average and standard deviation values. Hence, the SPSS 21 software package (Windows Evaluation Version, SPSS Inc., Waltham, USA) was used for statistical analysis of the empirical data. Besides, the Shapiro–Wilk test was carried out to determine data normality. In addition, independent t-test was performed to analyze normal data, whereas the Mann–Whitney test was undertaken for abnormal data. If the probability (P) is <0.05, then the data are considered as statistically significant.[17]


   Results Top


Cytotoxicity effects

The cytotoxicity of H. sabdariffa Linn fruit methanolic extract and fractions had been tested against 3T3F442A fibroblasts and L6 myoblast cells at eight varied concentrations, as depicted in [Table 1] and [Table 2], respectively. The butanol fraction revealed a higher cytotoxic effect on 3T3F442A and L6 myoblast cells with IC50 values of 489 μg/mL and 112 μg/mL, respectively. Nevertheless, the methanolic extract displayed lower toxicity effect on 3T3F442A and L6 myoblast cells with the corresponding IC50 values of 616 μg/mL and 125 μg/mL. The value of IC50 denotes 50% decrease in cells that are viable, as illustrated in the line graph plotted for extract logarithmic concentration against percentage viability. Hence, the nontoxic methanolic extract and fractions obtained from H. sabdariffa Linn fruit were used for further analyses.
Table 1: Percentages of cell viability of 3T3F442A fibroblasts cells treated with varied concentrations of Hibiscus sabdariffa Linn fruit methanolic extract and fractions

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Table 2: Percentages of cell viability of L6 myoblast cells treated with varied concentrations of Hibiscus sabdariffa Linn fruit methanolic extract and fractions

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Glucose uptake activity of Hibiscus sabdariffa Linn fruit methanolic extract and fractions in 3T3F442A adipocytes

[Figure 1] illustrates the impacts of H. sabdariffa Linn fruits methanolic extract and fractions, as well as rosiglitazone maleate, on glucose uptake activity of 3T3F442A adipocytes. It was discovered that the methanolic extract and fractions did not exert any significant impact on the activity of glucose uptake in 3T3F442A adipocytes. In fact, it was found that only insulin (100 nM) could enhance uptake of glucose by 1.51-fold (P<0.001) when compared to control. [Figure 1]a portrays the enhanced glucose uptake activity displayed by methanolic extract at a higher concentration (600 μg/mL) in 3T3F442A adipocytes cells under basal and insulin-mediated conditions had been 0.72- and 0.77-fold, respectively. In fact, when the concentration of insulin was lower than 100 nM, the uptake was 1.51-fold (P<0.001), when compared to that of control.
Figure 1: The impacts of Hibiscus sabdariffa Linn fruit (a) methanolic extract, (b) ethyl acetate, (c) n-hexane, (d) butanol, and (e) rosiglitazone maleate, on glucose uptake in 3T3F442A adipocytes under basal and insulin-mediated conditions. Values are mean ± SD from three replicates with **P<0.01 and ***P<0.001

Click here to view


Nevertheless, the n-hexane fraction revealed the highest magnification of glucose uptake under both insulin-mediated and basal states in 3T3F442A adipocytes, as portrayed in [Figure 1]c. Under the basal state, the enhancement was recorded at 50 μg/mL concentration (0.99-fold), while the glucose uptake for that of insulin-mediated improved by a factor of 0.81 at 300 μg/mL concentration, when compared to control. Both concentrations were <100 nM of insulin and induced uptake of glucose by 1.51-fold (P< 0.001), in comparison to control. On the other hand, the ethyl acetate fraction exhibited the highest capacity of enhancement for both basal and insulin-mediated conditions by 0.74- and 0.85-fold, respectively, at 50 μg/mL concentration, when compared to that of control.

Meanwhile, the butanol fraction demonstrated improved uptake of glucose under insulin-mediated and basal setting in 3T3F442A adipocytes, as displayed in [Figure 1]d. The enhancement effect, however, seemed lower, when compared to that of 100 nM insulin, with glucose uptake of 1.51-fold (P< 0.001), as compared to that of control. In short, all the studied samples exemplified insignificant effects, especially when compared to those of untreated control and 100 nM of insulin.

Finally, [Figure 1]e illustrates the impact of rosiglitazone maleate on glucose intake under insulin-mediated and basal settings in 3T3F442A adipocytes. The recorded levels of glucose uptake showed improvement with basal and insulin-mediated states, as given in the following: 1.98- (P< 0.001), 1.61- (P< 0.01), and 1.59-fold (P< 0.001); as well as 2.08 - (P< 0.001), 2.14- (P< 0.001), and 3.10-fold (P< 0.001), respectively, in comparison to those of untreated control and 100 nM insulin.

Effect of Hibiscus sabdariffa Linn fruit methanolic extract and fractions on regulation of glucose transporter 4 in L6 myotubes

As illustrated in [Figure 2], the impacts of H. sabdariffa Linn fruits methanolic extract and fractions (n-hexane, butanol, and ethyl acetate) on regulatory activity in L6 myotubes are displayed by GLUT4, which had been assessed by employing the ELISA kit through in vitro quantitative approach . The samples exhibited increment in the GLUT4 surface distribution at minimal dose-dependent setting, in comparison to that of the negative control. As a result, the magnitude of activity at 100 μg/mL concentration for methanolic extract and fractions (ethyl acetate, n-hexane, and butanol) are as follows: 3.3%, 3.33%, 2.92%, and 3.45%, respectively. On the contrary, 200 nM insulin (positive control) also seemed to increase the surface distribution of GLUT4 in a significant manner (P< 0.01) by 7.15%. However, all extract and fractions revealed some level of activity on the regulation of GLUT4 at concentrations above IC50, which is primarily attributable to cytotoxicity-mediated inhibition.
Figure 2: The effects of Hibiscus sabdariffa Linn fruits methanolic extract and fractions on glucose transporter.4 regulation in L6 myotubes. Values are mean ± SD from three replicates with **P<0.01

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   Discussion Top


If untreated or uncontrolled, diabetes mellitus can cause many metabolic disorders. The current therapy for type 2 diabetes primarily emphasizes on glucose homeostasis restoration, which is performed through enhancement of glucose uptake into insulin-responsive tissues.[18]

The current study reports the effect of H. sabdariffa Linn fruit methanolic extract and fractions on in vitro basal and insulin-mediated glucose uptake activity into 3T3F442A adipocytes and L6 myotubes muscle cells as the model of the systems. The 3T3F442A adipocyte cells were used widely as a model system for studying the glucose uptake activity into adipocytes,[19] on the other hand, the L6 myotube cells was found as the best-characterized cellular model of skeletal muscle to study GLUT4 translocation.[20]

This study illustrates the impacts of H. sabdariffa Linn fruit methanolic extract and fractions as well as rosiglitazone maleate, on glucose uptake activity of 3T3F442A adipocytes and regulating GLUT4 at the surface of L6 myotube cell. In fact, it was found that only insulin 100 nM could enhance uptake of glucose by 1.51-fold (P< 0.001) when compared to negative control. It was reported that insulin significantly increased basal glucose uptake in adipocytes cells,[10],[21] and muscle cells through glucose uptake study.[22] These reports agreed with the results of the present study that 100 nM insulin significantly enhanced the taken up of glucose by 1.51-fold (P< 0.001) in adipocytes cells. The same concentration of insulin was used to mediate the glucose uptake activity of H. sabdariffa Linn fruit methanolic extract and fractions into 3T3F442A adipocytes. This concentration was widely used to mediate glucose disposal into adipocytes cells.[19],[23]

In short, all methanolic extract and fractions from H. sabdariffa Linn fruit exemplified insignificant effects, and less potential of basal and insulin-mediated glucose uptake activity as compared to untreated control, 100 nM of insulin, and rosiglitazone maleate (positive control) could possibly be due to that the extracts contain mixture of bioactive and nonbioactive compounds and there is possibility that the nonbioactive would reduce the concentration of active compounds in the extract and decrease the ability of the extracts to enhance glucose uptake into 3T3F442A adipocytes.

Furthermore, this study illustrated the impacts of H. sabdariffa Linn fruit methanolic extract and fractions on regulatory activity in L6 myotubes are displayed by GLUT4, which had been assessed by employing the ELISA kit through in vitro quantitative approach . The samples exhibited insignificant increment in the GLUT4 surface distribution at minimal dose-dependent setting, in comparison to that of (negative and positive control).

In addition, we describe the consequences of this abnormality on glucose uptake in L6 myotube cell, a major site of insulin resistance in diabetes. However, we show that in cells which possess both GLUT1 and GLUT4 such as the L6 myotube cell, the increased GLUT1 presence at the cell surface is responsible for the small increase in GLUT4 and due to the block in GLUT4 translocation to the plasma membrane.[24] The increased presence of GLUT1 at the cell surface has also been reported to be accompanied by inhibition of insulin-stimulated glucose transport and GLUT4 translocation.[25]

Apart from H. sabdariffa Linn, other medicinal plants also have exhibited positive activities toward glucose uptake and GLUT4 regulation in L6 myotubes. Based on a prior study, the L. speciosa L. extract had been reported to stimulate the uptake of glucose in 3T3 adipocytes with the dose-dependent response and induction time, which appears to be similar in the case of insulin.[21] On top of that, it was also found that the methanolic extract derived from Cichorium intybus displayed a rather significant uptake of glucose with dose-dependent response in 3T3-L1.[26] Furthermore, it had been proven that quercetin and kaempferol, which are isolated from Euonymus alatus, can significantly enhance the uptake of glucose in 3T3 adipocytes.[27] In addition, extracts of Momordica charantia fruit showed significant regulatory effects of GLUT4, thus displaying the capability of enhancing glucose uptake, in comparison to control.[28],[29]


   Conclusion Top


This study was undertaken mainly to determine the regulatory impacts exerted by H. sabdariffa Linn fruit methanolic extract and fractions on the mechanism of glucose uptake in 3T3F442A adipocytes, as well as the GLUT4 regulation at the surface of L6 myotubes cells. The outcomes retrieved showed that the methanolic extract and fractions of H. sabdariffa Linn fruit were insensitive toward the stimulation of glucose uptake in 3T3F442A adipocytes when compared to control. On the other hand, these derivatives did not significantly increase the distribution of GLUT4 at the surface of L6 myotube cells. As a conclusion, the systematic empirical works performed in this study conclude that the H. sabdariffa Linn derivatives are unsuitable potentials for the progression of new anti-diabetic drugs.

Acknowledgment

The authors would like to express their sincere gratitude to the Ministry of Higher Education and Scientific Research of Iraq for their financial assistance, as well as the laboratory facilities of FBME, IBD, at Universiti Teknologi Malaysia.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2]



 

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