{"id":60387,"date":"2024-09-30T11:02:40","date_gmt":"2024-09-30T11:02:40","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=60387"},"modified":"2024-10-09T18:23:44","modified_gmt":"2024-10-09T18:23:44","slug":"role-of-hydroalcoholic-extracts-of-aegle-marmelos-am-leaves-and-tamarindus-indica-ti-seeds-on-high-fat-high-sugar-diet-induced-hyperlipidemia-in-rodent-models","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no3\/role-of-hydroalcoholic-extracts-of-aegle-marmelos-am-leaves-and-tamarindus-indica-ti-seeds-on-high-fat-high-sugar-diet-induced-hyperlipidemia-in-rodent-models\/","title":{"rendered":"Role of Hydroalcoholic Extracts of Aegle marmelos (AM) Leaves and Tamarindus indica (TI) Seeds on High Fat High Sugar Diet Induced Hyperlipidemia in Rodent Models"},"content":{"rendered":"\n

Introduction <\/strong><\/p>\n\n\n\n

Mortality due to cardiovascular disorders is an important\nhealth related problem in both developing and developed countries. Hyperlipidemia,\na prime cause of atherosclerosis, is\nassociated with an increase in plasma levels of cholesterol, triglycerides and\nvarious lipoproteins1<\/sup>. The exact cause and relation of\nhyperlipidemia and atherosclerosis is quite complex –  may be primary (genetic) or secondary\n(environmental or secondary to diabetes)2<\/sup>. Many\ndrugs have been used for treating hyperlipidemia. At present, five classes of\nhypolipidemic drugs are used for treatment of hyperlipidemia. They are HMG COA\nreductase (3-Hydroxy-3-methylglutaryl\u2013coenzyme A) inhibitors,\nbile acid sequestrants, fibric acid derivatives (fibrates), nicotinic acid\nderivatives (niacin) and selective cholesterol absorption inhibitor (ezetimibe).\nThe success of these drugs is limited on long term use as they are associated\nwith serious side effects like myopathy, osteoporosis, renal damage, gall\nstones etc1,3<\/sup>.\nHence, there is a need for drugs, which can be used effectively as\nhypolipidemics with minimum side effects. <\/p>\n\n\n\n

Use of plants for the treatment of various\ndiseases has been practiced since Vedic era. Currently, worldwide, around 70%\nof the population is dependent on herbal formulation for\ntheir health issues4<\/sup>. Herbal formulations\nhave gained importance around the globe owing to their lower adverse effects\nand cost effectiveness. Those\ncontaining more than one plant extract are being increasingly used for the\ntreatment of various diseases. They contain multiple phytoconstituents\nwhich may provide synergistic action for the management of diseases as compared\nto a single herb which may have less quantity of active principles. Such\npolyherbal formulations (PHF)\nare an important mode of therapy in Ayurveda, Chinese and Siddha\nsystem of medicine5<\/sup>.<\/p>\n\n\n\n

Aegle marmelos <\/em>(Family-Rutaceae<\/em>), commonly called as \u2018\u2018Bael\u2019\u2019 in Hindi and “Bilva”in Kannada, is indigenous to India. All parts of Aegle marmelos <\/em>is of medicinal importance. Leaves have antidiabetic, hypolipidemic, hepatoprotective, anticancer, mild laxative, antifungal, antibacterial, cardioprotective, chemoprotective, radioprotective, anti-oxidant, anti-spermatogenic and antithyroid activities. They have been used for asthma, fever, jaundice, ophthalmia, bronchitis, ulcers, abdominal disorders, vomiting, beriberi, cholera and diarrhea6-7<\/sup>. Tamarindus indica (family-Caesalpiniacae), <\/em>commonly called \u2018\u2018Imli\u2019\u2019 in Hindi and \u201cHunase Hannu \/ Huliis\u201d, in Kannada is found throughout India. The fruit of this tree has dark brown pods which is almost the size of a human finger, with soft sticky sour pulp containing up to 10 seeds per pod. These seeds have biologically active compounds with antidiabetic, hypolipidemic, hepatoprotective, antimalarial, antimicrobial, antioxidant, antiasthmatic, laxative, and antivenomic activities8\u201311<\/sup>. <\/p>\n\n\n\n

Aegle\nmarmelos <\/em>(AM)and Tamarindus\nindica <\/em>(TI)are\ntree-type plants which are abundantly found all over India.\nIt has been reported that the aqueous and alcoholic extract of Aegle marmelos<\/em> leaves and Tamarindus indica<\/em>\nseeds have individually shown antihyperglycemic\nand hypolipidemic effects6,12\u201318<\/sup>. However,\nthere are no reports on hypolipidemic effect of hydroalcoholic extract of AM\nleaves and TI seeds. Therefore, this study was designed to\ncheck the hypolipidemic activity of these hydroalcoholic extracts,\nalone and in combination, in different doses on High Fat High Sugar (Fructose) Diet (HFHSD) induced hyperlipidemia in\nrats.<\/p>\n\n\n\n

Materials and Methods<\/strong><\/p>\n\n\n\n

Preparation\nof hydroalcoholic extract <\/strong><\/strong><\/p>\n\n\n\n

Mature leaves of Aegle marmelos <\/em>and seeds of Tamarindus indica <\/em>were collected from Udupi district of Karnataka. The identification of Aegle marmelos<\/em> (family Rutaceae)<\/em> and Tamarindus indica (family Caesalpiniacae) <\/em>was carried out by the faculty of Department of Pharmacognosy, Manipal College of Pharmaceutical Sciences, Manipal. The materials of each plant were cleaned, dried, powdered and extraction was done separately. 100 g powdered extract of each i.e Aegle marmelos <\/em>leaves and Tamarindus indica<\/em> seeds were subjected to maceration for 7 days at room temperature using 500 mL ethanol and 500 mL distilled water (50:50 proportion). The extracts were filtered, solvent was evaporated under vacuum and further dried on water bath following which a paste residue of Aegle marmelos <\/em>leaves andpowdered residue of Tamarindus indica <\/em>seeds were obtained8,19<\/sup>.<\/p>\n\n\n\n

High-Fat and High- Sugar (Fructose) Diet (HFHSD)<\/strong><\/p>\n\n\n\n

High fat diet (HFD) cakes were prepared by mixing coconut oil (30 % v\/w), cholesterol (2% w\/w) and cholic acid (2 % w\/w) with powdered standard rat feed. Each HFD cake weighed 20 g. High sugar diet consisted of 25% fructose in drinking water20<\/sup>.<\/p>\n\n\n\n

Animals<\/strong><\/p>\n\n\n\n

Adult male Sprague Dawley<\/em> rats (n = 54) were used for the study conducted at Central Animal Research Facility Manipal, India. The experiment was conducted following the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA)21<\/sup>. Study approval was obtained from Institutional Animal Ethics Committee, Kasturba Medical College, Manipal; (IAEC\/KMC\/81\/2020). The animals were maintained in conducive environment (temperature 22\u2013240<\/sup>C, with 12–<\/strong>h light – dark cycle, 40\u201360% humidity, with a standard rat feed (VRK Nutritional solution, Pune, India) and water ad libitum.<\/em> Well acclimatized animals were used for the experiment. <\/p>\n\n\n\n

Out of 54 male Sprague Dawley <\/em>rats, six were in normal control group and received standard diet consisting of protein (22.85%), fat (4.55%), carbohydrate (63%), fiber (2.70%), phosphorus (0.665), total ash (5.10%) and moisture (8.20%) (VRK Nutritional solution, Pune, India) and water ad libitum<\/em> The remaining 48 rats were fed orally with HFHSD i.e., HFD as a 20 g cake combined with high sugar (25 % fructose in drinking water) for 1 month for inducing hyperlipidemia. Plasma cholesterol level above 200 mg\/dL on day 30 was an indicator for the induction of hyperlipidemia in the HFHSD fed rats. Animals were weighed on 0, 10th<\/sup>, 20th<\/sup> and 30th<\/sup> day and HFHSD dose was adjusted according to body weight22<\/sup>. <\/sup>The study was then continued from day 31 for next thirty days and rats were treated orally as follows – Group I, normal control (n=6), received standard diet. The 48 rats which had earlier received HFHSD were divided into 8 groups (n=6 in each). Group II- HFHSD control received standard rat diet, Group III -positive control (Niacin, 100 mg\/kg\/day),23<\/sup> Group IV – AM25 and Group V-AM50 (25 & 50mg\/kg\/day, respectively), Group VI TI25 and Group VII TI50 (25 and 50 mg\/kg\/day, respectively), Group VIII AM25+TI25 (25 mg\/kg\/day AM +25 mg\/kg\/day TI).and Group IX AM50+TI50 (50 mg\/kg\/day AM + 50mg\/kg\/day TI). Sodium carboxymethyl cellulose 1% (Na-CMC) was used as vehicle for administering the standard drug and extracts. Animals in all groups also received standard rat feed and water ad libitum<\/em>. <\/p>\n\n\n\n

After overnight fasting, blood samples were collected on day 61 by retro-orbital puncture from all animals and centrifuged at 4400 rpm for 15 minutes. Plasma triglycerides (TGs), total cholesterol, Low-Density Lipoprotein (LDL-cholesterol), Very Low-Density Lipoprotein (VLDL- cholesterol), High Density Lipoprotein (HDL-cholesterol) and random blood glucose were estimated as per the standard protocols given along with respective colorimetric kits (Aspen Laboratories, New Delhi, India). After collection of blood for biochemical investigations, rats were sacrificed. The liver was excised and rinsed with ice-cold saline and liver homogenates (10% w\/v) were prepared in cold potassium phosphate buffer (pH 7.4). The resulting supernatant were stored at -20\u02daC for the estimation of catalase (CAT) activity, malondialdehyde (MDA) and reduced glutathione (GSH) levels as per the standard protocols given along with respective colorimetric kits (Aspen Laboratories, New Delhi, India).<\/p>\n\n\n\n

Statistical analysis<\/strong><\/p>\n\n\n\n

 Results were analyzed using Statistical Package for the Social Sciences (SPSS version 20.0; SPSS Inc., Chicago, USA). Normally distributed data is expressed as mean \u00b1 standard error of mean and analyzed by one-way analysis of variance (ANOVA) followed by post hoc  Bonferroni test. Independent samples \u2018t\u2019 test was used to compare the body weight. A p value \u2264 0\u00b705 was considered as statistically significant.<\/p>\n\n\n\n

Results<\/strong><\/p>\n\n\n\n

Establishment of Hyperlipidemia Model <\/strong><\/p>\n\n\n\n\n\n
\"\"<\/td>\nFigure 1: Comparison of Plasma Cholesterol levels with Normal control vs HFHS induced hyperlipidemia among different groups (One-way ANOVA followed by post hoc test \u2013 Bonferroni).<\/strong>

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Figure 1. Shows the establishment of hyperlipidemia model (Plasma cholesterol level >200 mg\/dl), where in all groups except the normal control have significantly (p<0.0001) higher plasma cholesterol level induced by one month of HFHS diet.<\/p>\n\n\n\n

Effect of HFHS diet and treatment with extracts on body weight of rats (Figure 2A and 2B)<\/strong><\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 2A: Comparison of body weight (pretreatment) between HFHS vs NORmal control during induction Hyperlipidemia.<\/strong><\/p>\n

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Figure 2B: Comparison of Body weight in HFHS inducced hyperlipidemia among different groups followed by post hoc test \u2013 Bonferroni).<\/strong><\/p>\n

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Figure 2 A and 2B: depicts a significant increase in the body weight of rats fed with HFHS diet for 30 days. There was 25.89 % increase in the body weight in normal control while HFHS fed rats showed 89.76 % increase in body weight at the end of one month (figure 2A) (p<0.001). However, there was a significant reduction in body weight (figure 2B) in rats treated with extracts (AM25, AM50, TI25, TI50, AM25+TI25, AM50+TI50)   or niacin from day 31 to day 60. <\/p>\n\n\n\n

Effect of treatment with extracts and niacin on lipid profile of the HFHS fed rats (Figure 3A to 3E)<\/strong><\/p>\n\n\n\n

Figure 3A: Plasma cholesterol level in the HFHS group was significantly high (60.61%) in comparison to the normal control (p<0.001). Following treatment with extracts (AM25, AM50, TI25, TI50, AM25+TI25, AM50+TI50) or niacin, the plasma cholesterol level was significantly reduced (35%, 30%,39%, 33%, 40%, 50% and 51% respectively) (p<0.001).  In the groups which received combination of extracts (AM25+T125 and AM50+T150), the cholesterol levels were significantly lower as compared to the positive control group (Niacin, p<0.001). <\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 3A: Comparison of Plasma Cholesterol levels in  HFHS induced hyperlipidemia among different groups following treatment with extracts and Niacin(One-way ANOVA followed by post hoc test \u2013 Bonferroni).<\/strong><\/p>\n

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Figure 3B: Comparison of Plasma Triglyceride levels in  HFHS induced hyperlipidemia among different groups following treatment with extracts and Niacin(One-way ANOVA followed by post hoc test \u2013 Bonferroni).<\/strong><\/p>\n

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Figure 3C: Comparison of Plasma LDL levels  in HFHS induced hyperlipidemia among different groups following treatment with extracts and Niacin(One-way ANOVA followed by post hoc test \u2013 Bonferroni).<\/strong><\/p>\n

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Figure 3D: Comparison of Plasma VLDL levels in HFHS induced hyperlipidemia among different groups following treatment with extracts and Niacin (One-way ANOVA followed by post hoc test \u2013 Bonferroni).<\/strong><\/p>\n

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Figure. 3B to 3D: HFHS diet induced model of hyperlipidemia showed a significant increase in plasma TG levels (150.64%), LDL-cholesterol (231.09%) and VLDL- cholesterol (285%) as compared to normal control (p<0.001). However, following 30 days of treatment with extracts (AM25, AM50, TI25, TI50, AM25+TI25, AM50+TI50) or niacin, there was a significant decrease in TG, LDL and VLDL in all groups. <\/p>\n\n\n\n

Rats treated with high dose of combination of extracts (AM50+T150) had significantly lower triglycerides levels when compared to the other treatment groups (51.97%) (p<0.001).  <\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 3E: Comparison of Plasma HDL levels in HFHS induced hyperlipidemia among different groups following treatment with extracts and Niacin (One-way ANOVA followed by post hoc test \u2013 Bonferroni).<\/strong><\/p>\n

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Figure 3E: There was a significant decrease in the HDL levels (56.88%) in the HFHS group as compared with that of normal control (p<0.001). All the treated groups (Niacin, AM25, AM50, TI25, TI50, AM25+TI25 and AM50+TI50), showed significant increase in HDL-cholesterol (p<0.05), however, the higher dose in combination group i.e., AM50+T150 resulted in a significant increase as compared to positive control Niacin (p<0.05). <\/p>\n\n\n\n

Overall, the high dose combination of extracts (AM50+T150) significantly reduced plasma cholesterol and TGs but increased HDL levels as compared to treatment with niacin. <\/p>\n\n\n\n

Effect of treatment with extracts and niacin on random blood glucose level in HFHS fed rats (Figure:4)<\/strong><\/p>\n\n\n\n\n\n
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Figure 4: Comparison of Random Blood levels in HFHS induced hyperlipidemia among different groups following treatment with extracts and Niacin (One-way ANOVA followed by post hoc test \u2013 Bonferroni).<\/strong><\/p>\n

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Figure 4:\nThe HFHS diet induced an increase in random blood sugar (p<0.001) level as\ncompared to normal control group. Treatment with extracts (alone and in\ncombination) or niacin resulted in significant reduction of blood glucose\nlevels in comparison to HFHS diet treated animals (p<0.001).<\/p>\n\n\n\n

Effect of treatment with extracts and niacin on hepatic catalase activity, GSH levels and MDA levels in HFHS fed rats (Figure 5A to 5C)<\/strong><\/p>\n\n\n\n

Figure 5A & 5B: There was a significant reduction in hepatic catalase level and reduced GSH levels of the HFHS group (61.39%, 43.15% respectively) when compared with the normal control group (p<0.001). All the treatment groups (Niacin, AM25, AM50, TI25, TI50, AM25+TI25 and AM50+TI50) showed significant improvement in catalase levels (p<0.001) & reduced GSH levels (p<0.05) in comparison to HFHS group. <\/p>\n\n\n\n\n\n
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Figure 5A: Comparison of Catalase levels in HFHS induced hyperlipidemia among different groups following treatment with extracts and Niacin (One-way ANOVA followed by post hoc test \u2013 Bonferroni).<\/strong><\/p>\n

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Figure 5B: Comparison of GSH levels in HFHS induced hyperlipidemia among different groups following treatment with extracts and Niacin (One-way ANOVA followed by post hoc test \u2013 Bonferroni).<\/strong><\/p>\n

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Figure 5C: HFHS fed rats had a significant increase in plasma MDA levels (p value < 0.001) as compared to normal control group. A significant decrease in MDA levels in all treated groups in comparison with the HFHS group was noticed (p<0.001). <\/p>\n\n\n\n\n\n
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Figure 5C: Comparison of MDA levels in HFHS induced hyperlipidemia among different groups following treatment with extracts and Niacin (One-way ANOVA followed by post hoc test \u2013 Bonferroni).<\/strong><\/p>\n

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Discussion<\/strong><\/p>\n\n\n\n

Evaluation of cardiovascular risk helps to control and minimize cardiovascular diseases24<\/sup>.  Hyperlipidemia is a cluster of symptoms, which includes high cholesterol and triglyceride levels that predispose to cardiovascular diseases. It preludes the process of atherosclerosis, coronary artery disease, myocardial infarction and ischemic stroke by inducing oxidative stress25<\/sup>. Further, hypertriglyceridemia can contribute to metabolic syndrome and pancreatitis 26\u201329<\/sup>. Although many drugs are prescribed for controlling hyperlipidemia, most of the drugs have unpleasant side effects on chronic use. Though statins continue to be the major anti-hyperlipidemic agents prescribed by clinicians for the management of elevated plasma cholesterol and triglycerides, yet their prolonged use is associated with severe side effects like myopathy and hepatotoxicity 30-31<\/sup>. Hence, the need to look for newer hypolipidemic drugs which are effective but have less adverse effects.<\/p>\n\n\n\n

High fat diet increases lipid peroxidation resulting in oxidation of fatty acids in the liver leading to malondialdehyde (MDA) formation. Oxidative stress is an initial incident in the progression of hyperlipidemia32-33<\/sup>. The free radicals generated oxidise LDL cholesterol. This attracts the macrophages, which accumulate in the wall of the blood vessels after phagocytosing the LDL-C particles leading to atherosclerotic plaques34<\/sup>. Increase in serum triglycerides is consistent in coronary artery diseases35<\/sup>.  HDL-C decreases cholesterol levels and prevents atherosclerotic plaques by transporting cholesterol and cholesterol esters from different parts of the body to liver, where they are metabolized into bile acids36<\/sup>. All these suggests that high levels of triglycerides, LDL-cholesterol and total cholesterol are important factors in the pathogenesis of cardiovascular diseases.<\/p>\n\n\n\n

Increase in cholesterol levels alters the physical properties of cell\nmembrane resulting in seepage of reactive oxygen species (ROS) from electron\ntransport chain of mitochondria or NADPH oxidase (nicotinamide\nadenine dinucleotide phosphate oxidase) activation\nleading to lipid peroxidation and oxidation of proteins37<\/sup>. Lipid\nperoxidation damages the integrity of cell membrane, which results in\ninactivation of the receptors and enzymes present in cell membrane ensuing in\naugmented cell permeability and cell death. Further, reactive oxygen species\n(ROS) interact with antioxidant enzymes resulting in diminished cellular\nantioxidant defence38<\/sup>. These antioxidant enzymes are important\nduring oxidative stress as they minimize lipid peroxidation.<\/p>\n\n\n\n

There are\nvarious studies for the role of plant extracts in the treatment of\nhyperlipidemia. The importance of polyherbal formulations in\nAyurveda literature, Sarangdhar\nSamhita, is well documented \u2013 a combination of herbs has better\ntherapeutic efficacy and safety. A single active compound of an individual\nplant may be inadequate in producing a desirable therapeutic outcome5,39<\/sup>. Multiple active compounds in\nextracts have synergistic benefits for protection of cells. Hydroalcoholic extracts of Aegle Marmelos<\/em> leaves and Tamarindus indica<\/em> seeds were able\nto control hyperlipidemia in rodents when\nused individually and in combination but\nthe effect was better with the latter.  The\ncombination in higher dose (AM50+TI50) was more effective than the positive\ncontrol (Niacin) in controlling plasma triglyceride, cholesterol and improving\nHDL cholesterol levels. This could be due to effects of various constituents in\nthe combination.<\/p>\n\n\n\n

Studies have revealed the presence of phenolic compounds and flavonoids in the extracts of Aegle marmelos<\/em> (AM) leaves and Tamarindus indica<\/em> (TI)<\/a> seeds act as hypolipidemic and antioxidant agents40<\/sup>. It has been reported that the hydroalcoholic extracts of AM leaves contain rutin, quercetin and gallic acid whereas Tamarindus indica<\/em> (TI) consists of antioxidants epicatechin, taxifolin, apigenin and luteolinalong with other constituents10,17,41<\/sup> Rutin, quercetin and gallic acid are known to possess antioxidant and hypolipidemic activities42-45<\/sup>. Rutin and quercetin suppress lipid peroxidation, protect cell membrane and prevent liver injury. Quercetin also inhibits 3-Hydroxy-3-methylglutaryl\u2013coenzyme A (HMG CoA) reductase. Both quercetin and gallic acid decrease absorption of cholesterol, and lower plasma as well as hepatic cholesterol levels46<\/sup>. These compounds in the extracts could have exerted a beneficial effect on HFHS induced hyperlipidemia in the current study. <\/p>\n\n\n\n

Epicatechin, present in TI seed extract, is reported to augment the levels of phospho-AMPK \/total-AMPK (adenosine monophosphate-activated protein kinase) in muscle and liver. It also increases GLUT 4 (glucose transporter type 4) levels in membranes, which facilitates peripheral utilization of glucose47<\/sup>. This could be the one of the reasons for decrease in glucose levels in all treated groups. Taxifolin has an inhibitory effect on enzymes, \u03b1-glucosidase and \u03b1-amylase, which helps in controlling postprandial hyperglycemia. By inhibiting pancreatic lipase, taxifolin decreases the absorption of triglycerides48<\/sup>. Apigenin improves the lipid balance by promoting the low-density lipoprotein cholesterol absorption and increasing the conversion of hepatic cholesterol to bile acid49<\/sup>. Luteolin promotes the elimination of cholesterol by upregulating ABCG1 (ATP-binding cassette sub-family G member 1) transporters and SR-B1(Scavenger receptor class B type 1) receptors50-51<\/sup>.<\/p>\n\n\n\n

The antioxidants\nwe investigated in this study were catalase and glutathione (GSH). The present\nstudy showed a significant increase in hepatic lipid peroxidation and a decrease\nin catalase activity and GSH levels which, could be, associated with\nHFHS diet induced hyperlipidemia. Treatment with\nthese extracts, either alone or in combination, decreased lipid peroxidation\nand increased antioxidant levels. This could be due to antioxidant activity of\nthe extracts as well as inhibition of the absorption and synthesis of cholesterol\nand reuptake of bile acids52<\/sup>. The differential mode of action of various constituents in the\ncombination could result in synergistic hypolipidemic effect. This would be an\nadvantage of polyherbal formulations in hyperlipidemia. <\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

The hydroalcoholic\nextracts of Aegle Marmelos<\/em> leaves and Tamarindus indica<\/em> seeds,\nalone and in combination, reduced triglycerides, total cholesterol, LDL and\nVLDL but increased HDL levels in serum. In combination, the higher dose\n(AM50+TI50) has shown better result in controlling plasma triglyceride\nand cholesterol while improving HDL cholesterol levels which could be due\nto synergistic actions of the active constituents of Aegle Marmelos<\/em>\nleaves and Tamarindus indica<\/em> seeds. This type of polyherbal formulations\nmay help when translated to clinical scenario. <\/p>\n\n\n\n

Acknowledgements<\/strong><\/p>\n\n\n\n

The authors would like to thank Manipal Academy of Higher Education,\nManipal, India for the infrastructure provided to carry out this research.<\/p>\n\n\n\n

Conflict of\nInterest<\/strong><\/p>\n\n\n\n

The authors declare no conflict of interest, financial or otherwise with\nthis work.<\/p>\n\n\n\n

Funding sources<\/strong><\/p>\n\n\n\n

There is no funding Sources<\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

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