Journal of Pharmacy And Bioallied Sciences

ORIGINAL ARTICLE
Year
: 2019  |  Volume : 11  |  Issue : 3  |  Page : 240--247

Effect of Litsea lancifolia leaf extract on glucose transporter 4 translocation and glucose uptake in 3T3L1 cell line


Murad Alsawalha1, Abeer Mohammed Al-Subaie2, Reem Yousuf Al-Jindan3, Srinivasa Rao Bolla4, Janardhana Papayya Balakrishna5, Padma Kanchi Ravi6, Shiva Shankar Reddy Gollapalli5, Vishnu Priya Veeraraghavan7, Aruthra Arumugam Pillai5, Joel Palpath Joseph5, Surapaneni Krishna Mohan8,  
1 Department of Chemical and Process Engineering Technology, Jubail Industrial College (JIC), P.O.Box 10099, Jubail Industrial City 31961, Kingdom of Saudi Arabia
2 Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Imam Abdulrahman Bin Faisal University, P.O.Box 2435, Dammam 31441, Kingdom of Saudi Arabia
3 Department of Microbiology, College of Medicine, Imam Abdulrahman Bin Faisal University, P.O.Box 2114, Dammam 31451, Kingdom of Saudi Arabia
4 Department of Anatomy, College of Medicine, Imam Abdulrahman Bin Faisal University, P.O.Box 2114, Dammam 31451, Kingdom of Saudi Arabia
5 Department of Biotechnology, Stellixir Biotech Private Ltd, Peenya 2nd Stage Industrial Area, Bangalore-560058, Karnataka, India
6 Department of Biotechnology, Sri Padmavati Mahila Visvavidyalayam, Padmvathi Nagar, Tirupati, Andhra Pradesh, India
7 Department of Biochemistry, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences (SIMATS), Saveetha University, 162, P. H. Road, Velappanchavadi, Chennai – 600 077, Tamil Nadu, India
8 Department of Medical Biochemistry, College of Applied Medical Sciences in Jubail (CAMSJ), Imam Abdulrahman Bin Faisal University, P.O.Box 4030, Al Ansar Rd, Deffi, Jubail Industrial City, Al Jubail 35816, Kingdom of Saudi Arabia

Correspondence Address:
Dr. Surapaneni Krishna Mohan
Department of Medical Biochemistry, College of Applied Medical Sciences–Jubail (CAMSJ), Imam Abdulrahman Bin Faisal University, P. O. Box 4030, Al Ansar Road, Deffi, Jubail Industrial City, Al Jubail 35816
Kingdom of Saudi Arabia

Abstract

Background: Numerous synthetic drugs have been recommended as a remedy for diabetes, but their role in hypoglycemic effects are diverse. The side effects associated with these drugs due to their extended use led scientists to find unconventional medicines with no or little side effects. Aim: This study was aimed at assessment of in vitro antidiabetic activities of methanolic extract of Litsea lancifolia leaves by using 3T3L1 cell line. Materials and Methods: The cytotoxic effect of the leaf extract was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The glucose uptake–inducing capabilities and its correlation with glucose transporter 4 (GLUT4) translocation were measured by flow cytometry in 3T3L1 cells. In addition, the inhibitory effect of L. lancifolia leaf extract on α-amylase activity and α-glucosidase activity was determined by colorimetric methods. Results: Different concentrations of L. lancifolia leaf extract did not show any toxicity on 3T3L1 cells, after the treatment for 24h. On stimulation with leaf extract, 60.22% and 86.26% of 3T3L1 cells showed glucose uptake and GLUT4 expression, respectively. The colorimetric assays showed that the methanolic leaf extract of L. lancifolia has a significant inhibitory effect on the activity of α-amylase enzyme and α-glucosidase enzyme with inhibitory concentration (IC50) value of 248.65 µg/mL and 229.61 µg/mL, respectively. Conclusion: On the basis of the results of this study, it is evident that L. lancifolia leaf extract showed promising anti-diabetic effect when compared to the standard drugs metformin and acarbose and was nontoxic to 3T3L1 cells. Thus, it can be further investigated to recommend as a possible alternative treatment in antidiabetic applications.



How to cite this article:
Alsawalha M, Al-Subaie AM, Al-Jindan RY, Bolla SR, Balakrishna JP, Ravi PK, Gollapalli SS, Veeraraghavan VP, Pillai AA, Joseph JP, Mohan SK. Effect of Litsea lancifolia leaf extract on glucose transporter 4 translocation and glucose uptake in 3T3L1 cell line.J Pharm Bioall Sci 2019;11:240-247


How to cite this URL:
Alsawalha M, Al-Subaie AM, Al-Jindan RY, Bolla SR, Balakrishna JP, Ravi PK, Gollapalli SS, Veeraraghavan VP, Pillai AA, Joseph JP, Mohan SK. Effect of Litsea lancifolia leaf extract on glucose transporter 4 translocation and glucose uptake in 3T3L1 cell line. J Pharm Bioall Sci [serial online] 2019 [cited 2019 Dec 11 ];11:240-247
Available from: http://www.jpbsonline.org/text.asp?2019/11/3/240/262195


Full Text



 Introduction



The World Health Organization (WHO) comprehends diabetes mellitus as an emerging widespread epidemic disease. It is a general endocrine disorder impinging on greater than 150 million people worldwide and this number will probably multiply to 300 million by the year 2025.[1],[2] Diabetes is recognized by augmented plasma glucose concentrations, due to inadequate insulin or insulin resistance, or together, as a result of metabolic abnormalities in carbohydrates, lipids, and proteins.[3] Diabetes is characterized by hyperglycemia with modified lipid, carbohydrate, and protein metabolism, which influences the patient’s state of life in all terms of social, psychological well-being, including physical ill health.[4],[5] The WHO expressed that the people of Southeast Asia and Western Pacific fall under the category of higher risk, and the majority of these patients have type 2 diabetes. Insulin resistance is an indicator of the onset of type 2 diabetes and is frequently associated with several other cardiovascular risk factors such as dyslipidemia, hypertension, and prothrombotic factors.[6] Diabetes-coupled with cardiovascular problems raises the risk due to the abnormal lipoprotein metabolism-mediated atherosclerosis, and patients with diabetes are two to four times more liable to experience stroke.[7] Natural environment is a major resource for alternative medicines. The use of herbal medicine has been described in ancient literature, such as the Vedas and the Bible, which highlights the therapeutic potential of a wide range of wild plants. Plants produce a diverse group of bioactive molecules that serve as an affluent resource for several kinds of medicines.[8],[9] Currently, the medicinal and nutraceutical herbs have garnered massive scientific consideration for their holistic effects.[10],[11]

Since the existence, search for diabetes therapeutics and management has been relentless. It has propelled immense examinations attempting at identification as well as clinical trials of natural products obtained from plants and their analogs in drug discovery studies.[12],[13],[14],[15],[16],[17]

However, no scientific records and reports were obtainable demonstrating the antidiabetic activity of Litsea lancifolia leaf extract in type 2 diabetes. The genus Litsea is one of the widest, distinct genera of evergreen shrubs that comprises approximately 400 species of shrubs. They are dispersed profusely all through tropical and subtropical Asia and North and South America. Litsea species have been used widely in traditional medicine for the treatment of several diseases including influenza, diarrhea, vomiting, stomach aches, diabetes, bone pain, inflammation, illness related to the central nervous system, and other ailments.[18] The comprehensive and characterized reports in the ethnobotany, phytochemistry, and pharmacological research about the Litsea species enlighten us to explore their therapeutic potential and evaluate future research opportunities.[18][Figure 1] shows an image of L. lancifolia, a Himalayan shrub.{Figure 1}

This study was aimed to investigate in vitro antidiabetic activity of the methanolic leaf extract of L. lancifolia, which was collected from thickets, streamsides, and forests, in 3T3L1 cell line model by performing assays such as glucose uptake activity, glucose transporter 4 (GLUT4) expression studies, and the inhibition of carbohydrate-metabolizing enzymes: pancreatic α-amylase and α-glucosidase.

 Materials and Methods



Chemicals and reagents

The chemicals and reagents used in this study are as follows: Dulbecco’s Modified Eagle’s medium (DMEM) high glucose (#AL219A, HiMedia, India), DMEM without glucose (#AL186, HiMedia), adjustable multichannel pipettes and a pipettor (BenchTop Lab Systems, Saint Louis, Missouri, USA), fetal bovine serum (FBS) (#RM10432, HiMedia), Dulbecco’s phosphate-buffered saline (D-PBS) (#TL1006, HiMedia), antibiotic solution (#A018, HiMedia), 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) (#13195, Invitrogen, USA), metformin (#PHR1084, Sigma-Aldrich, USA), acarbose (#A8980, Sigma-Aldrich), anti-mouse GLUT4–fluorescein isothiocyanate (FITC) antibody (#NBPI-49533F, Novus Biologicals, USA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (#4060, HiMedia), and dimethyl sulfoxide (DMSO) (#PHR1309, Sigma-Aldrich).

Collection of plant material

Leaves of L. lancifolia were collected from Uttarakhand, India. The plant material was identified by Dr. Kotresha, Assistant Professor in Botany, East West College, Bengaluru, Karnataka, India. The collected leaves were washed thoroughly, shade dried, and then further processed.

Preparation of plant crude extract

The leaves of L. lancifolia were air dried and minced into a powder by using mortar and pestle. Methanolic extract was prepared by using Soxhlet extraction method. Under reduced pressure, the solvent was evaporated using rotary evaporator (Buchi R-210, Marshall Scientific, USA). The dried extract was dissolved in DMSO at different concentrations and was used for in vitro antidiabetic assays.

Cell culture

3T3L1 cell line was obtained from the National Centre for Cell Science (NCCS), Pune, Maharashtra, India, and cultured in DMEM (high glucose) supplemented with 10% FBS, 10,000 units of penicillin G, 10,000 μg/mL streptomycin sulfate, and 10mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) at 37°C in 5% CO2 atmosphere.

Cytotoxicity assay

The cytotoxicity of leaf extract of L. lancifolia was determined by MTT assay. The yellow-colored tetrazolium salt, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is converted to formazan crystals by the action of lactate dehydrogenase enzyme, produced by live cell mitochondria.[19],[20] 3T3L1 cells at a density of 20 × 104 cells per well/200 µL were seeded in a 96-well plate and cultured overnight. The spent medium was replaced with different concentrations of leaf extract (25–400 µg/mL) diluted in DMEM, and incubated for 24h at 5% CO2, and 37°C temperature. After 24h of incubation, the cells were treated with 0.5mg/mL of MTT reagent and incubated at 37°C temperature for 2h. Further, MTT reagent was removed and the formed formazan crystals were dissolved by adding 20 µL of DMSO. The purple-colored solution was measured at 570nm by microplate reader. The percentage of cell viability was calculated by considering the untreated cells as 100% viable population and using the following formula:

[INLINE:1]

Glucose uptake assay

In a six-well plate, 3T3L1 cells were seeded at a density of 2 × 105 cells/2mL and incubated in 5% CO2 overnight at 37°C. Later, the spent medium was removed and the cells were washed with D-PBS and treated with experimental compounds and controls in 2mL glucose-free culture medium containing 100 μM 2-NBDG and incubated for 2h. At the end of the treatment, medium was removed from all the wells and washed with D-PBS. Cells were harvested by trypsinization and washed with D-PBS, followed by centrifugation for 5min at 300 × g at 25°C. The supernatant was aspirated and the cells were resuspended in 0.5mL of D-PBS. FACSCalibur, BD Biosciences, USA was used to analyze the cellular uptake of 2-NBDG, by measuring fluorescence intensity in FL1 channel, and CellQuest Pro software, BD Biosciences, USA was used for data analysis.

Glucose transporter 4 expression studies

GLUT4 expression studies were performed by using flow cytometry technique. 3T3L1 cells were seeded in a six-well plate at a density of 2 × 105 cells/2mL and incubated in an incubator (5% CO2) overnight at 37°C. The spent medium was aspirated and the cells were treated with 100 µg/mL of test compound showing 94.6% of cell viability in MTT assay and 100 µM of positive control, metformin. After 24h of the treatment, the spent medium was removed from all the wells and the cells were washed with D-PBS. The cells were harvested by trypsinization and were stained with anti-mouse GLUT4–FITC antibody (Novus Biologicals) for 30min in dark and the unbounded antibodies were washed with D-PBS. FACSCalibur was used to analyze the translocation of GLUT4 to cell membrane, by measuring fluorescence intensity in FL1 channel and CellQuest Pro software was used for data analysis.

Measurement of α-amylase activity

Amylase activity was measured according to the standard protocol of Miller,[21] by using dinitrosalicylic acid (DNSA) method and by using soluble starch as substrate. A total of 10% DMSO was used to prepare the desired stock concentration of leaf extract, and further dilutions were made using buffer ([Na2HPO4/NaH2PO4, 0.02 M and NaCl, 0.006 M] at pH 6.9) to get 31.25–500 μg/mL concentrations. Equal volumes of α-amylase solution (2 units/mL) and leaf extract (200 μL each) were mixed and incubated at 30°C for 10min. The reaction mixture was incubated with 200 μL of 1% starch solution for 3min. A total of 200 μL of DNSA reagent was added to the tubes and kept in boiling water bath for 10min at 85°C–90°C to terminate the reaction. After cooling the reaction mixture to room temperature, further dilutions were made by using distilled water and the absorbance was measured at 540nm using a microplate reader.

The wells with 200 μL of buffer alone served as blanks. Acarbose was used as positive control and 31.25–500 μg/mL of acarbose concentrations were prepared. The enzyme inhibition studies were performed similarly as aforementioned procedure. The percent inhibition of α-amylase was calculated using the equation given below. All the experiments were carried out in triplicates. A graph was plotted against the % inhibition of α-amylase and the concentration of leaf extract and acarbose, and the IC50 values were obtained from the graph.

[INLINE:2]

α-Glucosidase inhibition assay

The assay to analyze the α-glucosidase enzyme inhibitory activity of leaf extract was conducted based on the procedure previously described by Shibano et al.[22] with minor modifications.

Test compounds at various concentrations from 31.25 to 500 μg/mL, prepared by using PBS (pH, 7.0), were mixed with 50 μL of 0.1 M phosphate buffer (pH, 7.0), 25 μL of 0.5mM 4-nitrophenyl α-d-glucopyranoside (dissolved in 0.1 M phosphate buffer with a pH of 7.0), and 25 μL of α-glucosidase solution (0.1 unit/mL), and then incubated for 30min at 37°C. Later, 100 μL of 0.2 M sodium carbonate solution was added to terminate the reactions. The quantity of p-nitrophenol liberated into the reaction mixture by the hydrolysis of the substrate was examined at 410nm using microplate reader. Wells with buffer alone were considered as blanks and were used to correct the background absorbance. Acarbose was used as positive control, and the leaf extract was taken in different concentrations ranging from 31.25 to 500 μg/mL. The percent inhibition of α-glucosidase was calculated using the equation given below. All the experiments were carried out in triplicates. A graph was plotted against the % inhibition of α-glucosidase and the concentration of leaf extract and acarbose, and the IC50 values were obtained from the graph.

[INLINE:3]

Statistical analysis

IC50 value of percentage inhibition of enzymes was determined using linear regression graph (concentration vs. percentage enzyme inhibition). All the experiments were conducted in triplicates and the results were expressed as mean percentage inhibition ± standard deviation (SD) (n = 3). All statistical analyses and determination of IC50 values were carried out in GraphPad Prism (version 3.1) software (San Diego, CA).

 Results



Cytotoxicity effect of Litsea lancifolia on 3T3L1 cell line

The given test compound, L. lancifolia leaf extract, at varying concentrations (25–400 µg/mL) did not show cytotoxicity on 3T3L1 cells after the treatment for 24h. The concentrations of L. lancifolia leaf extract used to treat the 3T3L1 cells and the respective percentage of viability are given in [Table 1]. As metformin is a standard antidiabetic drug and has been used to conduct in vitro cell-based antidiabetic activities, the toxicity of metformin was also tested against 3T3L1 cells and it showed 91.52% of viability at 100 µM concentration. Untreated cells were considered as 100% viable cells. Each experiment was performed thrice and the results are represented as mean values ± SD [Table 1] [Figure 2] and [Figure 3].{Table 1}, {Figure 2}, {Figure 3}

Inhibition of α-amylase activity

L. lancifolia leaf extract showed 19.77% inhibition of α-amylase activity at 31.25 µg/mL and 73.29% at 500 µg/mL concentrations, respectively, and its IC50 value was found to be 248.65 µg/mL. The standard drug acarbose showed 34.16% inhibition of α-amylase activity at 31.25 µg/mL and 84.95% at 500 µg/mL concentrations, respectively, and its IC50 was found to be 47.54 µg/mL [Table 2] [Figure 4].{Table 2}, {Figure 4}

Inhibition of α-glucosidase activity

L. lancifolia leaf extract showed 22.30% inhibition of α-glucosidase activity at 31.25 µg/mL and 74.51% at 500 µg/mL concentration, respectively, and its IC50 value was found to be 229.61 µg/mL. The standard drug, acarbose, showed 31.82% inhibitory effect on α-glucosidase activity at 31.25 µg/mL and 85.39% at 500 µg/mL concentrations, respectively, and its IC50 value was found to be 50.26 µg/mL [Table 3] [Figure 5].{Table 3}, {Figure 5}

Glucose transporter 4 expression study on 3T3L1 cell line by flow cytometry

The observation in statistical data of GLUT4 expression study by flow cytometry suggests that 0.12% of cells in untreated population, 99.67% cells in metformin (100 µM) treated population, and 86.26% of cells in L. lancifolia leaf extract–treated population showed GLUT4 expression. The results are shown in [Figure 6] and [Figure 7].{Figure 6}, {Figure 7}

Glucose uptake assay

Glucose uptake–inducing capabilities of the leaf extract was performed by treating 3T3L1 cells with 2-NBDG, a fluorescent deoxyglucose analog, in the presence and absence of test compounds. FACSCalibur was used to analyze the fluorescence intensity of 2-NBDG in FL1 channel. The overlaid fluorescence intensities of untreated cells, metformin, and leaf extract–treated cells are shown in [Figure 8] and the percentage of cells with 2-NBDG uptake is shown in [Figure 9], respectively.{Figure 8}, {Figure 9}

 Discussion



The controlling mechanisms of blood glucose varied based on the type of antidiabetic drug used. Mechanism of action of some drugs involves stimulation of insulin secretion or synthesis of insulin in adequate amounts by the pancreatic beta cells, regeneration of damaged pancreatic beta cells, increase of insulin sensitivity, which triggers glucose uptake by fat and muscle cells, impersonating the action of insulin, and finally, modification of the activity of some of the enzymes, especially related in glucose metabolism, and diminishing the absorption of sugars from the gut.[23],[24] However, the usage of synthetic drugs for longer time span caused side effects, which led to the investigation and use of traditionally available natural plant species with no toxicity. Litsea is one among those naturally available plants shown to have promising, intense antidiabetic activity.

A preliminary cytotoxicity study was performed to analyze the cytotoxicity of L. lancifolia leaf extract against 3T3L1 cells by MTT assay. The results revealed that after 24h of incubation, the leaf extract did not show any toxicity. This screening of plants have been of great interest to scientists as plant extracts with no exhibited cytotoxicity may be useful in therapeutic applications.

Under regular conditions, glucose transport activity in muscle cells augments three- to fourfold pursing insulin stimulation, and a greater part of this outcome is related with a net translocation of the GLUT4 from intracellular compartment to the cell surface, where it makes it possible for the uptake and reduction of plasma glucose.[25],[26],[27]

Experiments on cellular uptake of glucose revealed that L. lancifolia has the capability to induce glucose utilization by 3T3L1 cells. After incubation of cells with 100 μg/mL of L. lancifolia leaf extract for 2h at 37°C, 2-NBDG uptake was observed in 60.22% of cells, whereas it was observed in 99.94% of cells in the presence of 100 µM of the standard drug metformin. Moreover, these results can be correlated with GLUT4 expression studies, which showed the GLUT4 translocation–inducing capabilities of L. lancifolia leaf extract. 3T3L1 cells were treated with 100 μg/mL of L. lancifolia leaf extract and 100 µM of the standard drug metformin for 24h and were examined for the GLUT4 expression on the surface of cells by using flow cytometry. Both metformin and L. lancifolia leaf extract induced the translocation of GLUT4 in 99.67% and 86.26% of cells, respectively, after 24h of treatment. Thus, it indicates that cellular uptake of 2-NBDG might have occurred in 3T3L1 cells because of GLUT4 translocation–inducing capabilities of L. lancifolia leaf extract.

Carbohydrate-degrading digestive enzymes are considered to be one of the major targets in treating diabetic condition. These enzymes aid in the breakdown of complex carbohydrates of food and break them into smaller simple sugar units and increase their concentration in the bloodstream. Inhibition of these digestive enzymes can reduce the blood glucose levels in patients with diabetes mellitus.[28] Thus, this study was envisioned to examine the inhibitory effect of L. lancifolia on the activity of these enzymes (i.e., α-amylase and α-glucosidase). Our results showed that methanolic leaf extract of L. lancifolia has a significant inhibitory effect on the activity of α-amylase enzyme and α-glucosidase enzyme with IC50 value of 248.65 µg/mL and 229.61 µg/mL, respectively. This inhibitory effect is comparable with the inhibitory effect of acarbose, which lowers the blood glucose levels in patients with diabetes.[29],[30],[31],[32]

These observations and findings clearly proposed that the L. lancifolia leaf extract has possible therapeutic and antidiabetic properties. Voglibose, acarbose, and miglitol are examples of α-glucosidase and α-amylase inhibitors and are currently being used in clinical therapy.[33] However, they are expensive and have a risk of clinical side effects such as abdominal distention, flatulence, meteorism, and diarrhea on long-term usage. Hence, use of natural resources such as L. lancifolia leaf extract, which show no toxicity can be recommended to diminish the hyperglycemic conditions with no side effects.

 Conclusion



The possible antidiabetic activity of methanolic leaf extract of L. lancifolia has been studied through In vitro studies. The experiments on glucose uptake and GLUT4 expression studies by flow cytometry in 3T3L1 cell line and colorimetric assays on α-amylase and α-glucosidase enzyme inhibition showed promising results when compared to that of the standard drugs metformin and acarbose, respectively. Moreover, methanolic extract of L. lancifolia leaf revealed no cytotoxicity against 3T3L1 cells, which concludes that L. lancifolia leaf extract could be recommended as a possible candidate for antidiabetic applications.

Summary

The effect of L. lancifolia leaf extract was evaluated in vitro on 3T3L1 cell line.

We performed biochemical (α-amylase and α-glucosidase inhibition assays) and cellular assays (cellular glucose uptake and GLUT4 expression studies) to evaluate the antidiabetic activities of the extract.

L. lancifolia had no cytotoxic effect on 3T3L1 cells.

L. lancifolia extract showed significant inhibitory effect on the activity of α-amylase enzyme and α-glucosidase enzyme.

On treating 3T3L1 cells with L. lancifolia extract, 60.223% of the cells took up glucose and 86.263% expressed GLUT4.

L. lancifolia extract showed promising antidiabetic activity and could be a potential source of antidiabetic agents.

Financial support and sponsorship

Nil.

Conflicts of interests

There are no conflicts of interest.

References

1Chakrabarti R, Rajagopalan R. Diabetes and insulin resistance associated disorders: disease and the therapy. Curr Sci 2002;83:12.
2Sharma G, Kumar S, Sharma M, Upadhyay N, Ahmed Z, Mahindroo N. Anti-diabetic, anti-oxidant and anti-adipogenic potential of quercetin rich ethyl acetate fraction of Prunus persica. Pharmacogn J 2018;10:463-9.
3Hashim A, Khan MS, Ahmad S. Alleviation of hyperglycemia and hyperlipidemia by Phyllanthus virgatus forst extract and its partially purified fraction in streptozotocin induced diabetic rats. Excli J 2014;13:809-24.
4Davis SN. Insulin, oral hypoglycemic agents, and pharmacology of the endocrine pancreas. In: Brunton LL, Lazo JS, Parker KL. editors. Goodman and Gilman`s The Pharmacological Basis of Therapeutics. 11th ed. United States of America McGraw‑Hill; 2006. p. 1613-45.
5Dewanjee S, Das AK, Sahu R, Gangopadhyay M. Antidiabetic activity of Diospyros peregrina fruit: effect on hyperglycemia, hyperlipidemia and augmented oxidative stress in experimental type 2 diabetes. Food Chem Toxicol 2009;47:2679-85.
6Gray RS, Fabsitz RR, Cowan LD, Lee ET, Howard BV, Savage PJ. Risk factor clustering in the insulin resistance syndrome. The strong heart study. Am J Epidemiol 1998;148:869-78.
7Oranje WA, Wolffenbuttel BH. Lipid peroxidation and atherosclerosis in type II diabetes. J Lab Clin Med 1999;134:19-32.
8Durga RK, Karthikumar S, Jegatheesan K. Isolation of potential anti bacteria and antioxidant compounds from Acalypha indica and Ocimum basilicum. Afr J Plant Sci 2009;4:163-16.
9Fan C, Wang W, Wang Y, Qin G, Zhao W. Chemical constituents from Dendrobium densiflorum. Phytochemistry 2001;57:1255-8.
10Cousins MM, Adelberg JW. In vitro plant and organ culture of medicinal and neutriceutical species in laboratory and industrial scales. Acta Physiologiae Plantarum 2009;31:961-7.
11Cragg GM, Newman DJ, Snader KM. Natural products in drug discovery and development. J Nat Prod 1997;60:52-60.
12Moller DE. New drug targets for type 2 diabetes and the metabolic syndrome. Nature 2001;414:821-7.
13Gupta PD, Amartya D. Diabetes mellitus and its herbal treatment. Int J Res Pharm Bio Sci 2012;3:706-12.
14Rutebemberwa E, Lubega M, Katureebe SK, Oundo A, Kiweewa F, Mukanga D. Use of traditional medicine for the treatment of diabetes in eastern Uganda: a qualitative exploration of reasons for choice. BMC Int Health Hum Rights 2013;13:1.
15Gessler MC, Msuya DE, Nkunya MH, Schär A, Heinrich M, Tanner M. Traditional healers in Tanzania: sociocultural profile and three short portraits. J Ethnopharmacol 1995;48:145-60.
16Sharma AK, Kumar R, Mishra A and Gupta R. Problems associated with clinical trials of Ayurvedic medicines. Brazilian J Pharmacogn 2010;20:276-81.
17Hall V, Thomsen RW, Henriksen O, Lohse N. Diabetes in sub-Saharan Africa 1999-2011: epidemiology and public health implications. A systematic review. BMC Public Health 2011;11:564.
18Wang YS, Wen ZQ, Li BT, Zhang HB, Yang JH. Ethnobotany, phytochemistry, and pharmacology of the genus Litsea: an update. J Ethnopharmacol 2016;181:66-107.
19Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55-63.
20Patel S, Patel NJ. Spectrophotometric and chromatographic simultaneous estimation of amitriptyline hydrochloride and chlordiazepoxide in tablet dosage forms. Indian J Pharm Sci 2009;71:472-6.
21Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 1959;1:426-8.
22Shibano M, Kitagawa S, Nakamura S, Akazawa N, Kusano G. Studies on the constituents of Broussonetia species. II. Six new pyrrolidine alkaloids, broussonetine A, B, E, F and broussonetinine A and B, as inhibitors of glycosidases from Broussonetia kazinoki sieb. Chem Pharm Bull (Tokyo) 1997;45:700-5.
23Tiwari AK, Rao JM. Diabetes mellitus and multiple therapeutic approaches of phytochemicals: present status and future prospects. Curr Sci 2002;83:30-8.
24Tiwari BK, Pandey KB, Abidi AB, Rizvi SI. Therapeutic potential of Indian medicinal plants in diabetic condition. Ann Phytomed 2013;2:37-43.
25Takazawa K, Noguchi T, Hosooka T, Yoshioka T, Tobimatsu K, Kasuga M. Insulin-induced GLUT4 movements in C2C12 myoblasts: evidence against a role of conventional kinesin motor proteins. Kobe J Med Sci 2008;54:E14-22.
26Hussey SE, McGee SL, Garnham A, McConell GK, Hargreaves M. Exercise increases skeletal muscle GLUT4 gene expression in patients with type 2 diabetes. Diabetes Obes Metab 2012;14:768-71.
27Bryant NJ, Govers R, James DE. Regulated transport of the glucose transporter GLUT4. Nat Rev Mol Cell Biol 2002;3:267-77.
28Tundis R, Loizzo MR, Menichini F. Natural products as alpha-amylase and alpha-glucosidase inhibitors and their hypoglycaemic potential in the treatment of diabetes: an update. Mini Rev Med Chem 2010;10:315-31.
29DiNicolantonio JJ, Bhutani J, O’Keefe JH. Acarbose: safe and effective for lowering postprandial hyperglycaemia and improving cardiovascular outcomes. Open Heart 2015;2:e000327.
30Lebovitz HE. Alpha-glucosidase inhibitors as agents in the treatment of diabetes. Diabetes Rev 1998;6:132-45.
31Scott LJ, Spencer CM. Miglitol: A review of its therapeutic potential in type 2 diabetes mellitus. Drugs 2000;59:521-49.
32Mogale, MA, Lebelo SL, Thovhogi N, de Freitas AN, Shai LJ. α-amylase and α-glucosidase inhibitory effects of Sclerocarya birrea [A. Rich. Hochst] subspecies caffra (sond) Kokwaro (Anacardiaceae) stem-bark extracts. Afr J Botany 2011;10:15033-9.
33Bischoff H. Pharmacology of alpha-glucosidase inhibition. Eur J Clin Invest 1994;24:3-10.