Siamese neem flower extract suppresses cholesterol absorption by interfering NPC1L1 and micellar property in vitro and in intestinal Caco-2 cells

Acharaporn Duangjai , Atcharaporn Ontawong, Chutima Srimaroeng

Abstract


Siamese neem (Azadirachta indica A. Juss var. siamensis Valeton) (A. indica) leaf extract, a traditional ayurvedic medicine, has been reported to exhibit antipyretic, antibacterial, antidyslipidemic, and antihyperglycemia effects. This study investigated the mechanism of hypocholesterolemic effect of methanolic extract of Siamese neem flowers in in vitro studies and in Caco-2 cells. Pancreatic cholesterol esterase and 3-hydroxy 3-methylglutaryl-CoA (HMG-CoA) reductase activities were assessed. Cholesterol micelle formation was prepared for in vitro cholesterol physicochemical property analyses, micelle size  and solubility, and transport of cholesterol into the Caco-2 cells. The expression of niemann-pick C1 like 1 (NPC1L1), and its major regulator, peroxisome proliferator-activated receptor d (PPARd), were determined by western blot and real time polymerase chain reaction, respectively. A. indica flower extract inhibited pancreatic cholesterol esterase activity and increased cholesterol micelles size. Uptake of cholesterol  into Caco-2 cells was inhibited by A. indica flower extract in a dose-dependent manner. In addition,  A. indica extract inhibited HMG-CoA reductase activity, resulting in low level of intracellular cholesterol accumulation, together with increased cytosolic NPC1L1 protein expression and decreased PPARd gene expression. In conclusion, A. indica flower extract has cholesterol-lowering effects by inhibiting intestinal cholesterol absorption, interfering micellar cholesterol formation, and attenuating cholesterol synthesis. As such, A. indica flower extract has potential for developing into nutraceutical product for prevention of hypocholesterolemia.


Keywords


A. indica; Cholesterol; HMGR; Micelle; NPC1L1.

Full Text:

PDF

References


Wang DQ. New concepts of mechanisms of intestinal cholesterol absorption. Ann Hepatol. 2003;2(3):113-121.

Altmann SW, Davis HR Jr, Zhu LJ, Yao X, Hoos LM, Tetzloff G, et al. Niemann-Pick C1 Like 1 protein is critical for intestinal cholesterol absorption. Science. 2004;303(5661):1201-1204.

Telford DE, Sutherland BG, Edwards JY, Andrews JD, Barrett PH, Huff MW. The molecular mechanisms underlying the reduction of LDL apoB-100 by ezetimibe plus simvastatin. J Lipid Res. 2007;48(3):699-708.

Davis HR Jr, Zhu LJ, Hoos LM, Tetzloff G, Maguire M, Liu J, et al. Niemann-Pick C1 Like 1 (NPC1L1) is the intestinal phytosterol and cholesterol transporter and a key modulator of whole-body cholesterol homeostasis. J Bio Chem. 2004;279(32):33586-33592.

Iwayanagi Y, Takada T, Suzuki H. HNF4α is a crucial modulator of the cholesterol-dependent regulation of NPC1L1. Pharm Res. 2008;25(5):1134-1141.

Iwayanagi Y, Takada T, Tomura F, Yamanashi Y, Terada T, Inui K, et al. Human NPC1L1 expression is positively regulated by PPARα. Pharm Res. 2011;28(2):405-412.

Pramfalk C, Jiang ZY, Cai Q, Hu H, Zhang SD, Han TQ, et al. HNF1α and SREBP2 are important regulators of NPC1L1 in human liver. J Lipid Res. 2010;51(6):1354-1362.

Mathur SN, Watt KR, Field FJ. Regulation of intestinal NPC1L1 expression by dietary fish oil and docosahexaenoic acid. J Lipid Res. 2007;48(2):395-404.

McKenney JM. Pharmacologic characteristics of statins. Clin Cardio. 2003;26(4 Suppl 3):32-38.

Garcia-Calvo M, Lisnock J, Bull HG, Hawes BE, Burnett DA, Braun MP, et al. The target of ezetimibe is Niemann-Pick C1-Like 1 (NPC1L1). Proc Natl Acad Sci U S A. 2005;102(23):8132-8137.

Florentin M, Liberopoulos EN, Elisaf MS. Ezetimibe‐associated adverse effects: what the clinician needs to know. Int J Clin Pract. 2008;62(1):88-96.

Golomb BA, Evans MA. Statin adverse effects : a review of the literature and evidence for a mitochondrial mechanism. Am J Cardiovasc Drugs. 2008;8(6):373-418.

Feng D, Ohlsson L, Duan RD. Curcumin inhibits cholesterol uptake in Caco-2 cells by down-regulation of NPC1L1 expression. Lipids Health Dis. 2010;9(1):40-44.

Abbasi Oshaghi E, Mohammadi A, Zynolebadi N, Nabi Moradi M, Ghasemi H, Totonchi A, et al. New insight in ezetimibe/garlic combination in hypercholesterolemic mice. Res Pharm Sci. 2012;7(5):S757.

Ngamukote S, Mäkynen K, Thilawech T, Adisakwattana S. Cholesterol-lowering activity of the major polyphenols in grape seed. Molecules. 2011;16(6):5054-5061.

Su J, Wang H, Ma C, Liu C, Gao C, Nie R, et al. Hypocholesterolaemic mechanism of bitter melon aqueous extracts via inhibition of pancreatic cholesterol esterase and reduction of cholesterol micellar solubility. Int J Food Sci Nutr. 2016;67(1):20-28.

Reddy Palvai V, Urooj A. Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (ex vivo) by Morus indica (Mulberry). Chinese Journal of Biology. 2014;2014. Article ID: 318561.

Nishan M, Subramanian P. Pharmacological and non pharmacological activity of Azadirachta indica (Neem) -A review. Int J Biosci. 2014;5:104-112.

Mahmoud DA, Hassanein NM, Youssef KA, Abou Zeid MA. Antifungal activity of different neem leaf extracts and the nimonol against some important human pathogens. Braz J Microbiol. 2011;42(3):1007-1016.

Bisht S, Sisodia S. Anti-hyperglycemic and antidyslipidemic potential of Azadirachta indica leaf extract in STZ-Induced diabetes mellitus. J Pharm Sci Res. 2010;2(10):622-627.

Chattopadhyay RR, Bandyopadhyay M. Effect of Azadirachta indica leaf extract on serum lipid profile changes in normal and streptozotocin induced diabetic rats. Afr J Biomed Res. 2005;8:101-104.

Singleton VL, Rossi JA. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Vicult. 1965;16(3):144-158.

Yamanashi Y, Takada T, Suzuki H. Niemann-Pick C1-like 1 overexpression facilitates ezetimibe-sensitive cholesterol and β-sitosterol uptake in CaCo-2 cells. J Pharmacol Exp Ther. 2007;320(2):559-564.

Kirana C, Rogers PF, Bennett LE, Abeywardena MY, Patten GS. Naturally derived micelles for rapid in vitro screening of potential cholesterol-lowering bioactives. J Agric Food Chem. 2005;53(11): 4623-4627.

Stępień AE, Gonchar M. A simple method for the determination of the cholesterol esterase activity. Acta Biochim Pol. 2013;60(3):401-403.

Heidrich JE, Contos LM, Hunsaker LA, Deck LM, Vander Jagt DL. Inhibition of pancreatic cholesterol esterase reduces cholesterol absorption in the hamster. BMC Pharmacol. 2004;4:5. DOI: 10.1186/1471-2210-4-5.

Johnson UE, Akwaji PI, Effiong US, Aniedi-Abasi M, Effiom OE. Phytochemical composition, antimicrobial effect of Azadirachta indica and carica papaya extracts on fungi isolated from gmelina arborea seedling. Int J Phytopathol. 2014;3(3): 109-115.

Muhammad H, Kabiru A, Makun H, Adefolalu F, Fasiku O, Abdullah A. Evaluation of methanolic and ethanolic extracts of Azadirachta indica seed oil for hypoglycaemic properties and effects on some biochemical parameters in diabetic mice. Iosr J Pharmacy. 2013;3(3):52-58.

Duangjai A, Nuengchamnong N, Lee LH, Goh BH, Saokaew S, Suphrom N. Characterisation of an extract and fractions of Azadirachta indica flower on cholesterol lowering property and intestinal motility. Nat Prod Res. 2017:1-4.

Sitasiwi AJ, Isdadiyanto S, Mardiati SM. Effect of ethanolic Neem (Azadirachta indica) leaf extract as an herb contraceptive on Hepato-somatic Index of the male mice (Mus musculus). J Phys: Conf Series; 2018. DOI :10.1088/1742-6596/1025/1/012043.

Maithani A, Parcha V, Pant G, Dhulia I, Kumar D. Azadirachta indica (neem) leaf: a review. J Pharm Res. 2011;4(6):1824-1827.

Koo SI, Noh SK. Green tea as inhibitor of the intestinal absorption of lipids: potential mechanism for its lipid-lowering effect. J Nutr Biochem. 2007;18(3):179-183.

Jesch ED, Carr TP. Sitosterol reduces micellar cholesterol solubility in model bile. Nutr Res. 2006;26(11):579-584.

Nekohashi M, Ogawa M, Ogihara T, Nakazawa K, Kato H, Misaka T, et al. Luteolin and quercetin affect the cholesterol absorption mediated by epithelial cholesterol transporter niemann–pick c1-like 1 in caco-2 cells and rats. PloS one. 2014;9(5):e97901.

Leifert WR, Abeywardena MY. Grape seed and red wine polyphenol extracts inhibit cellular cholesterol uptake, cell proliferation, and 5-lipoxygenase activity. Nutr Res. 2008;28(12):842-850.

Chaisawangwong W, Gritsanapan W. Quality assessment and scavenging activity of Siamese neem flower extract. Nat Prod Res. 2013;27(4-5):394-401.

Kitdamrongtham W, Ishii K, Ebina K, Zhang J, Ukiya M, Koike K, et al. limonoids and flavonoids from the flowers of Azadirachta indica var. siamensis, and their melanogenesis‐inhibitory and cytotoxic activities. Chem Biodivers. 2014;11(1): 73-84.

Xie C, Zhou ZS, Li N, Bian Y, Wang YJ, Wang LJ, et al. Ezetimibe blocks the internalization of NPC1L1 and cholesterol in mouse small intestine. J Lipid Res. 2012;53(10):2092-2101.

Tremblay AJ, Lamarche B, Lemelin V, Hoos L, Benjannet S, Seidah NG, et al. Atorvastatin increases intestinal expression of NPC1L1 in hyperlipidemic men. J Lipid Res. 2011;52(3): 558-565.

Gross B, Hennuyer N, Bouchaert E, Rommens C, Grillot D, Mezdour H, et al. Generation and characterization of a humanized PPARδ mouse model. Br J Pharmacol. 2011;164(1):192-208.

Friesen JA, Rodwell VW. The 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductases. Genome Biol. 2004;5(11):248-254.

Ademosun AO, Oboh G, Passamonti S, Tramer F, Ziberna L, Boligon AA, et al. Phenolics from grapefruit peels inhibit HMG-CoA reductase and angiotensin-I converting enzyme and show antioxidative properties in endothelial EA. Hy 926 cells. Food Sci Hum Wellness. 2015;4(2):80-85.

Reddy VP, Ahmed F, Urooj A. Inhibition of 3-hydroxy-3-methylglutaryl coenzyme a (hmg co-a) reductase in liver microsomes by Moringa Oleifera L. polyphenols. Int J Pharm Sci Res. 2012;3(8):2510-2516.

Islam B, Sharma C, Adem A, Aburawi E, Ojha S. Insight into the mechanism of polyphenols on the activity of HMGR by molecular docking. Drug Des Devel Ther. 2015;9:4943-4951.


Refbacks

  • There are currently no refbacks.


Creative Commons LicenseThis work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.