|
 |
| RESEARCH ARTICLE |
|
|
|
| Year : 2013 | Volume
: 45
| Issue : 1 | Page : 30-33 |
| |
Insulin sensitizing effect of 3 Indian medicinal plants: An in vitro study
Samidha A Kalekar1, Renuka P Munshi1, Supriya S Bhalerao1, Urmila M Thatte2
1 Department of Clinical Pharmacology, TN Medical College and BYL Nair Charitable Hospital, Mumbai Central, Mumbai - 400 008, India
2 Department of Clinical Pharmacology, Seth GS Medical College & KEM Hospital, Parel, Mumbai - 400 012, India
| Date of Submission | 04-Mar-2012 |
| Date of Decision | 30-Aug-2012 |
| Date of Acceptance | 29-Oct-2012 |
| Date of Web Publication | 24-Jan-2013 |
Correspondence Address:
Renuka P Munshi
Department of Clinical Pharmacology, TN Medical College and BYL Nair Charitable Hospital, Mumbai Central, Mumbai - 400 008
India
 Source of Support: Partial Financial support from Ministry of Earth Sciences
(MoES)., Conflict of Interest: None  | Check |
DOI: 10.4103/0253-7613.106431
Objective: Measurement of glucose uptake into peripheral tissue is an important mechanism to assess Insulin sensitivity. The present in vitro study was conducted to evaluate the Insulin sensitizing activity of Phyllanthus emblica (Pe), Tinospora cordifolia (Tc) and Curcuma longa (Cl) by assessing glucose uptake activity in a 3T3L1 adipocyte model.
Materials and Methods: The 3T3 L1 fibroblast cells were differentiated to adipocytes, using a cocktail of insulin, isobutyl-1-methylxanthine and dexamethazone. These adipocytes were initially treated with different concentrations of the selected plants following which 2-deoxy glucose uptake was estimated using a radioactive assay. The effects of plants on glucose uptake both in the presence and absence of insulin was evaluated and compared with pioglitazone, a known insulin sensitizer.
Results: Pe and Tc per se significantly stimulated glucose uptake in 3T3-L1 adipocytes in a dose dependent manner with maximal effect at higher concentrations (200μg/ml). The effect of both Pe and Tc at 200μg/ml was comparable to insulin and greater than pioglitazone. Cl per se stimulated glucose uptake with maximal effect at 50μg/ml. However, this effect was lesser as compared to insulin with higher concentrations inhibiting glucose uptake. When combined with insulin, an antagonist effect was observed between Pe, Tc and insulin indicating a possible plant-drug interaction while Cl in combination with insulin showed an increase in the glucose uptake as compared to Cl alone.
Conclusion: The results suggest that one of the mechanisms for the anti-diabetic effect of Pe, Cl and Tc may be through an insulin sensitizing effect (stimulation of glucose uptake into adipocytes). Further studies using other target sites viz. skeletal muscle and hepatocytes models and in an insulin resistant state would help substantiate this conclusion.
Keywords: Glucose uptake, 3T3 L1 adipocytes, Phyllanthus emblica, Curcuma longa, Tinospora cordifolia
How to cite this article:
Kalekar SA, Munshi RP, Bhalerao SS, Thatte UM. Insulin sensitizing effect of 3 Indian medicinal plants: An in vitro study. Indian J Pharmacol 2013;45:30-3 |
How to cite this URL:
Kalekar SA, Munshi RP, Bhalerao SS, Thatte UM. Insulin sensitizing effect of 3 Indian medicinal plants: An in vitro study. Indian J Pharmacol [serial online] 2013 [cited 2021 Dec 1];45:30-3. Available from: https://www.ijp-online.com/text.asp?2013/45/1/30/106431 |
| » Introduction | |  |
Glucose uptake and storage in peripheral tissues such as skeletal muscles and adipose tissue is a major regulatory process in the homeostatic control of blood glucose levels. [1] It is widely accepted that skeletal muscle, by virtue of its large contribution to body mass, represents the major site of insulin-mediated glucose disposal. However, both the tissues contribute toward the lowering of blood glucose. [2] Glucose uptake is mediated through the translocation of the Glut4 receptor from the interior to the cell surface which is stimulated by the insulin signaling pathway initiated by activation of the insulin receptor. Any defect in this pathway triggers the development of hyperglycemia in type II diabetes. Thus, measurement of glucose uptake into peripheral tissues is an important mechanism to assess insulin sensitivity. Insulin sensitizers like thiazolidinediones (TZDs) are effective in improving insulin resistance; however their adverse effect profile limits their long term use. Hence, the demand for new anti-diabetic compounds continues.
Medicinal plants offer a rich, yet inadequately explored source of potentially useful anti-diabetic drugs. Phyllanthus emblica (amla), Tinospora cordifolia (guduchi) and Curcuma longa (haldi) are extensively used by ayurvedic practitioners for the treatment of diabetes. [3],[4],[5] Their anti-diabetic activity has been explored in various animal models of diabetes and its complications. [6],[7],[8],[9],[10],[11],[12],[13],[14],[15],[16],[17],[18],[19] Most of these studies have focused on the glucose lowering effects of these plants in experimental models. However their potential mechanism(s) of action have not been clearly elucidated at the molecular level. Keeping in view that glucose uptake into the peripheral tissues is an important action of insulin in maintaining glucose homeostasis and a decrease in the glucose uptake is one of the major signs of type II diabetes, the present study was conducted to evaluate the effect of the selected plants on glucose uptake by the adipocytes, signifying an insulin sensitizing effect. Pioglitazone, a known insulin sensitizer, was used as a comparator. Additionally the effect of the plants on glucose uptake in the presence of insulin was also evaluated.
| » Materials and Methods | | |
The study was carried out using 3T3-L1 cell line which is a well-defined system to study insulin action and factors regulating glucose transport. [20] When differentiated into adipocytes, these cells express increased insulin receptors and insulin stimulated glucose transport and oxidation. [21] The 3T3-L1 fibroblast cell line was procured from National Centre for Cell Sciences (NCCS), Pune and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% new born calf serum (NBCS). DMEM, NBCS and fetal bovine serum (FBS) were obtained from Invitrogen Corporation, USA. Trypsin, dexamethazone (DEX), iso-butyl methyl xanthine (IBMX) and insulin were obtained from Sigma-Aldrich Chemicals Co., St. Louis Mi, USA. 2-deoxy-D-[≥H] glucose was obtained from Board of Radiation and Isotope Technology (BRIT), Unit of Department of Atomic Energy, Government of India at Deonar, Mumbai.
Standardized hydro-alcoholic extracts of Phyllanthus emblica (fruit) and Curcuma longa (rhizome) and aqueous extract of Tinospora cordifolia (stem) were procured from Natural Remedies, Bangalore. The authentication report and certificate of analysis is available on file. Pioglitazone in the pure form, used in the present study as the positive control, was donated by Glenmark Pharmaceuticals, Mumbai.
The plant extracts were evaluated over a concentration range of 10 to 200μg/ml. These concentrations were calculated from the therapeutic dose of the plants and were within the concentration range commonly used for in vitro studies. Insulin was evaluated at a dose of 1 μmol/L; dose selected on the basis of previous studies [22],[23] and pioglitazone was evaluated at a dose of 6 μg/ml; a dose extrapolated from the median therapeutic human dose.
The extracts of Phyllanthus emblica and Tinospora cordifolia (in powder form) were dissolved in Krebs-ringer hepes buffer (KRHB) and diluted further. Curcuma longa and pioglitazone was first dissolved in DMSO and then reconstituted in KRHB to achieve the required concentration. The concentration of DMSO in the extract and standard drug did not exceed 0.2%, which did not affect the rate of 2-deoxy glucose uptake into the cell.
Viability Test
Viability test were carried out to eliminate cytotoxic concentrations of plant extracts using the trypan blue dye exclusion method. [24] The plant concentrations studied ranged from 10μg/ml to 200μg/ml.
Cell Culture and Adipocyte Differentiation
3T3L1 fibroblasts were grown in DMEM medium, supplemented with 10% FBS, in a humidified atmosphere of 5% CO 2 at 37°C. On attaining 75-80% confluency, the cells at a concentration of 1 × 10 4 cells/ml, were seeded in 24 well plates. As described previously [21] the differentiation was induced by supplementing the media with a combination of 1 mg/L insulin, 100 mg/L isobutyl-1-methylxanthine (IBMX) and 0.1 mg/L dexamethazone for 48 hrs followed by insulin alone for an additional 48 hrs. The media was then replaced with fresh culture medium (DMEM supplemented with 10% FBS) after two days and then every three days thereafter.
Insulin Sensitizing Effect: Glucose Uptake Activity Assay
Insulin sensitizing effect was analyzed by measuring the uptake of 2-deoxy-D-[3H] glucose as described previously. [21] Briefly, the confluent 3T3-L1 adipocytes grown in 24 well plates were washed twice with serum-free DMEM and incubated with 1 ml of the same medium at 37°C for 2 hrs. The cells were then washed three times with KRHB and incubated with 0.9ml KRHB at 37°C for 30 minutes. Insulin, standard drug and the plant extracts in the required concentrations were added respectively and adipocytes were further incubated at 37°C for 20 minutes. Glucose uptake was initiated by addition of 0.1 ml KRHB buffer containing 37 MBq /L 2-deoxy-D-[ [3] H] glucose. After 15 minutes, the assay was terminated by washing the cells thrice with cold PBS. The cells were then lysed with 0.7 ml of 0.1% Triton X -100 at 37°C for 20 minutes. The radioactivity retained by the cell lysate was determined by a scintillation counter. The uptake measurement was done in duplicate and repeated thrice. Potential interaction between insulin and plant extracts was studied in only those concentrations which exhibited maximum glucose uptake.
Statistical Analysis
The results were expressed as mean ± SD. The data was analyzed using one way analysis of variance (ANOVA) followed by post-hoc tests. A value of p<0.05 was considered as statistically significant.
| » Results | | |
Viability Assay
The viability was not affected at any of the concentrations tested (ranging from 10-200 μg/ml) of the selected plant extracts hence concentrations varying over this range were selected for the glucose uptake assay.
Glucose Uptake Assay
Differentiated adipocytes, after experimental incubation, were analyzed for insulin sensitizing effect using glucose uptake assay. The effect of the plant extracts were compared to that of insulin (1 μmol/L) and pioglitazone (6 μg/ml) [Figure 1]. To test the effect of plant extracts in presence of insulin, the three extracts were individually combined with insulin and then added to the cell culture medium [Figure 2]. Insulin significantly stimulated glucose uptake. Pioglitazone showed a significant increase in glucose uptake as compared to the control cells.  | Figure 1: The effect of the different concentrations of plant extracts (Pe, Tc and Cl) or Insulin or Pioglitazone on glucose uptake in 3T3-L1 adipocytes
Click here to view |
 | Figure 2: Combined effects of Insulin and plant extracts (Pe, Tc and Cl) on glucose uptake in 3T3-L1 adipocytes
Click here to view |
Effect of Phyllanthus Emblica
Phyllanthus emblica alone stimulated glucose uptake in a dose dependent manner, with significant increase at higher concentrations. The effect at concentration of 200 μg/ml was comparable to that of insulin and greater than that seen with pioglitazone. Assuming the percentage response of Insulin as 100%, the response of Phyllanthus emblica per se on glucose uptake was 123.82% at 200μg/ml [Figure 1]. In the presence of insulin however, Phyllanthus emblica decreased the amount of glucose taken up into the cells [Figure 2].
Effect of Tinospora Cordofolia
Tinospora cordifolia per se also stimulated glucose uptake in the adipocytes in a dose dependent manner with the maximum effect seen at 200μg/ml which was significantly greater than the stimulation shown by the cells in the presence of insulin alone. The response of Tinospora cordifolia at 200μg/ml was 110.69 % when compared to that seen with insulin [Figure 1]. However, the effect of Tinospora cordifolia on glucose uptake was decreased in presence of insulin [Figure 2].
Effect of Curcuma Longa
Curcuma longa per se stimulated glucose uptake, but its effect was lesser than that seen with insulin. The effect was maximal at 50μg/ml; with higher concentrations, a decrease in the effect was observed [Figure 1]. Curcuma longa exhibited the least effect on glucose uptake (45.67%) which was much lower than that seen with insulin. However, in the presence of insulin, its effect on glucose uptake was enhanced especially at higher concentrations (100-200 μg/ml) [Figure 2].
| » Discussion | | |
Non-insulin dependent diabetes mellitus (NIDDM) is characterized by three major defects: abnormal pancreatic insulin secretion, enhanced hepatic glucose output and peripheral insulin resistance. Peripheral insulin resistance at the molecular level is associated with defects in the glucose transport system which reduces the intracellular pool of transporters. These results in fewer transporter molecules available for translocation to the plasma membrane in response to insulin, thus affecting the process of glucose uptake and leading to insulin resistance. [25] Hence compounds that increase the peripheral sensitivity to insulin are useful in the treatment of type II diabetes mellitus. The present in vitro study was thus planned to evaluate the insulin sensitizing effect of Phyllanthus emblica (Pe), Tinospora Cordifolia (Tc) and Curcuma longa (Cl).
Insulin sensitizing effect was evaluated by assessing the glucose uptake activity of the differentiated adipocytes both in presence and absence of insulin. Phyllanthus emblica significantly stimulated glucose uptake in 3T3-L1 adipocytes in a dose dependent manner with maximal effect at the highest concentration tested i.e., 200 μg/ml. Although there are various reports about the anti-diabetic activity of Phyllanthus emblica,[6],[7],[8],[9],[10],[11]
studies with respect to insulin sensitizing effect have not been documented till date. This study thus documents for the first time the glucose stimulatory activity and hence insulin sensitizing effect of Phyllanthus emblica.
Similarly Tinospora cordifolia also dose dependently stimulated glucose uptake. A review of available literature showed that most studies have demonstrated the anti-diabetic activity of extracts prepared from either the roots or leaves of Tinospora cordifolia. [15],[16],[17] Only one recently published in vitro study have demonstrated the glucose uptake-stimulatory activity of this extract in Ehrlich ascites tumor cell model system. [26] Thus the observation in our study is in agreement with the results of this published study stating that prevention of hyperglycemia may be due to its insulin sensitizing effect on the peripheral tissues.
Although Curcuma longa per se stimulated glucose uptake (maximal at 50μg/ml), its effect was lesser than that seen with insulin. However, in the presence of insulin, its effect on glucose uptake was enhanced especially at higher concentrations (100-200 μg/ml). This suggests that Curcuma longa may be beneficial in treating type II diabetes patients in which the target cells become resistant to the action of insulin.
When insulin was added in the cell medium along with the plant extracts, a reduction in the amount of glucose taken up was observed with Phyllanthus emblica and Tinospora cordifolia which could be due to antagonism between the plant extracts and insulin at some point in the insulin signaling pathway.
On the basis of these observations, a mechanism of action of these plants may be hypothesized. These plants may be interacting with one/more of the factors involved in the insulin-mediated glucose transport signaling pathway. In presence of insulin, the interaction between plants and these factors may result in the structural alteration (either physical, chemical or biological) of the factor, thus blocking the insulin signaling pathway at one or more steps and the translocation of Glut4, and hence inhibiting glucose uptake into the cell. Thus, further interaction studies between the plants extracts and insulin involving insulin receptor binding and intracellular protein phosphorylation are needed to understand the glucose uptake mechanism and identify the site of action of these plants.
Additionally as the treatment period of the plants was 20 minutes, a concentration and time dependent kinetic study is advisable to confirm the insulin sensitizing effect demonstrated by these plants. Additionally, the effect of the selected plants on glucose uptake has been demonstrated only on naοve 3T3L1 adipocytes. A study to explore the efficacy of these plants in a model of insulin resistance, a major characteristic feature of NIDDM, would be the next logical step forward followed by in vivo studies. Further, the insulin sensitizing effect demonstrated by these plants can be further evaluated in other sites of glucose uptake i.e., liver and skeletal muscles.
| » Acknowledgement | | |
We thank Natural Remedies for providing the standardized plant extracts, Glenmark Pharmaceuticals for providing the standard drug and Ms. Tanushree Joag for her technical assistance.
| » References | | |
| 1. | Kahn BB, Flier JS. Obesity and Insulin resistance. J Clin Invest 2000;106:473-81. 
[PUBMED] |
| 2. | Turban S, Beardmore VA, Carr JM, Sakamoto K, Hajduch E, Arthur JS. Insulin- Stimulated Glucose Uptake Does Not Require p. 38 Mitogen-Activated Protein Kinase in Adipose Tissue or Skeletal Muscle. Diabetes 2005;54:3161-8.
|
| 3. | Gogte VM. Part II: Medicinal plants. In: Ramakrishnan S, editor. Ayurvedic Pharmacology and Therapeutics Uses of Medicinal plants (Dravyagunavigyan). 1 st ed. Mumbai: Bharatiya Vidya Bhavan Publication; 2000. p. 309-11.
|
| 4. | Gogte VM. Part II: Medicinal plants. In: Ramakrishnan S, editor. Ayurvedic Pharmacology and Therapeutics Uses of Medicinal plants (Dravyagunavigyan). 1 st ed. Mumbai: Bharatiya Vidya Bhavan Publication; 2000. p. 359-61.
|
| 5. | Gogte VM. Part II: Medicinal plants. In: Ramakrishnan S, editor. Ayurvedic Pharmacology and Therapeutics Uses of Medicinal plants (Dravyagunavigyan). 1 st ed. Mumbai: Bharatiya Vidya Bhavan Publication; 2000. p. 514-5.
|
| 6. | Rao TP, Sakaguchi N, Juneja LR, Wada E, Yokozawa T. Amla (Emblica officinalis Gaertn.) extracts reduce oxidative stress in streptozotocin-induced diabetic rats. J Med Food 2005;8:362-8.
[PUBMED] |
| 7. | Babu PS, Stanely M, Prince P. Anti-hyperglycaemic and antioxidant effect of hyponidd, an ayurvedic herbomineral formulation in streptozotocin-induced diabetic rats. J Pharm Pharmacol 2004;56:1435-42.
|
| 8. | Mehta S, Singh RK, Jaiswal D, Rai PK, Watal G. Anti-diabetic activity of Emblica officinal is in animal models. Pharmaceu Biol 2009;47:1050-5.
|
| 9. | Qureshi SA, Asad W, Sultana V. The Effect of Phyllantus emblica Linn on Type - II Diabetes, Triglycerides and Liver - Specific Enzyme. Pak J Nutr 2009;8:125-8.
|
| 10. | Suryanarayan P, Saraswat M, Petrash JM, Reddy GB. Emblica officinal is and its enriched tannoids delay streptozotocin-induced diabetic cataract in rats. Mol Vis 2007;24:1291-7.
|
| 11. | Suryanarayana P, Kumar PA, Saraswat M, Petrash JM, Reddy GB. Inhibition of aldose reductase by tannoid principles of Emblica officinalis: Implications for the prevention of sugar cataract. Mol Vis 2004;12:148-54.
|
| 12. | Nwozo S, Adaramoye O, Ajaiyeoba E .Oral Administration of Extract from Curcuma longa Lowers Blood Glucose and Attenuates Alloxan-Induced Hyperlipidemia in Diabetic Rabbits. Pak J of Nutrition 2009;8:625-8.
|
| 13. | Arun N, Nalini N. Efficacy of turmeric on blood sugar and polyol pathway in diabetic albino rats. Plant Foods Hum Nutr 2002;57:41-52.
[PUBMED] |
| 14. | Halim EM, Hussain A. Hypoglycemic, Hypolipidemic and Antioxidant properties of combination of Curcumin from Curcuma Longa, Linn, and partially purified product from Abroma Augusta, Linn. in Streptozotocin induced Diabetes. Ind J Clin Biochem 2002;17:33-43.
|
| 15. | Stanely M, Prince P, Menon VP. Antioxidant action of Tinospora cordifolia root extract in alloxan diabetic rats. Phytother Res 2001;15:213-8.
|
| 16. | Stanely M, Prince P, Menon VP, Gunasekaran G. Hypolipidaemic action of Tinospora cordifolia roots in alloxan diabetic rats. J Ethnopharmacol 1999;64:53-7.
|
| 17. | Stanely M, Prince P, Menon VP. Hypoglycaemic and other related actions of Tinospora cordifolia roots in alloxan-induced diabetic rats. J Ethnopharmacol 2000;70:9-15.
|
| 18. | Gupta SS, Varma SC, Garg VP, Rai M. Anti-diabetic effect of Tinospora cordifolia.I. Effect on fasting blood sugar level, glucose tolerance and adrenaline induced hyperglycemia. Indian J Med Res 1967;55:733-45.
|
| 19. | Rathi SS, Grover JK, Vikrant V, Biswas NR. Prevention of experimental diabetic cataract by Indian Ayurvedic plant extracts. Phyto Res 2002;16:774-7.
[PUBMED] |
| 20. | Knutson VP, Balba Y. 3T3-L1 Adipocytes as a cell culture model of Insulin resistance. In Vitro Cell Dev Biol Anim 1997;33:77-81.
[PUBMED] |
| 21. | Saltiel AR, Kahn CR. Insulin signaling and the regulation of glucose and lipid metabolism. Nature 2001;414:799-806.
[PUBMED] |
| 22. | Liu F, Kim J, Li Y, Liu X, Li J,Chen X. An Extract of Lagerstroemia speciosa L. has Insulin-like glucose uptake-stimulatory and adipocyte differentiation-inhibitory activities in 3T3-L1 Cells. J Nutri 2001;131:2242-7.
[PUBMED] |
| 23. | Kang Y, Kim HY. Glucose uptake-stimulatory activity of Amomi Semen in 3T3-L1 adipocytes. J Ethnopharmacol 2004;92:103-5.
[PUBMED] |
| 24. | Hanks JH, Wallace JH. Determination of cell viability. Proc Soc Exp Biol Med 1958;98:188-92.
[PUBMED] |
| 25. | Kreutter DK, Andrews KM, Gibbs EM, Hutson NJ, Stevenson RW. Insulin like activity of new anti-diabetic agent CP 68722 in 3T3-L1 adipocytes. Diabetes 1990;39:1414-9.
[PUBMED] |
| 26. | Joladarashi D, Chilkunda ND, Salimath PV. Glucose uptake-stimulatory activity of Tinospora cordifolia stem extracts in Ehrlich ascites tumor cell model system. J Food Sci Technol 2011 [In Press].
|
[Figure 1], [Figure 2]
| This article has been cited by | | 1 |
Tinospora cordifolia (Willd.) Miers: Protection mechanisms and strategies against oxidative stress-related diseases |
|
| Karuppusamy Arunachalam, Xuefei Yang, Thae Thae San | | Journal of Ethnopharmacology. 2022; 283: 114540 | | [Pubmed] | [DOI] | | | 2 |
Advances in biotechnology of Emblica officinalis Gaertn. syn. Phyllanthus emblica L.: a nutraceuticals-rich fruit tree with multifaceted ethnomedicinal uses |
|
| Saikat Gantait, Manisha Mahanta, Soumen Bera, Sandeep Kumar Verma | | 3 Biotech. 2021; 11(2) | | [Pubmed] | [DOI] | | | 3 |
Medicinal plants utilized in Thai Traditional Medicine for diabetes treatment: Ethnobotanical surveys, scientific evidence and phytochemicals |
|
| Catarina Andrade, Nelson G.M. Gomes, Sutsawat Duangsrisai, Paula B. Andrade, David M. Pereira, Patrķcia Valentćo | | Journal of Ethnopharmacology. 2020; 263: 113177 | | [Pubmed] | [DOI] | | | 4 |
Polypharmacy in diabetes: A boon or a bane |
|
| Geetika Pahuja, Prashant Kumar, Shivani Ghildiyal, Tanuja Nesari | | Indian Journal of Health Sciences and Biomedical Research (KLEU). 2020; 13(1): 11 | | [Pubmed] | [DOI] | | | 5 |
Comprehensive Pharmacology Review of Guduchi [Tinospora cordifolia (Willd.) Miers] |
|
| Lalrinpuia Kawlni, Sachchidanand Upadhayay, Koyel Mukherjee | | Journal of Drug Research in Ayurvedic Sciences. 2018; 3(1): 48 | | [Pubmed] | [DOI] | | | 6 |
Anti-diabetic effects of the Indian indigenous fruit Emblica officinalis Gaertn: active constituents and modes of action |
|
| Jason Jerome Dęsouza,Prema Pancy Dęsouza,Farhan Fazal,Ashish Kumar,Harshith P. Bhat,Manjeshwar Shrinath Baliga | | Food & Function. 2014; 5(4): 635 | | [Pubmed] | [DOI] | | | 7 |
An in vitro study reveals the nutraceutical potential of punicic acid relevant to diabetes via enhanced GLUT4 expression and adiponectin secretion |
|
| S. S. Anusree,A. Priyanka,V. M. Nisha,Arya A. Das,K. G. Raghu | | Food Funct.. 2014; 5(10): 2590 | | [Pubmed] | [DOI] | |
|
|
|
|
|
|
|
|
