IJCRR - 10(10), May, 2018
Date of Publication: 30-May-2018
Download XML Download PDF
Effect of Terminalia chebula and Gallic Acid on Increased Adiposity of High-Fat Diet Induced Hyperlipidemic Mice
Author: Sarada S., Padmini E.
Category: Life Sciences
Abstract: Objective: To elucidate the role of Terminalia chebula and Gallic Acid on increased adiposity and as a regulator of Lipogenesis and its associated factors in high-fat diet induced hyperlipidemic mice.
Methods: The study was performed on C57BL/6J Male mice aged 6-8 weeks. Control was fed with normal diet and the other 3 groups were fed with commercial hyperlipidemic diet. Adipose tissue markers Peroxisome proliferator-activated receptorgamma, CCAAT/enhancer-binding proteins-beta, 4-Hydroxy-2-nonenal, Thioredoxin, Tumour necrosis factor-?, adiponectin and
leptin were estimated using adipose tissue by ELISA.
Results: The diet-induced hyperlipidemic C57BL/6J Male mice showed a marked reduction in the levels of transcription factors, oxidative stress markers and pro-inflammatory markers when treated with Terminalia chebula (Peroxisome proliferator-activated receptor-gamma (P< 0.001), CCAAT/enhancer-bindingproteins-beta (P< 0.001), 4-Hydroxy-2-nonenol (P< 0.001), Thioredoxin (P< 0.01) and Tumour necrosis factor-? (P< 0.001) which was more effective than Gallic Acid (Peroxisome proliferator-activated receptor-gamma (0.05), CCAAT// enhancer-binding proteins-beta (P< 0.01), 4-Hydroxy-2-nonenal (P< 0.01), Thioredoxin (P< 0.05) and Tumour necrosis factor-? (P< 0.01) and an elevation in adipokines- adiponectin and leptin in Terminalia chebula treated group- adiponectin (P< 0.001) and leptin (P< 0.001) was seen which was more significant than Gallic Acid- adiponectin (P< 0.01) and leptin (0.05) when compared to group II HFD.
Conclusion: The study indicates the efficacy of Terminalia chebula as a potent antihyperlipidemic herbal drug which helps in controlling the increased adiposity by regulating the key markers in adipose tissue.
Keywords: Terminalia chebula, Hyperlipidemia, Adiposity, Gallic Acid
Hyperlipidemia is a metabolic syndrome which includes lipid abnormalities (1,2). Regular intake of high-fat diet can result in hyperlipidemia (3). Hyperlipidemia is associated with an increase in the number of adipocytes due to enhanced adipogenesis. Adipose tissue is an important organ involved in dynamic regulation of metabolism in living body (4), our present study monitored the biochemical changes in adipose tissue by examining the expression of 4-Hydroxy-2-nonenal, Thioredoxin, adipogenic transcriptional factors, and adipokines. The important transcriptional factors Peroxisome proliferator-activated receptor-gamma and CCAAT/enhancer-binding proteins-beta required for adipogenesis and lipid metabolism are up-regulated in individuals whose diet composed of fatty foods (5,6) demanding the need for their screening during Hyperlipidemia. 4-Hydroxy-2-nonenal (HNE) an, α β-unsaturated aldehyde generated due to the lipid peroxidation (7) may alter the expression of antioxidants. One of the antioxidant playing a vital role in regulating redox status is Thioredoxin (8). Adipokines are peptides produced by adipose tissue involved in the regulation of glucose and lipid metabolism (9). Since adipokines were bound to fluctuate in the hyperlipidemic condition associated with adiposity (10,11), the expression of pro-inflammatory adipokine such as leptin, tumor necrosis factor-alpha, and anti-inflammatory adipokine- adiponectin was observed in adipose tissue
In the present study, Gallic acid and Terminalia chebula were used for assessing their effect during hyperlipidemia. Gallic acid is 3, 4, 5, - trihydroxybenzoic acid, used for the treatment of a vast number of diseases such as cancer, cardiovascular diseases, ulcers, arthritis, gout etc. It is found to be antihyperglycemic, antihyperlipidemic, antioxidant, and cardioprotective (12). Terminalia chebula is a common herbaceous plant used in ayurvedic preparations as it possesses diverse medicinal properties which include antimicrobial, antioxidant, antiviral, anticarcinogenic, antihyperlipidemic, etc. Ellagic acid, gallic acid, chebulinic acid, chebugalic acid and corilagin present in this fruit have a beneficial effect in the treatment of hyperlipidemia (13,14).
MATERIALS AND METHODS
Preparation of T. chebula aqueous powder :
Terminalia chebula [Haritaki] dried fruits were collected from the Deciduous forest area in Kombankaddu area [Kodaikanal to Palani route]. It was authenticated at CAPTAIN SRINIVASA MURTI RESEARCH INSTITUTE OF AYURVEDA AND SIDDHA DRUG DEVELOPMENT, Arignar Anna Government Hospital Campus, Arunbakkam, Chennai, Tamil Nadu. Seeds from the individual fruit were removed, washed thoroughly and then air-dried on a drier table at room temperature. Then the dried pulp crushed in an electrical mixer-grinder into a coarse powder. The powder was stored in a closed vessel for future use.
Mice -C57BL/6J Male aged 6-8 weeks obtained from CPCSEA approved Breeder were taken for this experiment and kept at Centre for Toxicology and Developmental Research (CEFT) Sri Ramachandra University (SRU). All animal experiments were conducted as per the instructions of Institutional Animal Ethics Committee (IAEC/XLVIII/SRU/495/2016).
Animals were randomised and grouped based on the stratified body weight.
Induction of hyperlipidemia
Hyperlipidemia was induced to all the animals (GII – GIV) by giving commercially procured High fat diet (HFD) for a period of 15 weeks except Group I. Group I animals received standard rodent pellet diet for 15 weeks.
Solubility / Suspendability, Stability, and Homogeneity of Formulation
Solubility / Suspendability of the test and reference item (Gallic acid and Terminalia chebula) were ensured one day before dosing. 0.5% CMC Vehicle was selected based on the nature of test and reference drug. 0.5% CMC as a vehicle used for the preparation of test and reference drug. The homogeneity of test item in 0.5 % CMC was ensured using glass rod while dosing.
Liver, adipose were collected from all animals of the all dose groups and preserved in 10% neutral buffered formalin. The fixed tissues were processed, embedded in paraffin wax, sectioned approximately at 3-5 microns thick and stained with Haematoxylin and eosin for histopathological examination.
200 mg of adipose tissue was homogenized in standard homogenization buffer with anti-proteases (20 mM Tris-HCl, 1 mM EDTA, 255 mM sucrose, pH 7.4, anti-protease). The homogenates were centrifuged at 1,000 g for 10 min and the subnatant (whole tissue extract) below the lipid cake was aspirated and denatured by adding SDS to a final concentration of 0.06% and boiling for 10 min at 100ºC. Total protein concentrations of the tissue extracts were measured using the method of Bradford (1976). Based on the requirement, the sample was diluted to acquire 100 µg of protein by using PBS buffer and used for the ELISA.
PPAR-γ was quantified using ELISA kit (MBS005886, MyBioSource, USA)
C/EBP-β was quantified using ELISA kit (MBS006925, MyBioSource, USA)
Leptin was quantified using Leptin ELISA kit (11-LEPHU-E01, ALPCO, India)
Adiponectin was quantified using Total Adiponectin ELISA kit (47-ADPHUT-E01, ALPCO, India)
Tumour necrosis factor-α was quantified using ELISA kit (K0331131, Koma Biotech, Korea)
4-Hydroxy-2-nonenal was quantified using 4 hydroxy nonenol ELISA kit (MBS161454 96T, My Biosource, USA)
Thioredoxin was quantified using Trx ELISA kit (MBS009625, MyBioSource, USA).
The results were expressed as mean value standard deviation. Statistical analysis of the data was carried out using SPSS software. Statistical significance was arrived by comparing results of One Way ANOVA of Group I and II with III and IV.
Histopathology of adipose tissue of Group I (Control) showed the normal size of adipose cells. However, the adipose of Group II (HFD) showed marked hypertrophy of adipose cells with a minimal degree of severity of inflammatory cell infiltration in crown shaped structure between adipose cells. In Group III (HFD + Gallic acid treatment group) Histopathology of adipose tissue revealed decrease in the size of adipose cells and no infiltration in crown shaped structure when compared to high-fat diet (G2) group animals and Group IV (HFD + Terminalia chebula treatment group) revealed normalization of adipose cells when compared with normal control group animals.
The remarkable decrease in severity and incidence of lesions in adipose tissues of animals was observed in Group 4 animals as compared to the high-fat diet (G2) and gallic acid (G3) treated animals.
Transcription Factors in Adipose
From the ELISA studies conducted on the adipocytes of all the 4 groups the levels of Peroxisome proliferator-activated receptor-gamma and CCAAT/enhancer-binding proteins-beta were found to be significantly increased in Group compared to Group I. The levels of Peroxisome proliferator-activated receptor-gamma and CCAAT/enhancer-binding proteins-beta were found to decrease significantly compared to HFD in Group IV (HFD +Terminalia chebula ) than the Group III (HFD + Gallic Acid ) ( Table I).
From the Elisa studies conducted on the adipocytes of all groups, the levels of Adiponectin and Leptin were found to be normal in Group I which was remarkably reduced in Group II (HFD). On receiving treatment the levels of Adiponectin and Leptin were found to increase considerably compared to HFD in Group IV (HFD +Terminalia chebula) than the Group III (HFD + Gallic Acid). However the levels of Tumour necrosis Factor -α which was found to be less in Group I (Normal) and increased in Group II (HFD) decreased remarkably in Group IV (HFD +Terminalia chebula ) than the Group III (HFD + Gallic Acid ) as compared to HFD. (Table II)
Oxidative stress in Adipose Tissue
The ELISA studies conducted on the adipocytes of 4 groups revealed normal levels of 4-Hydroxy-2-nonenal and Thioredoxin in Group I (Normal) which was notably increased in Group II (HFD). On treatment, the levels of 4-Hydroxy-2-nonenal and Thioredoxin were found to decrease appreciably in Group IV (HFD +Terminalia chebula ) than the Group III (HFD + Gallic Acid )( Table III).
Adipose tissue is an important organ where excess fat in our body is stored. It is a dynamic organ that plays an important role in energy balance and homeostasis of our body. It acts as a sensor for lipid levels in our body and controls hunger, satiety and sleep patterns. Adipocytes are considered a major endocrine organ as they secrete lipid and protein factors which produce an impact on the metabolism of other tissues, regulation of appetite, immunological responses and vascular disease etc (15). The development of hyperlipidemia as a result of High fat diets (HFD) lead to an expansion of adipose tissue along with increase in size and number. The Adipose tissue inflammation is an important marker for hyperlipidemia. Our study observed changes in the structure of adipocyte in group II (HFD) mice which shows marked hypertrophy of adipose cells with minimal degree of inflammatory cell infiltration in crown shaped structure in the histopathological report of the adipose tissue. Herbal treatment by Terminalia chebula has resulted in decrease in hypertrophy of adipose tissue as assimilation of fat is decreased in the body. The results are more predominant than Group III –Treatment with pure compound Gallic as Terminalia chebula is rich in fibre which reduces lipid absorption in the small intestine. Adipose tissue is composed of more of fatty acids and phospholipids and less of triglyceride in the form of neural fat. The presence of redox systems in microsomes in the adipose tissue results in high activity of lipid peroxidation (16). Oxidative stress and inflammation go hand-in-hand in the many tissues that are affected because oxidative stress induces the production of inflammatory cytokines, and the cytokines in turn induce free radical production. Lipid peroxidation (LP), an autocatalytic process is initiated by free radical attack on the unsaturated bonds of membrane fatty acids which leads to 4-Hydroxy-2-nonenal . In our study the levels of 4-Hydroxy-2-nonenal were found to increase significantly in Group II (HFD) as compared to Group I (Normal diet) as a result of high incidence of lipid peroxidation due to hyperlipidemia. The increase in HNE levels indicates damage to adipose tissue which is confirmed by the histopathological report (17). The levels of 4-Hydroxy-2-nonenal were found to be reduced in group IV (HFD+ Terminalia chebula) as inflammation and oxidative stress is found reduced by the herbal drug. ( Table III) The results are far more appreciable than treatment with Gallic acid as Terminalia chebula is a better drug in controlling fat assimilation in the adipose ( evident from histopathological reports and Fig 6). Thioredoxin (TRX) is a redox protein that is involved in many biological functions which controls a number of transcription factors (18). Increased lipid peroxidation associated with increase in the levels of LDL results in the formation of oxLDL. This oxLDL further aggravates the problem by increasing its uptake by macrophages in adipocytes. This increases Thioredoxin expression and so the levels of Thioredoxin were found to be increased by the consumption of high fat diet as compared to mice that was given normal diet (Table III). The treatment by the herbal drug reduced the levels of LDL and lipid peroxidation causing decrease in the levels of Thioredoxin and this effect was more pronounced than treatment with Gallic Acid, pure compound suggesting that Terminalia chebula is a better drug in treating oxidative stress and its associated complications caused by hyperlipidemia (19) (Table III and Fig 7). Further increase in the levels of lipids leads to increased Adipogenesis. Fat cell formation or adipogenesis is a process characterised by changes in factors that determine the structure of adipocyte (20). It is a process in which preadipocytes develop into mature adipocytes. This is a multistep process controlled by various transcriptional factors (15). The two factors that have a pronounced effect on this synthesis are Peroxisome proliferator-activated receptor-gamma and CCAAT/enhancer-binding proteins-beta (21). CCAAT/enhancer-binding proteins-beta genes are rapidly induced to express which then activate Peroxisome proliferator-activated receptor-gamma (22). Peroxisome proliferator-activated receptor-gamma can initiate adipogenesis program, giving rise to fat cells by activating the adipogenic genes responsible for adipogenesis (23). In our study increased levels of both Peroxisome proliferator-activated receptor-gamma and CCAAT/enhancer-binding proteins-beta were observed in group II( HFD) compared to group I (Normal diet) which has lead to increased adipogenesis evident from the histopathological studies which indicate increase in fat depots of adipose tissue (Table I and Fig 1 and 2) . The adipokines which keep the adipogenesis at check by reducing lipogenesis and favouring lipolysis are leptin and adiponectin which are drastically reduced in hyperlipidemia (24) (Table II). The deficiency of leptin or leptin receptor is seen in animals fed with high fat diet which leads to extreme obesity (25) (Fig 3). Adiponectin is a peptic hormone which decreases serum FFA, glucose, and triacylglycerol concentrations in the adipose tissue (26). However adiponectin concentrations fall with increasing obesity which was observed in group II (HFD) mice (Table II, Fig 4) which indicated both decreased adiponectin and leptin levels. The herbal drug Terminalia chebula is more efficient than Gallic Acid in increasing the levels of both adiponectin and leptin which is evident from their levels in group III (HFD+GA) and group IV (HFD+TC) and thereby decreasing adipogenesity. ( Fig 3 and 4) However, the Tumor necrosis factor-alpha (TNF alpha) a multifunctional cytokine which exerts a series of biological actions on adipose and regulates or interfere with adipocyte metabolism by regulating fatty acid metabolism (27) is downregulated more effectively by Terminalia chebula than pure compound Gallic Acid. (Table II and Fig 5). Our study indicates that herbal treatment with Terminalia chebula brings about a reduction in fat content of adipose as it reduces adipogenesis by down-regulating the transcription factors, Tumour necrosis factor α, and oxidative stress markers and brings about lipid breakdown or lipolysis by increased production of adiponectin and leptin more effective than pure compound Gallic Acid.
These results suggest that Terminalia chebula could decrease adipose tissue mass by down-regulation of transcriptional factors- Peroxisome proliferator-activated receptor-gamma, CCAAT/enhancer-binding proteins-beta, Pro-inflammatory cytokine-TNF α, Oxidative stress markers - 4-Hydroxy-2-nonenal, Thioredoxin and upregulation of adipokines that favor lipolysis - adiponectin and leptin. This effect has been far more pronounced than group III mice treated with Pure compound Gallic Acid (50 mg-Kg body wt)). Further, to confirm the anti-adipogenic activity of Terminalia chebula more effective than Gallic Acid by histopathological examination of adipose tissue. The following results prove Terminalia chebula a more potent antihyperlipidemic factor than pure compound Gallic Acid.
The authors acknowledge the immense help received from the scholars whose article cited and included in references of this manuscript. The authors are also grateful to authors/editors/publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed. Authors acknowledge the immense help received from the scholars whose articles are cited and included in references of this manuscript. The authors are also grateful to authors / editors / publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed.
Conflict of interest : nil
Financial Support : nil
Ethical Clearance : (IAEC/XLVIII/SRU/495/2016)
Asmare Amuamuta Bahir Dar, 2014, A Review on Risk Factors/Indicators and Effects of Hyperlipidemia. Middle-East Journal of Scientific Research 22 (6): 886-893.
Bragg DA, Walling A , 2015, Metabolic Syndrome: Hyperlipidemia. FP Essent.;435:17-23.
Jong Weon Choi, 2003, Association Between Elevated Serum Hepatic Enzyme Activity and Total Body Fat in Obese Humans. Ann Clin Lab Sci Summer vol. 33 no. 3 257-264.
Liang Guo, Xi Li, and Qi-Qun Tang, 2015, Transcriptional Regulation of Adipocyte Differentiation: A Central Role for CCAAT/Enhancer-binding Protein (C/EBP) β. J Biol Chem. 2015 Jan 9; 290(2): 755–761
Gretchen J. Darlington et al, 1998, The ROLE of C/EBP Genes in Adipocyte Differentiation .. J Biol Chem. 13; 273(46):30057-60.
Martina I. Lefterova et al, 2008, PPARγ and C/EBP factors orchestrate adipocyte biology via adjacent binding on a genome-wide scale. Genes Dev. 22(21): 2941–2952.
Kalavathi Dasuri et al, 2013, Role of Physiological Levels of 4-Hydroxynonenal on Adipocyte Biology: Implications for Obesity and Metabolic Syndrome. Free Radic Res. 47(1):8–19.
Betul Catalgol and Nesrin Kartal Ozer, 2010, Lipid Rafts and Redox Regulation of Cellular Signaling in Cholesterol Induced Atherosclerosis. Curr Cardiol Rev, 6(4): 309-324.
Benson T. Akingbemi , 2013, G Protein-Coupled Receptors in Energy Homeostasis and Obesity Pathogenesis. Progress in Molecular Biology and Translational Science Volume 114, Pages 317-342
William P. Cawthorn and Jaswinder K. Sethi, 2008, TNF-α and adipocyte biology. FEBS Lett. 582(1): 117-131.
Sanet van Zyla et al, 2017, The relationship between obesity, leptin, adiponectin and the components of metabolic syndrome in urban African women, Free State, South Africa. South African Journal of Clinical Nutrition 30(3):68–73.
Snehal S. Patel and Ramesh K. Goyal , 2011, Cardioprotective effects of gallic acid in diabetes-induced myocardial dysfunction in rats. Pharmacognosy Res 3(4): 239–245.
Rubaiat Nazneen Akhand et al , 2013 , Sub-chronic oral administration of the ethanolic extracts of dried Terminalia chebula mature fruits in streptozotocin (STZ)-induced type 2 diabetes mellitus (T2DM) model of Long-Evans (L-E) rats improve glycemic, lipidemic and anti-oxidative status. Journal of Applied Pharmaceutical Science Vol. 3 (05), pp. 027-032.
Priya F, Velpandian V, Ayyasamy S, Pitchiahkumar M , 2013 , Hypolipidemic activity of Kadukkai Chooranam (Terminalia chebula) in Triton WR - 1339 induced hyperlipidemic rats. Journal of Pharmacy and Biological Sciences (IOSR-JPBS) Volume 5, Issue 5 PP 77-84.
Aus TariqAli , 2013 ,Adipocyte and adipogenesis, Volume 92, Issues 6–7, Pages 229-236.
Hai-LuZhao et al, 2008, Fat redistribution and adipocyte transformation in uninephrectomized rats. Kidney International Volume 74, Issue 4, 2 August 2008, Pages 467-477.
Mark P. Mattson, 2009, Roles of the Lipid Peroxidation Product 4-Hydroxynonenal in Obesity, the Metabolic Syndrome, and Associated Vascular and Neurodegenerative Disorders. Exp Gerontol. 44(10):625–633.
Song JS, 2011, Role of thioredoxin 1 and thioredoxin 2 on proliferation of human adipose tissue-derived mesenchymal stem cells. Stem Cells Dev. 2011 Sep;20(9):1529-37.
Betul Catalgol and Nesrin Kartal Ozer, 2010, Lipid Rafts and Redox Regulation of Cellular Signaling in Cholesterol Induced Atherosclerosis. Curr Cardiol Rev, 6(4): 309–324.
S. Niemela, S. Miettinen, J.R. Sarkanen and N. Ashammakh, 2008, Adipose Tissue and Adipocyte Differentiation: Molecular and Cellular Aspects and Tissue Engineering and applications. Topics in Tissue Engineering, Vol. 4. Eds. N.
Farmer SR , 2005, regulation of ppargamma activity during adipogenesis. Int J Obes (Lond).; 29 Suppl 1:S13-6.
Hishida T et al , 2009, The role of C/EBPdelta in the early stages of adipogenesis.. Biochimie.;91(5):654-7.
Stephen R. Farmer, 2006, Transcriptional control of adipocyte formation. Cell Metab. 2006 Oct; 4(4): 263–273.
MatthiasBluhe, 2014, Adipokines – removing road blocks to obesity and diabetes therapy. Molecular Metabolism Volume 3, Issue 3, Pages 230-240.
Wasim M, 2015, Role of Leptin in Obesity. J Obes Weight Loss Ther 5:258. doi:10.4172/2165-7904.1000258
Andrew S and Greenberg Martin S Obin, 2006, Obesity and the role of adipose tissue in inflammation and metabolism. The American Journal of Clinical Nutrition, Volume 83, Issue 2, 1 February 2006, Pages 461S-465S.