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Modulation of adipogenesis and glucose uptake by Curcuma longa extract in 3T3L1 and L6 cell lines -An in vitro study

DOI:10.1016/S2222-1808(12)60144-3
Authors:
Prathapan Ayyappan at Sanford Research
Prathapan Ayyappan
Mahesh S. Krishna at Rajiv Gandhi Centre for Biotechnology
Mahesh S. Krishna
P C Lekshmi
P C Lekshmi
P C Lekshmi
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Raghu K G at National Institute for Interdisciplinary Science and Technology
Raghu K G
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Abstract and Figures

Objective To evaluate the effects of ethyl acetate extract of Curcuma longa against modulation of glucose uptake and adipogenesis in cell line models.Methods We used 3T3L1 and L6 cells to investigate cytotoxicity, glucose uptake with 2-NBDG as probe and adipogenesis. All the analysis was done with flowcytometry.ResultsThe results showed that the extract did not possess any significant glucose uptake activity but it exhibited significant adipocyte differentiation potential.Conclusions Ethyl acetate extract of Curcuma longa exhibits significant antiadipogenesis and can be used as an effective drug for the treatment of obesity and other associated complications.
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S163
Document heading
Modulation of adipogenesis and glucose uptake by Curcuma longa
extract in 3T3L1 and L6 cell lines - An in vitro study
A. Prathapan, Mahesh S Krishna, P.C Lekshmi, K.G Raghu, A. Nirmala Menon*
Agroprocessing and Natural Products Division, National Institute for Interdisciplinary Science and Technology (NIIST), CSIR, Trivandrum, Kerala,
India 695019
Asian Pacific Journal of Tropical Disease (2012)S163-S165
Asian Pacific Journal of Tropical Disease
journal homepage:www.elsevier.com/locate/apjtcm
*Corresponding author: A. Nirmala Menon, Agroprocessing and Natural Products
Division, National Institute for Interdisciplinary Science and Technology (NIIST), CSIR,
Trivandrum, Kerala, India 695019.
E-mail: nirmalamenon2000@gmail.com
Foundation project: It is supported by CSIR (SIP 004).
1. Introduction
Metabolic syndrome describes the human condition
characterized by the presence of coexisting traditional risk
factors for cardiovascular disease, such as hypertension,
dyslipidemia, glucose intolerance, and obesity, in addition
to non traditional risk factors, such as inflammatory
processes and abnormalities of the blood coagulation
system[1]. Obesity currently affects over a billion of people. It
is the growing health problem in many of the richest nations
of the world and should now be considered as a chronic
disease that is reaching epidemic proportions[2]. It acts as
a major risk factor for type 2 diabetes and cardiovascular
diseases. Most drugs used for treating type 2 diabetes
causes obesity as a side effects by reducing blood glucose
level and inducing adipogenesis. Traditional medicinal
can serve as an ideal candidate in treating obesity and
type 2 diabetes by acting on adipocytes and can act as a
better alternative for the treatment of metabolic syndromes.
Curcuma longa (Family: Zingiberacea) is reported to possess
many medicinal properties including cardioprotective,
hypolipidemic, antibacterial, anti-HIV, anti-tumour, anti-
carcinogenic and antiarthritic activities[3].
So the present study aims to evaluate the modulatory
effects of Curcuma longa extract against glucose uptake
and adipocyte differenciation in 3T3L1 and L6 cells
2. Materials and methods
2.1. Chemicals
All the chemicals were purchased from Sigma-Aldrich
(Saint Louis, MO, USA) unless or otherwise stated.
2.2. Preparaton of Curcuma longa extract
Fresh turmeric rhizomes were dried at 40-45 曟, ground
into powder and extracted (250 g) successively with hexane,
ethyl acetate, methanol and water at room temperature.
Ethyl acetate fraction contains highest amount of
curcuminoids was further taken to evaluate its biological
ART ICLE INFO ABSTRACT
Article history:
Received 15 June 2012
Received in revised form 27 June 2012
Accepted 18 October 2012
Available online 28 October 2012
Keywords:
Adipocytes
2-NBDG
Flow cytometry
Metabolic syndrome
Objective: To evaluate the effects of ethyl acetate extract of Curcuma longa against modulation
of glucose uptake and adipogenesis in cell line models. Methods: We used 3T3L1 and L6 cells to
investigate cytotoxicity, glucose uptake with 2-NBDG as probe and adipogenesis. All the analysis
was done with flowcytometry. Results: The results showed that the extract did not possess any
significant glucose uptake activity but it exhibited significant adipocyte differentiation potential.
Conclusions: Ethyl acetate extract of Curcuma longa exhibits significant antiadipogenesis and
can be used as an effective drug for the treatment of obesity and other associated complications.
Contents lists available at ScienceDirect
A. Prathapan et al ./Asian Pacific Journal of Tropical Disease (2012)S163-S165
S164
properties.
2.3. Determination of the subtoxic concentration of Curcuma
longa by MTT assay
Cytotoxicity of the extract was evaluated against L6 and
3T3L1 after 24 h incubation by MTT assay. The method was
carried out as per previously described protocol[4].
2.4. Glucose uptake assay by 2- NBDG
Glucose uptake assay was performed according to the
previous method[5] in L6 rat skeletal muscle cells with
fluorescent analogue of glucose, 2-NBDG (10 毺M) using
flow cytometry (BD FACS Aria II, USA). Insulin (100 U) and
rosiglitazone (100 nM) served as control.
2.5. Adipocyte differentiation assay
Effect of Curcuma longa extract on inhibiting
differentiation of 3T3-L1 preadipocyte to mature adipocyte
was carried out based on reported protocol[6] using flow
cytometry (BD FACS Aria II, USA).
2.5. Statistical analyses
The experimental results were expressed as mean暲
standard deviation (SD) of triplicate measurements. The
data were subjected to one way analysis of variance and the
significance of differences between means were calculated
by Duncan’s multiple range test using SPSS for Windows,
standard version 7.5.1, and the significance accepted at P <
0.05.
3. Results
3.1. MTT assay
To determine the sub toxic concentrations against 3T3L1
and L6 cell lines, Curcuma longa extract was evaluated at
10, 50, 100 and 200 毺g/mL concentrations and cell viability
was determined by MTT assay. Results showed Curcuma
longa extract exhibited toxicity in both the cell lines after a
concentration of 100 毺g/mL.
3.2. Glucose uptake assay
Effect of Curcuma longa on glucose uptake was checked
by 2-NBDG based assay. L6 cells were pre-incubated
with extract at different concentrations from 1-100 毺g/
mL. Results indicated that Curcuma longa was not able
to enhance uptake of 2-NBDG at subtoxic concentrations
(Figure 1). The effect was compared with standard drug
roziglitazone (100 nM), which showed 38.5% increase in probe
uptake. Uptake potential of cells showed a reduction at 100
毺g/mL concentration compared to control. This might be
due to slight toxic effect of extract against L6 cells.
3.3. Antiadipogenic effect of Curcuma longa extract
Around 10 000 events were recorded and analyzed by
flow cytometer based on difference in size and granularity
which gives a separation between undifferentiated cells
and differentiated cells. Results showed that within a
concentration 1 and 5 毺g/mL, the extract seemed to reduce
adipocyte differentiation and lipid droplet content by 22%
(Figure 2).
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Figure 1. Glucose uptake effect of Curcuma longa.
a) Unstained control; b) NBDG control; c) Insulin treated; d) Roziglitazone treated; e-h) Extract treated at 1, 10, 50 and 100 毺g/mL concentration.
A. Prathapan et al ./Asian Pacific Journal of Tropical Disease (2012)S163-S165 S165
4. Discussion
Adipocyte differentiation inhibitors may be effective
in preventing obesity, atherosclerosis, diabetes and
other associated complications. 3T3-L1 cells are known
to differentiate into adipocytes under the appropriate
conditions[2] and have been useful as a model for adipose
cells, which are one of the major sites of lipid and glucose
metabolism. Overweight and obesity are the result of
excessive adipogenesis and therefore inhibition of the
differentiation of 3T3-L1 cells to adipocytes is beneficial
for the prevention of obesity complicated by diabetes and
atherosclerosis[2]. The inhibition potential of Curcuma longa
was found to be dose dependant suggesting that Curcuma
longa may have altered the expression of any genes involved
in the adipocyte expression.
In conclusion, the present study reveals that Curcuma
longa extract didn’t exhibit any significant glucose uptake
activity but it can inhibit adipocyte diffrenciation and can
serve as an ideal candidate for the management of obesity
and associated metabolic syndromes.
Conflict of interest statement
We declare that we have no conflict of interest.
Acknowledgements
Financial support from CSIR (SIP 004) is greatly
acknowledged.
References
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87(suppl): 481S-487S.
[2] Arumughan M, Vijayan P, Raghu C, Ashok G, Dhanraj SA,
Kumarappan CT. Anti-adipogenic activity of Capsicum annum
(Solanaceae) in 3T3 L1. J Compl Int Med 2008; 5(1): 1-9.
[3] Prathapan A, Lukhman M, Arumughan C, Sundaresan A, Raghu
KG. Effect of heat treatment on curcuminoid, color value and total
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[4] Wilson AP, Cytotoxicity and viability assays. In: Masters JRW.
Animal cell culture: A practical approach. 2nd ed. Oxford: Oxford
University Press; 2000, p. 175-219.
[5] Chen QC, Zhang WY, Jin W, Lee IS, Min BS, Jung HJ. Flavonoids
and isoflavonoids from Sophorae Flos improve glucose uptake in
vitro. Planta Med 2010; 76:79-81.
[6] Kim JK, Kim Y, Na KM, Surh YJ, Kim TY. Gingerol prevents
UVB-induced ROS production and COX-2 expression in vitro and
in vivo. Free Rad Res 2007; 41: 603-614.
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Figure 2. Change in percentage of differentiated cells upon extract treatment.
a) Untreated control, b) MDI treated cells, c-f) cells treated with different concentration of extract (1-50 毺g/mL).
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Various extracts of Capsicum annum L were screened for their cytotoxicity and antiadipogenic activity in vitro. In cytotoxic studies all the extracts were screened against the 3T3 L1 cell line. Micro titre tetrazolium (MTT) and sulpho rhodamine B (SRB) assays against the 3T3 L1 cell line determined cytotoxicity. Methanol extract of fruit showed higher cytotoxicity with a lower CTC50 value of 167.46 µg/ml and the remaining extracts show CTC50 ranging from 180-282 µg/ml. In 3T3 L1 cells dexamethazone, 3- isobutyl-1-methyl xanthine (IBMX) and insulin, induced adipogenesis. All the four extracts (methanolic and water extracts of whole plant and fruit) were screened for their antiadipogenic activity in 3T3 L1 cell line; the methanolic fruit extract showed good antiadipogenic activity at 100 µg/ml.
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Anti-Adipogenic Activity of Capsicum annum (Solanaceae) in 3T3 L1
Article
Full-text available
Various extracts of Capsicum annum L were screened for their cytotoxicity and antiadipogenic activity in vitro. In cytotoxic studies all the extracts were screened against the 3T3 L1 cell line. Micro titre tetrazolium (MTT) and sulpho rhodamine B (SRB) assays against the 3T3 L1 cell line determined cytotoxicity. Methanol extract of fruit showed higher cytotoxicity with a lower CTC50 value of 167.46 μg/ml and the remaining extracts show CTC50 ranging from 180-282 μg/ml. In 3T3 L1 cells dexamethazone, 3- isobutyl-1-methyl xanthine (IBMX) and insulin, induced adipogenesis. All the four extracts (methanolic and water extracts of whole plant and fruit) were screened for their antiadipogenic activity in 3T3 L1 cell line; the methanolic fruit extract showed good antiadipogenic activity at 100 μg/ml.
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Botanicals and the metabolic syndrome
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Full-text available
  • Mar 2008
Metabolic syndrome describes the human condition characterized by the presence of coexisting traditional risk factors for cardiovascular disease, such as hypertension, dyslipidemia, glucose intolerance, and obesity, in addition to nontraditional cardiovascular disease risk factors, such as inflammatory processes and abnormalities of the blood coagulation system. Although the specific etiology for metabolic syndrome is not known, insulin resistance--a clinical state in which a normal or elevated insulin concentration reflects an impaired biological response--is present and is considered a key pathophysiologic abnormality. As such, metabolic syndrome can be considered to be a prediabetic state and contributes greatly to increased morbidity and mortality in humans. Given the public health significance of metabolic syndrome, successful strategies are direly needed to intervene in its development. As such, nutritional supplementation with botanicals that effectively address pathogenic mechanisms, combined with the acceptance and widespread use of botanical supplements by the general public, represents an attractive, novel, and potentially effective approach to the problem. Thus, the overall goal of our botanical research center is to comprehensively evaluate botanicals in addressing the pathophysiologic mechanisms that lead to the development of insulin resistance and metabolic syndrome. Currently, each of the 3 research projects evaluates a specific botanical [Russian tarragon (Artemisia dracunculus L), shilianhua (Sinocrassula indica), and grape (Vitus vinifera) anthocyanins] and assesses the effect on pathogenic mechanisms leading to the development of insulin resistance. With the completion of our research, we anticipate a better understanding of the cellular mechanisms by which insulin resistance develops and the role of botanicals in modulating the progression to metabolic syndrome.
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Botanicals and the metabolic syndrome
Article
  • Nov 2007
Metabolic syndrome describes the human condition characterized by the presence of coexisting traditional risk factors for cardiovascular disease, such as hypertension, dyslipidemia, glucose intolerance, and obesity, in addition to nontraditional cardiovascular disease risk factors, such as inflammatory processes and abnormalities of the blood coagulation system. Although the specific etiology for metabolic syndrome is not known, insulin resistance--a clinical state in which a normal or elevated insulin concentration reflects an impaired biological response--is present and is considered a key pathophysiologic abnormality. As such, metabolic syndrome can be considered to be a prediabetic state and contributes greatly to increased morbidity and mortality in humans. Given the public health significance of metabolic syndrome, successful strategies are direly needed to intervene in its development. As such, nutritional supplementation with botanicals that effectively address pathogenic mechanisms, combined with the acceptance and widespread use of botanical supplements by the general public, represents an attractive, novel, and potentially effective approach to the problem. Thus, the overall goal of our botanical research center is to comprehensively evaluate botanicals in addressing the pathophysiologic mechanisms that lead to the development of insulin resistance and metabolic syndrome. Currently, each of the 3 research projects evaluates a specific botanical [Russian tarragon (Artemisia dracunculus L), shilianhua (Sinocrassula indica), and grape (Vitus vinifera) anthocyanins] and assesses the effect on pathogenic mechanisms leading to the development of insulin resistance. With the completion of our research, we anticipate a better understanding of the cellular mechanisms by which insulin resistance develops and the role of botanicals in modulating the progression to metabolic syndrome
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Effect of heat treatment on curcuminoid, colour value and total polyphenols of fresh turmeric rhizome
Article
  • Jul 2009
Summary Studies were undertaken to examine the effect of heat treatment on total phenolic content (TPC), colour value (yellowishness and brightness), polyphenol oxidase (PPO) activity and curcuminoid of fresh turmeric rhizome. Fresh turmeric rhizomes were subjected to heat treatment at different temperatures (60-100 °C) for different durations (10-60 min), causing a reduction in browning which was evident from the improved yellowishness and brightness. Activity of PPO was also decreased during heat treatment and PPO was almost inactivated when heated at 80 °C for 30 min. TPC of heat-treated turmeric after drying (powder) is significantly higher than that after the fresh process. TPC values increased gradually when samples were heated from 60 to 80 °C. At 90 and 100 °C, TPC values were almost identical. Maximum brightness and yellowishness were obtained when the turmeric was heated above 80 °C. Quantitation of curcuminoids in the turmeric sample was made with high performance thin layer chromatography (HPTLC). There was no significant change in the concentration of curcuminoids among the heat-treated samples. But in the sun-dried samples, a significant reduction in curcuminoid concentration was observed.
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Flavonoids and Isoflavonoids from Sophorae Flos Improve Glucose Uptake in Vitro
Article
Glucose uptake assay-guided fractionations on the methanol extract of Sophorae Flos led to the isolation of the flavonoids rutin (1), narcissin (2), quercetin (3), tamarixetin (4), and kaempferol (5) and the isoflavonoids cajanin (6), genistein (7), orobol (8), and pratensein (9). Among them, 1, 4, 5, 6, 8, and 9 significantly improved basal glucose uptake in HepG2 cells. Their improving effects were concentration dependent. Compounds 4, 5, 6, and 9 exhibited effects stronger than that of rosiglitazone, which has been used as an antidiabetic drug. However, 2, 3, and 7 did not show any improving effects. Stimulating glucose uptake into peripheral cells may be responsible for reducing the level of blood glucose in the circulation. Therefore, these findings demonstrate a potential to develop these flavonoids and isoflavonoids as hypoglycemic drugs.
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[6]-Gingerol prevents UVB-induced ROS production and COX-2 expression in vitro and in vivo
Article
  • Jun 2007
[6]-Gingerol, a naturally occurring plant phenol, is one of the major components of fresh ginger (Zingiber officinale Roscoe, Zingiberaceae) and has diverse pharmacologic effects. Here, we describe its novel anti-oxidant, anti-apoptotic, and anti-inflammatory activities in vitro and in vivo. In vitro, pre-treatment with [6]-gingerol reduced UVB-induced intracellular reactive oxygen species levels, activation of caspase-3, -8, -9, and Fas expression. It also reduced UVB-induced expression and transactivation of COX-2. Translocation of NF-kappaB from cytosol to nucleus in HaCaT cells was inhibited by [6]-gingerol via suppression of IkappaBalpha phosphorylation (ser-32). Examination by EMSAs and immunohistochemistry showed that topical application of [6]-gingerol (30 microM) prior to UVB irradiation (5 kJ/m(2)) of hairless mice, also inhibited the induction of COX-2 mRNA and protein, as well as NF-kappaB translocation. These results suggest that [6]-gingerol could be an effective therapeutic agent providing protection against UVB-induced skin disorders.
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