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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 14  |  Issue : 3  |  Page : 190-200

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


1 Division of Physiology, School of Medical Sciences, University of Phayao, Phayao, Thailand
2 Division of Physiology, School of Medical Sciences, University of Phayao, Phayao; Department of Physiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand
3 Department of Physiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand

Date of Web Publication21-May-2019

Correspondence Address:
Chutima Srimaroeng
Department of Physiology, Faculty of Medicine, Chiang Mai University, Chiang Mai
Thailand
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1735-5362.258485

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  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 δ (PPARδ), 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 PPARδ 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


How to cite this article:
Duangjai A, Ontawong A, Srimaroeng C. Siamese neem flower extract suppresses cholesterol absorption by interfering NPC1L1 and micellar property in vitro and in intestinal Caco-2 cells. Res Pharma Sci 2019;14:190-200

How to cite this URL:
Duangjai A, Ontawong A, Srimaroeng C. Siamese neem flower extract suppresses cholesterol absorption by interfering NPC1L1 and micellar property in vitro and in intestinal Caco-2 cells. Res Pharma Sci [serial online] 2019 [cited 2019 Oct 19];14:190-200. Available from: http://www.rpsjournal.net/text.asp?2019/14/3/190/258485




  Introduction Top


Cholesterol homeostasis is maintained by intestinal absorption, endogenous biosynthesis, and removal of cholesterol from the blood circulation [1]. Intestinal cholesterol absorption is a multi-step process in which cholesterol is micellized in the lumen, taken up by the enterocytes, and transported to the blood circulation. Interestingly, inhibiting cholesterol absorption is the primary approach to reduce plasma cholesterol levels. Niemann-pick C1-like 1 (NPC1L1) protein is a cholesterol transporter localized on the brush border membrane of enterocytes [2]. As previously shown, a lack of NPC1L1 results in reduced plasma cholesterol and low-density lipoprotein (LDL) and improved fatty liver in high-cholesterol diet fed mice [3], while mice fed with cholesterol-restricted diet demonstrated upregulation of intestinal NPC1L1 expression [4]. Moreover, hepatocyte nuclear factors 4 α (HNF4α), sterol regulatory element-binding protein 2, and peroxisome proliferator-activated receptor a agonist, fenofibrate, positively activated NPC1L1 transcription in hepatocellular carcinoma (HepG2) cells [5],[6]. HNF1a also increased NPC1L1 promoter activity and mRNA expression in human hepatocyte derived cellular carcinoma cells [7].

On the other hand, activation of PPARδ down regulated intestinal NPC1L1 gene expression in hamsters [8]. Since, the liver controls whole body cholesterol homeostasis, this organ plays an important role in de novo cholesterol synthesis through a rate limiting step enzyme, 3-hydroxy-3-methyl-glutaryl- CoA reductase (HMGR), and through controlling reversed cholesterol transport pathway. At present, ezetimibe and statins are prescribed as lipid-lowering drugs which target NPC1L1 and HMGR, respectively [9],[10]; and nonresponsive individuals and adverse effects have been reported [11],[12].

Natural supplements are gaining attention as alternative treatments for hypercholesterolemia. Several natural plants show potential hypocholesterolemic and anti-atherosclerotic effects. For instance, curcumin inhibits cholesterol uptake by decreasing levels of NPC1L1 protein and mRNA expression in intestinal Caco-2 cells [13]. The combination of garlic and ezetimibe decreased cholesterol level, low-density lipoprotein cholesterol levels, liver weight and atherogenic index [14]. Moreover, major polyphenols in grape seed (e.g., gallic acid, catechin, and epicatechin) lower cholesterol by inhibiting pancreatic cholesterol esterase activity, binding to bile acids, and suppressing solubility of cholesterol in micelles as shown by delayed cholesterol absorption [15]. Recently, bitter melon aqueous extract was shown to decrease intestinal cholesterol absorption via inhibition of pancreatic cholesterol esterase and micelle formation [16], and Morus indica (mulberry) inhibited HMGR activity, suggesting it also has a role in reducing cholesterol levels [17].

Azadirachta indica A. Juss var. siamensis Valeton (A. indica) is well-known as neem and belongs to the Meliaceae family. Neem is native to East India and Burma, and grows widely in South East Asia including Thailand [18]. Each part of the neem tree is used in traditional ayurvedic medicine and several active compounds have been isolated from different parts including azadirachtin, nimbidin, nimbin, nimbinin, nimbidinin, nimbolide, nimbidic acid, nimbidin, sodium nimbidate [18], quercetin, and ß-sitosterol [19]. Neem shows a variety of pharmacological effects such as antipyretic, antiviral, analgesic, antibacterial, contraceptive, hepatoprotective [18], and anti-dyslipidemic [20]. In addition, neem’s ethanolic leaf extract reduces total cholesterol, LDL, VLDL, and triglyceride in streptozotocin-induced diabetic rats [20],[21]. However, the mechanisms behind the hypocholesterolemic effect of A. indica flower extract remains unclear. The present study investigated the effects of metanolic A. indica flower extract on cholesterol absorption and synthesis using intestinal Caco-2 cells and in vitro studies. The cholesterol-lowering mechanisms of A. indica flower extract were also identified.


  Materials and Methods Top


Chemicals

Dulbecco’s modified eagle medium (DMEM-F12), fetal bovine serum (FBS), penicillin and streptomycin solution, and trypsin-ethylenediaminetetra acetic acid were purchased from Life Technologies (Eugene, OR, USA). Phosphate buffered saline (PBS) was obtained from Biochrom AG (Berlin, Germany). HMG-CoA reductase assay kit was purchased from Sigma Chemical Co. (St. Louis, MO, USA). (1α,2α(n)-3H) Cholesterol (specific activity, 49 Ci/mmol) was purchased from Perkin-Elmer (Wellesley, MA, USA). NPC1L1 antibody was bought from Novus Biologicals (Littleton, CO, USA). Ezetimibe was purchased from Schering-Plough Research Institute (Kenilworth, NJ, USA). Folin-Ciocalteu reagent, sodium bicarbonate, 1,2-di-O-lauryl-rac-glycero-3-glutaric acid 6’-methylresorufin ester, taurocholic acid sodium salt hydrate, glycodeoxycholic acid, taurodeoxycholic acid, and hydrazine hydrate solution were received from Sigma-Aldrich Co. (St. Louis, MO, USA). All other chemical reagents used in this study were obtained from commercial sources.

Methanolic extract of A. indica preparation

A. indica was harvested at Tumbon Maeka, Phayao District Mueang Phayao, Phayao Province, Thailand. The plant was identified by a botanist, and plant specimens were collected into herbarium of the Faculty of Biology, Naresuan University, Phitsanulok, Thailand (voucher specimen No. 003805). Flower part of A. indica was collected and dried in a hot air oven at 37 °C. Dried materials were cut and ground into small pieces. One hundred g of the dried plant material were weighed and extracted twice with 300 mL methanol (95%) by reflux extraction. Subsequently, the extract was filtered and evaporated in a rotavapor apparatus at 55-60 °C. Crude extract was stored at -20°C and dissolved in dimethylsulfoxide (DMSO) prior to use.

Total phenolic content determination

The content of total phenolic compounds in A. indica methanolic extract was determined according to the Folin-Ciocalteu method [22]. Briefly, crude extract at 2 mg/mL was mixed with Folin-Ciocalteu reagent and Na2CO3 solution, and incubated at room temperature for 30 min. Total phenolic content of the mixture solution was measured at 750 nm using a spectrophotometer. Gallic acid was used as a standard phenolic content. Total phenolic content was expressed as gallic acid equivalents (GAE) in mg/g of dry material.

Pancreatic cholesterol esterase activity determination

Pancreatic cholesterol esterase activity was determined using p-nitrophenyl butyrate as described previously [15]. Briefly, A. indica extract at 0.01-10 mg/mL was incubated with mixtures containing 5.16 mM taurocholic acid, 0.2 mM p-nitrophenyl butyrate, and 100 mM NaCl at pH 7.0. The reaction was initiated by adding porcine pancreatic cholesterol esterase (1 mg/mL) and incubating for 5 min at 25 °C. Liberated p-nitrophenoxide was determined by measuring absorbance at 405 nm. Inhibition of pancreatic cholesterol esterase activity was compared with control (absence of A.indica extract). The data are shown as the half maximal inhibitory concentration (IC50).

Cholesterol micelle size determination

Cholesterol micelles were prepared by addition of 1 μM cholesterol and 50 μM phosphatidylcholine dissolved in chloroform, 2 mM sodium taurocholate dissolved in methanol, and evaporated with N2 gas which modified from Yamanashi et al. [23] and Kirana et al. [24]. Reconstituted cholesterol micelle with PBS was filtered through 0.22 μm membrane to obtain the similar size range of micelle cholesterol particles and A. indica extract was subsequently co-incubated at 37 °C for 3 h. Particle size was measured using a particle size analyzer (Zetasizer Nano, Malvern Instruments, Malvern, UK).

Micellar cholesterol solubility assay

The method to determine the solubility of cholesterol in micelle was modified from Kirana et al. [24]. Briefly, A. indica extract (12.5, 25, 50, 75, and 100 μg/mL) was incubated with micelle solution containing 10 mM cholesterol, 1 mM sodium taurocholate, and 0.6 mM phosphatidylcholine for 3 h at 37 °C. The mixed solution was filtered through a 0.22 μm membrane to eliminate nonmicellar fraction. Cholesterol content in the filtrate was measured, defining the micellar cholesterol solubility.

Cell culture preparation

Human Caco-2 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells at passages 2 to 22 were maintained in 25 cm 2 flasks in a 95% air, 5% CO2 atmosphere in the presence of media containing DMEM/F-12, 1.2 g/L NaHCO3, 10% FBS, and 100 unit/mL penicillin-streptomycin solution.

Cell viability assay

Viability of cells was determined using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT). Briefly, cells were plated at 2.5 × 104 cells/well in a 96-well plate for 24 h. Cells were treated with A. indica extract (12.5, 25, 50, 75, or 100 μg/mL) for 3, 24, and 48 h, respectively. Subsequently, MTT solution (5 mg/mL) was added and incubated for 4 h. At the end of experiment, MTT solution was aspirated and the formazan product was eluted from cells by the addition of DMSO for 30 min. Absorbance of dissolved formazan was measured at 595 nm using SynergyTM HT microplate reader (Biotek, VT, USA). Cell viability was calculated according to the following equation:

Cell viability (%) = (absorbance of treated group/absorbance of control group) × 100

Cholesterol uptake study

To determine the effect of A. indica extract on cholesterol transport in human intestinal epithelial cells, Caco-2 cells were seeded at a density of 5 × 104 cells/mL into a 24-well plate and were grown for 18-21 days. Differentiated Caco-2 cells were incubated in medium containing 1 μCi/mL of [3H]-cholesterol micelles in the presence or absence of A. indica extract at various concentrations (12.5, 25, 50, 75, or 100μg/mL), and 40 μg/mL of ezetimibe, cholesterol absorption inhibitor, for 3 h. At the end of the experiment, cells were washed three times with ice-cold PBS and were lyzed with 1 N sodium hydroxide and neutralized by 1 N hydrochloric acid. Radioactivity was quantified by liquid scintillation spectroscopy (Perkin Elmer, MA, USA). Uptake of cholesterol was calculated as fmole and normalized by mg protein using Bradford protein assay (Bio-Rad, CA, USA).

Subcellular fractionation and western blot analysis

To measure protein expression of cholesterol transporter, NPC1L1, subcellular fractions extracted from Caco-2 cells were prepared using differential centrifugation. Treated cells (with A. indica extract at 100 μg/mL) and control cells (without extract) were lysed using CelLytic MT mammalian tissue lysis/extraction reagent (Sigma-Aldrich Co., MO, USA) containing 1% complete protease inhibitor mixture (Merck, Darmstadt, Germany) according to the manufacturer’s protocol. Briefly, the sample was homogenized and centrifuged at 5,000 g at 4 °C for 10 min. Supernatant was designated as whole cell lysate. Half of the supernatant was recentrifuged at 100,000 g for 2 h. The supernatant fraction from this step was designated as cytosolic fraction while the pellet was resuspended by the same buffer and used as membrane fraction. Samples were stored at -80 oC prior to use. For western blot analysis, protein concentration was determined, resolved in 4× Laemmli solution, electrophoresed on 10% SDS-PAGE, and transferred onto polyvinylidene difluoride membranes (GE Healthcare, WI, USA). Non-specific binding was eliminated by blocking with 5% (w/v) non-fat dry milk in 0.05% Tween® 20 in tris-buffered saline for 1 h. Polyclonal anti-rabbit NPC1L1, monoclonal anti-mouse alkaline phosphatase, or antimouse β-actin antibody were incubated at 4 °C overnight. Polyvinylidene difluoride membrane was washed with tris-buffered saline and incubated with horseradish peroxidase-conjugated immunopure secondary goat antirabbit or antimouse IgG (Merck, Darmstadt, Germany) for 1 h. The target protein was detected using Super Signal West Pico Chemiluminescent substrate (GE Healthcare, WI, USA) and quantitatively analyzed with the Image J program from the Research Services Branch of the National Institute of Mental Health (Bethesda, MD, USA).

Real time polymerase chain reaction analysis

To evaluate the effect of A. indica extract on PPARδ expression, total RNA was extracted and purified from Caco-2 cells. The cells were incubated in the presence (treated cells) or absence (control cells) of A. indica extract at 100 μg/mL using TRIzol® reagent (Thermo Fisher Scientific, MA, USA), according to the manufacturer’s instruction. The first strand cDNA was obtained using iScript cDNA synthesis kit (Bio-Rad, CA, USA) and real time polymerase chain reaction (RT-PCR) was performed using SYBR RT-PCR master mix (Bioline, London, UK) on ABI 7500 (Life Technologies, NY, USA). Forward and reverse primers were purchased from Macrogen (Seoul, Korea) and used at a final concentration of 0.4 μM. Human PPARδ (forward primer: 5’- GTCACACAACGCTATCCGTTT-3’, reverse primer: 5 ' - AGGCATTGTAGATGTGCTT GG-3’) and human GAPDH (forward primer: 5’-AGCCTTC TCCATGGTGGTGAAAC-3 ', reverse primer: 5’-CGGAGTCAACGGATT TGGTCG-3’). Gene expression was normalized to GAPDH mRNA and reported as relative fold changes. RT-PCR amplification was performed in duplicate for each cDNA.

HMG-CoA reductase inhibitory activity

To determine the effect of A. indica extract on cholesterol synthesis, HMG-CoA reductase activity assay kit was used as recommended by the manufacturer (Sigma-Aldrich, MO, USA). The reaction containing A. indica extract at 100 μg/mL, NADPH, and different concentration of HMG-CoA substrate (400, 800, and 1600 mg/mL) was initiated by addition of the catalytic domain of human recombinant HMG-CoA reductase. Pravastatin (0.25 μM) was used as an inhibitory control for the activity of HMGR. Oxidation of NADPH by the catalytic subunit of HMGR was measured using Synergy™ HT microplate reader (Biotek, VT, USA) at the wavelength of 340 nm at 37 °C with a kinetic program. HMGR activity was expressed as a percentage of inhibition compared with control (absence of test compound) and HMG-CoA at 400 μM.

Statistical analysis

Data are expressed as mean ± SEM. Statistical differences were assessed using one-way ANOVA followed by Tukey’s post hoc test. Statistical analyses were conducted using SPSS statistical software version 23 (IBM Corp., NY, USA). Differences were considered significant at P < 0.05.


  Results Top


Percent yield and total phenolic content of metanolic A. indica extract

A. indica flowers were extracted with 95% methanol. The percent yield and total phenolic content of the crude extract were 11.43% (w/w) and 23.73 ± 0.69 mg/L of gallic acid, respectively.

Effect of A. indica extract on pancreatic cholesterol esterase activity, cholesterol micelle size and solubility

As shown in [Figure 1]A, A.indica extract’s potent inhibitory effect on pancreatic cholesterol esterase was dose-dependent with an IC50 value of 3.36 ± 1.24 mg/mL. In addition, methanolic A.indica extract increased micelle size in a dose dependent manner [Figure 1]B. However, A. indica extract at 12.5-100 μg/mL had no effect on cholesterol micelle solubility [Figure 1]C, indicating potential inhibition of cholesterol absorption by A. indica via partial modulation of pancreatic cholesterol esterase enzyme and cholesterol micelle size.
Figure 1: Effect of A. indica extract on physicochemical property of cholesterol micelles on (A) pancreatic cholesterol esterase activity represents as the inhibitory concentration at 50% (IC50) (n = 3), (B) cholesterol micelle particle size, and (C) intermicellar cholesterol levels. Values are represented as mean ± SEM; n = 4; * P < 0.05 compared with control. A. indica, Azadirachta indica.

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Effect of A. indica extract on cholesterol uptake and cell viability in Caco-2 cells

Differentiated Caco-2 cells were incubated with serum-free medium containing 1 μCi/mL of [3H]-cholesterol micelles in the presence or absence of either A. indica extract at 12.5, 25, 50, 75, and 100 μg/mL or 40 μg/mL of ezetimibe for 3 h. A. indica extract decreased cholesterol uptake in a dose-dependent manner at a maximum inhibition much like ezetimibe [Figure 2]A. Furthermore, A. indica extract at 12.5-100 μg/mL did not interfere cell viability at 3, 24, or 48 h, respectively [Figure 2]B, [Figure 2]C, [Figure 2]D, indicating A. indica extract inhibited cholesterol absorption without any cytotoxic effect.
Figure 2: Effect of A. indica extract on cholesterol uptake and cell viability in differentiated Caco-2 cells. (A) Cells were incubated with different concentration of A. indica extract at 12.5, 25, 50, 75, and 100 μg/mL or 40 μg/mL of ezetimibe for 3 h at 37 °C. The radioactivity of [3H]-micelle cholesterol was measured and expressed as percent of control. Viability of Caco-2 cells after exposure to either A. indica extract or ezetimibe for (B) 3 h, (C) 24 h, and (D) 48 h was determined using MTT assay. Each experiment was performed separately, n = 5. *** Indicates significant differences compared with control (P < 0.001). A. indica, Azadirachta indica.

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Effect of A. indica extract on niemann-pick C1 like 1 expression in Caco-2 cells

To further determine whether A. indica extract interferes NPC1L1 expression or trafficking, the maximum and effective dose of A. indica on cholesterol absorption inhibition at 100 μg/mL was used to determine cellular protein expression of NPC1L1 using western blot analysis. Membrane and cytosolic NPC1L1 protein expression significantly increased by A. indica extract when compared with control cells [Figure 3]. The findings indicated A. indica extract may have blocked NPC1L1 trafficking and internalization, consequently interfering with cholesterol absorption.
Figure 3: NPC1L1 expression in 100 ug of whole cell, 50 ug of membrane, and 50 ug of cytosolic fractions extracted from Caco-2 cells. Cells were incubated with 100 ug/mL of A. indica for 3 h. NPC1L1 proteins were detected using anti-NPC1L1 antibody. ALP and anti-β-actin antibodies were also used as apical membrane marker and loading control, respectively. Data are expressed as mean ± SEM, n = 5. (A) A representative blot of NPC1L1 and β-actin protein expressions and (B) indicated quantification of relative NPC1L1/β -actin. * Shows significant differences compared to control, P < 0.05. NPC1L1, niemann-pick C1 like 1; A. indica, Azadirachta indica; ALP, anti-alkaline phosphatase.

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Effect of A. indica extract on peroxisome proliferator-activated receptor δ in Caco-2 cells

Since PPAR-δ activation has previously been shown to decrease NPC1L1 mRNA expression in Caco-2 cells [8], the next step was to determine if A. indica extract at 100 μg/mL regulates PPAR-δ. As shown in [Figure 4], A. indica extract down-regulated PPARδ mRNA expression, leading to increased membrane and cytosolic NPC1L1 protein expression.
Figure 4: Effect of A. indica extract on PPAR d mRNA expression in Caco-2 cells. Cells were incubated with 100 μg/mL of A. indica extract for 3 h. Total RNAs were extracted from Caco-2 cells and PPAR d mRNA level was determined using quantitative polymerase chain reaction. Data are expressed as mean ± SEM from 3 separate experiments. ** Indicates significant differences compared to control, P < 0.01. A. indica, Azadirachta indica; PPAR, peroxisome proliferator-activated receptor.

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Effect of A.indica extract on HMG-CoA reductase activity

To determine if A. indica extract inhibits cholesterol synthesis, HMGR activity was evaluated. NADPH concentration was fixed and HMG-CoA substrate was used in the range of 400-1600 mg/mL in the presence of either A. indica extract (0.1 mg/mL) or pravastatin (0.25 μM). Oxidation of NADPH by the catalytic subunit of HMGR was measured. As shown in [Figure 5], A. indica extract inhibited HMGR activity in a dose-dependent manner, indicating A. indica methanolic extract directly inhibited HMGR activity resulting in decreased endogenous cholesterol synthesis.
Figure 5: Effect of A. indica extract on HMG-CoA reductase activity using in vitro cell-free based assay. A. indica extract at 100 μg/mL or pravastatin 0.25 μM was incubated with HMG-CoA at 400, 800, and 1600 mg/mL. Value represented as mean ± SEM, n = 3. **, *** Indicate significant differences compared with control, P < 0.01 and P < 0.001. ### Shows significant differences in comparison with HMG-CoA at 400 μM, P < 0.001. A. indica, Azadirachta indica; HMG-CoA, 3-hydroxy 3-methylglutaryl-CoA.

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  Discussion Top


This study examined the cholesterol-lowering mechanisms of methanolic A. indica extract, specifically, on inhibition of cholesterol transport and interference in cholesterol metabolism. Since cholesterol absorption is a multi-step process, factors interfering with digestion, micelle formation, or transport influence intestinal cholesterol absorption [1]. Previous studies report anti-dyslipidemic properties of ethanolic A. indica leaf extract in streptozotocin-induced diabetic rats [20],[21]. As pancreatic cholesterol esterase hydrolyzes cholesterol esters into un-esterified cholesterol and free fatty acids, a deficit of this enzyme leads to accumulation of cholesterol esters and triglycerides in cells and tissues [25]. Accordingly, inhibiting cholesterol esterase has been suggested as a useful agent to lower cholesterol levels [26]. In this study, methanolic A. indica extract demonstrated an inhibitory effect against pancreatic cholesterol esterase activity similar to a previous in vitro study where polyphenols present in grape seed inhibited pancreatic cholesterol esterase [15]. Another study, using phytochemical screening, showed A. indica contains secondary metabolites such as alkaloids, glycosides, flavonoids, and polyphenols [27]. Moreover, the methanolic A. indica seed oil extracts also revealed the presence of saponins, terpenes, tannins and steroids [18],[28]. Our recent study and other independent studies also revealed that A. indica plant contains steroids such as campesterol, beta-sitosterol, and stigmasterol [29],[30],[31]. Taken together, it is possible the polyphenols in A. indica extract enacted an inhibitory effect on pancreatic cholesterol esterase activity, subsequently diminishing lipid digestion and limiting intestinal cholesterol absorption.

Additionally, modulated cholesterol micelle size and solubility interfere with cholesterol transport and inhibition of its absorption. A major constituent of green tea catechins, epigallocatechin-3-gallate, alters the physicochemical properties of lipid emulsion by increasing particle size, leading to an interference of intestinal lipid absorption [32]. Sitosterol, the most common dietary plant sterol, also reduces micellar cholesterol solubility by displacing cholesterol from micelles [33]. Similar to A. indica, ß-sitosterol at a greater concentration demonstrated a greater reduction of micellar cholesterol solubility [33], and this compound subsequently inhibited intestinal cholesterol absorption [33],[34]. Thus, A. indica extract containing β-sitosterol [19] might reduce cholesterol absorption by disturbing micelle formation.

Previous study reports that cholesterol absorption in NPC1L1-deficient mice has been reduced while ezetimibe treatment does not produce the same effect. This indicates NPC1L1 plays a critical role in intestinal cholesterol absorption [2]. Consistently, the present study showed A. indica extract reduced cholesterol uptake in a dose-dependent manner similar to ezetimibe [Figure 2]A. Several pieces of evidence exhibited point to involvement of polyphenols in cholesterol absorption. For instance, curcumin and quercetin decreased NPC1L1 protein and mRNA expressions in Caco-2 cells, resulting in reduced cholesterol absorption [34] similar to the way grape seed and red wine, which are rich in polyphenols, inhibit cholesterol uptake in human epithelial cells – HT29, HepG2, and Caco-2 cells [35]. Previous studies reported that limonoids, flavonoids, rutin, and quercetin can be isolated from methanolic extract of A. indica flowers [36],[37]. Hence, A. indica extract may inhibit cholesterol transport via polyphenol activity. This study also further demonstrated that A. indica extract up-regulated NPC1L1 protein expression in membrane and cytosolic fractions [Figure 3] and exhibited a statin-like effect by inhibiting HMGR activity [Figure 4]. Likewise ezetimibe, a cholesterol absorption inhibitor, blocks internalization of NPC1L1 causing retention on plasma membrane [38] while atorvastatin, an HMGR inhibitor, induces total protein expression of NPC1L1 for 33% by likely activating sterol regulatory element-binding protein (SREBP)-2 and HNF4-a in hyperlipidemic patients [39]. Hence, A. indica may dominantly block NPC1L1/cholesterol internalization, leading to an increase in membrane NPC1L1 expression. Furthermore, it may also induce transcription factors regulated NPC1L1 expression, SREBP-2, and HNF4-a, similar to that of atorvastatin’s effect as previously shown, resulting in up-regulation of cytosolic NPC1L1 expression. In addition, activation of PPAR-δ reduces NPC1L1 mRNA expression in Caco-2 cells [8], and cholesterol levels significantly decrease in PPARδ knock-in mice [40]. Together, these data suggest that PPARδ controls cholesterol level partly through up-regulation of NPC1L1. In this study A. indica down-regulated PPAR5 mRNA expression. Thus, A. indica extract may have reduced cholesterol transport independently from PPAR-δ. Further study could identify the molecules responsible for A. indica mediated transcriptional regulation of NPC1L1.

Human HMGR contains three major domains, catalytic, linker, and anchor and the active sites in the catalytic domain reduce HMG-CoA substrate [41]. Here, A. indica extract exhibits direct inhibition of HMGR activity. Likewise, phenols extracted from grapefruit peels [42] and Moringa oleifera leaves [43] inhibit HMGR activity. By binding to HMGR and blocking nicotinamide adenine dinucleotide phosphate (NADP+) binding sites, the latter polyphenols partially occupy active sites, hence inhibiting HMGR activity [44]. Thus, A. indica extract not only interferes with cholesterol absorption, but also inhibits de novo cholesterol synthesis, suggesting A. indica extract could be developed as a nutraceutical to lower lipid levels due to its concomitant mechanisms of cholesterol homeostasis.


  Conclusion Top


Hypocholesterolemic action of methanolic A. indica flower extract appears to involve inhibition of cholesterol absorption and synthesis. Reducing cholesterol uptake, pancreatic cholesterol esterase activity as well as interfering with physiochemical properties of cholesterol micelles and blocking HMGR activity were identified. The data indicate a vital role for A. indica in cholesterol-lowering treatment; however, further in vivo study is needed to validate its hypocholesterolemic effects in humans.


  Acknowledgements Top


This research was financially supported by the Faculty of Medicine Endowment Fund, Chiang Mai University, Chiang Mai, Thailand (66/2558 to CS and AD) and the NSTDA Chair Professor Grant (the Fourth Grant) of the Crown Property Bureau Foundation and the National Science and Technology Development Agency to Professor Dr. Vatcharin Rukachaisirikul. The authors would like to thank Ms. Sineenart Santidherakul, Medical Science Research Equipment Center, Faculty of Medicine, Chiang Mai University, Thailand, for technical assistance.



 
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