Piperine Congeners as Inhibitors of Vascular Smooth Muscle Cell Proliferation^[*]

• Introduction
• Results and Discussion
• Materials and Methods
• Isolation of amide alkaloids (1–10) from Piper nigrum fruits
• Synthesis of piperine analogs
• Bioactivity evaluation
• Acknowledgements
• References

Abstract

Successful vascular healing after percutaneous coronary interventions is related to the inhibition of abnormal vascular smooth muscle cell proliferation and efficient re-endothelialization. In the search for vascular smooth muscle cell anti-proliferative agents from natural sources we identified piperine (1), the main pungent constituent of the fruits from Piper nigrum (black pepper). Piperine inhibited vascular smooth muscle cell proliferation with an IC₅₀ of 21.6 µM, as quantified by a resazurin conversion assay. Investigations of ten piperamides isolated from black pepper fruits and 15 synthesized piperine derivatives resulted in the identification of three potent vascular smooth muscle cell proliferation inhibitors: the natural alkaloid pipertipine (4), and the two synthetic derivatives (2E,4E)-N,N-dibutyl-5-(3,5-dimethoxyphenyl)penta-2,4-dienamide (14) and (E)-N,N-dibutyl-3-(naphtho[2,3-d][1,3]dioxol-5-yl)acrylamide (20). They showed IC₅₀ values of 3.38, 6.00, and 7.85 µM, respectively. Furthermore, the synthetic compound (2E,4E)-5-(4-fluorophenyl)-1-(piperidin-1-yl)penta-2,4-dien-1-one (12) was found to be cell type selective, by inhibiting vascular smooth muscle cell proliferation with an IC₅₀ of 11.8 µM without influencing the growth of human endothelial cells.

Introduction

Atherosclerosis is the worldwide leading cause of heart disease and stroke. In westernized societies this disease condition is the underlying cause of about 50 % of all deaths [1]. VSMC proliferation strongly contributes to initial atherosclerotic plaque formation as well as to restenosis. Restenosis means the pathological re-narrowing of the blood vessel lumen and is a severe complication occurring after angioplasty, stenting procedures or bypass surgery. The major function of VSMCs in their quiescent or contractile phenotype is to regulate blood vessel diameter and blood flow in normal mature blood vessels. After an initial injury, mediators like cytokines and growth factors, including PDGF, promote a change of the VSMC phenotype, which does no longer regulate contraction but vascular reconstruction [2]. Abnormal migration, proliferation and apoptosis of VSMCs at this stage, are key elements in the development of atherosclerosis and restenosis [3]. Drug-eluting stents are the state of the art treatment of coronary artery diseases and aim to overcome restenosis after percutaneous coronary interventions by inhibiting VSMC proliferation [4]. Paclitaxel (taxol), a natural, microtubules stabilizing compound, and sirolimus, everolimus and zotarolimus, i.e., rapamycin derivatives acting as mTOR inhibitors, are the most prominent drugs used therefore [5]. However, unresolved drug-related issues, like impaired re-endothelialization or delayed thrombosis induction, remain, since both drug classes not only inhibit VSMC proliferation but also endothelial cell growth [6], [7], [8], [9]. Hence, identifying new VSMC proliferation inhibitors is of high relevance.

Dried unripe fruits of Piper nigrum L. (Piperaceae), commonly called black pepper, are referred to as “king of spices” and are known in almost every cuisine worldwide. Responsible for its alluring odour are various monoterpenes, like linalool, limonene, or sabinene [10] while the typical pungency of black pepper and other Piper species is mainly attributed to the predominant piperamide piperine (1-[5-(1,3-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]piperidine) [11], [12]. Also other piperamides and diverse unsaturated, long-chain fatty acid amides are reported to contribute to the tingling sensation [13], [14], [15]. Further constituents of P. nigrum and related species include lignans and flavonoids [16]. Besides its use as spice, black pepper is described to be endowed with medicinal and disease preventing properties, e.g., anti-inflammatory, anti-oxidant, and bactericidal activity [11], [17], [18].

The main constituent piperine is reported to enhance drug bioavailability and to modulate GABA_A receptors, besides various other pharmacological activities, including analgesic, anti-inflammatory, anti-pyretic, anti-convulsive, anti-oxidant, anti-depressant, anti-apoptotic, anti-cancer, and anti-viral properties [12], [19], [20], [21]. In this study, we identified piperine (1) to be a promising inhibitor of VSMC proliferation, which was very recently also described by others [22]. We used piperine as lead compound to synthesize derivatives thereof and isolated structurally related amides from the fruits of P. nigrum to deduce a qualitative SAR with regard to the anti-proliferative effect in VSMCs.

Results and Discussion

In a biological assessment, we identified the piperamide piperine (1) ([Table 1]) from P. nigrum as a VSMC proliferation inhibitor ([Fig. 1]). In a concentration dependent manner, it revealed an anti-proliferative effect in the resazurin conversion assay, with an IC₅₀ of 21.6 µM.

Table 1 Inhibition of VSMC proliferation at 30 µM of isolated (2–10) and synthesized (11–25) congeners of piperine (1), and IC₅₀ values of the most potent identified compound. Results were obtained with the resazurin conversion method and presented as means ± SD in RU (n = 3 for all data groups).

Compound, cluster	Structure	Name	Inhibition of VSMC proliferation
			at 30 µM (relative units)	IC50 (µM)
** p < 0.01; *** p < 0.001
1, A		Piperine	0.747 ± 0.065**	21.6
2, A		Piperylin	0.938 ± 0.114
3, A		Piperoleine A	0.942 ± 0.043
4, A		Pipertipine	0.014 ± 0.018***	3.38
5, A		Pipernonaline	0.804 ± 0.059
6, A		Dehydropipernonaline	0.803 ± 0.094
7, A		(2E,4E,8E)-9-(Benzo[d][1, 3]dioxol-5-yl)-1-(pyrrolidin-1-yl)nona-2,4,8-trien-1-one	1.024 ± 0.089
8, B		Chabamide	0.819 ± 0.089
9, C		N-trans Feruloylpiperidine	1.118 ± 0.082
10, C		Feruperine	1.059 ± 0.113
11, C		(2E,4E)-5-Phenyl-1-(piperidin-1-yl)penta-2,4-dien-1-one	0.769 ± 0.075***	48.3
12, C		(2E,4E)-5-(4-Fluorophenyl)-1-(piperidin-1-yl)penta-2,4-dien-1-one	0.426 ± 0.079***	11.8
13, C		(2E,4E)-5-(4-Methoxyphenyl)-1-(piperidin-1-yl)penta-2,4-dien-1-one	0.810 ± 0.011***
14, C		(2E,4E)-N,N-Dibutyl-5-(3,5-dimethoxyphenyl)penta-2,4-dienamide	0.000 ± 0.004***	6.00
15, D		Naphtho[2,3-d][1, 3]dioxol-6-yl(piperidin-1-yl)methanone	0.716 ± 0.059***	26.1
16, D		Naphtho[2,3-d][1, 3]dioxol-5-yl(piperidin-1-yl)methanone	0.836 ± 0.176
17, D		(E)-3-(Naphtho[2,3-d][1, 3]dioxol-6-yl)-1-(piperidin-1-yl)prop-2-en-1-one	0.831 ± 0.097***
18, D		(E)-3-(Naphtho[2,3-d][1, 3]dioxol-5-yl)-1-(piperidin-1-yl)prop-2-en-1-one	0.828 ± 0.057***
19, D		(E)-3-(Naphtho[2,3-d][1, 3]dioxol-5-yl)-N,N-dipropylacrylamide	0.573 ± 0.064***	14.8
20, D		(E)-N,N-Dibutyl-3-(naphtho[2,3-d][1, 3]dioxol-5-yl)acrylamide	0.005 ± 0.010***	7.85
21, E		(5-(Benzo[d][1, 3]dioxol-5-yl)naphthalen-1-yl)(piperidin-1-yl)methanone	0.542 ± 0.119***	15.3
22, E		(5-(Benzo[d][1, 3]dioxol-5-yl)thiophen-2-yl)(piperidin-1-yl)methanone	0.899 ± 0.171
23, E		(2-(Benzo[d][1, 3]dioxol-5-yl)phenyl)(piperidin-1-yl)methanone	0.974 ± 0.055
24, E		(3-(Benzo[d][1, 3]dioxol-5-yl)phenyl)(piperidin-1-yl)methanone	0.790 ± 0.041***	46.0
25, E		3-(Benzo[d][1, 3]dioxol-5-yl)-N,N-dipropylbenzamide	0.682 ± 0.188***	22.3

In order to identify compounds with enhanced activity and to deduce a SAR, nine additional piperamides were isolated from black pepper (2–10). All of them have been described previously as compounds of P. nigrum (2, 5, 10 [23]; 3 [24]; 4 [25]; 6 [26]; 7 [27]; 8 [28]; 9 [29]) and their structures were elucidated by comparing spectral data (NMR, MS, and IR) with those published before (1, 3 [30]; 2, 5, 7, [27]; 4 [25]; 6 [26]; 8 [31]; 9, 10 [32]). In addition to the isolated compounds, 15 further congeners were synthesized (11–25). All piperine analogues were subjected to cell-based assays. Their chemical structures are shown in [Table 1] together with their anti-proliferative effect at 30 µM (expressed in RU).

In general, the chemical structure of the lead compound piperine (1) comprises three main parts: an aromatic moiety (1,3-benzodioxole), a four carbon chain linker region with two conjugated double bonds, and an amidic bound piperidinyl moiety. To derive a SAR, the congeners were clustered by their type of structural modification ([Table 1]).

Cluster A (2–7) focuses on alterations of the linker region, like carbon chain length and number of double bonds. Compound 4, differing from the other derivatives in this group by having seven C atoms with only one double bond in the linker chain, revealed a significant increase in activity (0.014 ± 0.018 RU) in contrast to 1 (0.747 ± 0.065 RU). All other compounds of this cluster, including two piperamides with a pyrrolidinyl (2 and 7) instead of the piperidinyl moiety, showed no significant enhancement of VSMC inhibition.

Cluster B consists of only one compound, the piperidin-dimer chabamide (8), which had no noteworthy inhibitory effect.

Cluster C (9–14) comprises compounds with variations of the aromatic function. Removal or opening of the dioxole ring did not significantly alter the VSMC inhibitory effect in comparison to 1 (11 and 13) or even decreased it (10). The same was observed for compound 9, which in addition has a shortened linker region. In contrast, when the dioxole was replaced by 3-fluoro (12), the proliferation was inhibited significantly (0.426 ± 0.079 RU). Intriguingly, supplementing the aromatic cycle with two methoxy moieties in meta positions and additional replacement of the piperidinyl ring by an n-dibutylamide (14) completely blocked the VSMC proliferation (0.000 ± 0.004 RU).

Derivatives from cluster D (15–20) are characterized by a naphtho dioxole moiety. This compound class does not enhance the VSMC inhibitory activity, unless the piperidinyl moiety was altered to noncyclic substituents, like n-dipropyl- (19) or n-dibutylamide (20), which clearly improved the anti-proliferative activity (0.573 ± 0.064 and 0.005 ± 0.010 RU, respectively). Similar to compound 14, compound 20 was able to decrease VSMC proliferation at 30 µM to nearly zero. Both are characterized by the n-dibutylamide feature.

Compounds from cluster E (21–25) were modified in the linker region by replacing the carbon chain by an aryl spacer. Only 21 and 25, the first containing a naphthalene and the second a benzene cycle as aryl spacer, revealed an increase in anti-proliferative activity with values of 0.542 ± 0.119 and 0.682 ± 0.188 RU, respectively. In the case of 25, the piperidinyl moiety was additionally altered in analogy to compound 19 (cluster D).

To sum up, at a concentration of 30 µM, the natural isolate 4 and the two synthetic compounds 14 and 20, both comprising noncyclic n-dibutylamide substituents at the tertiary amide, were able to stop VSMC proliferation nearly completely. Also 12 showed promising activity below 0.49 RU, whereas 11, 15, 19, 21, 24, and 25 inhibited proliferation in the range of 0.50 to 0.79 RU and had therefore comparable efficacy to 1 (0.747 ± 0.065 RU). Subsequently, IC₅₀s were determined for all derivatives with values below 0.79 RU ([Table 1]). Compounds 11, 15, 24, and 25 showed similar or higher IC₅₀ values than piperine (1) and were therefore not further investigated. Compounds 4, 14, and 20 were highly active with IC₅₀s of 3.38, 6.00, and 7.85 µM, respectively. Also the synthetic derivatives 12 (11.8 µM), 19 (14.8 µM), and 21 (15.3 µM) decreased metabolic activity with lower IC₅₀ values than that of 1.

The used resazurin conversion assay is based on the ability of viable cells to metabolize added resazurin into the highly fluorescent resorufin. Measured fluorescence correlates generally well with cell number. However, redox-active chemicals or treatments modulating the cellular metabolic capacity can potentially interfere with this assay.

Therefore, to confirm the anti-proliferative effect, the two most active compounds, the natural derivative 4 and the synthesized compound 14, were additionally assayed with the 5-bromo-2′-deoxyuridine (BrdU) incorporation assay, which detects integration of BrdU into newly synthesized DNA of dividing cells by immunoassay. As shown in [Fig. 2], the compounds blocked DNA synthesis in treated cells with IC₅₀ values of 4.39 (4) and 6.03 µM (14), which is in accordance to the IC₅₀ values obtained by the resazurin conversion assay ([Table 1]).

An intact and functional endothelium is crucial for vascular health. Optimal vasoprotection is featured by a compound that potently inhibits VSMC proliferation and moreover does not interfere with endothelial cell viability. Hence, we investigated our six most active compounds (4, 12, 14, 19, 20, and 21) as to whether they affect proliferation of HUVECs.

As shown in [Fig. 3], none of the investigated derivatives interacted with HUVEC proliferation at 10 µM or below. Only at a concentration of 30 µM, compounds 14, 19, 20, and 21 influenced HUVEC growth with values above 0.6 RU. The piperamide 4 inhibited HUVEC proliferation at 30 µM stronger than the other compounds. However, the IC₅₀ of 3.38 µM in VSMCs indicates a much higher efficacy in this cell type ([Fig. 3]). Interestingly, compound 12 did not influence HUVEC viability at all up to 30 µM, which renders it a promising cell selective candidate.

All compounds with IC₅₀ values below 16 µM (4, 12, 14, 19, 20, and 21) were additionally tested for their cytotoxic effects by measuring LDH inside cells and in cell supernatants. All compounds were well tolerated up to 30 µM, except compounds 4 and 14, which showed some cytotoxic effects at 30 µM ([Fig. 4]).

In this study, we identified six compounds (4, 12, 14, 19, 20, and 21) out of 24 investigated piperine derivatives with enhanced anti-proliferative activity in VSMC compared to the natural scaffold piperine (1) itself (IC₅₀ of 21.6 µM). This major alkaloid from black pepper was just recently also described by Lee et al. to inhibit VSMC migration at a concentration of 100 µM [22]. Piperine and congeners are reported to modulate GABA_A receptors and to activate transient receptor potential vanilloid type 1 receptors [33], [34]. In pharmacokinetic studies performed in Wistar rats, 1 revealed an absolute oral bioavailability of 24 % [35], and is reported to be a potent bioavailability enhancer for drugs by inhibiting CYP3A4 and human P-glycoprotein [36], [37], [38], [39]. Beside its multitude of described activities, e.g. analgesic, anti-pyretic, anti-convulsive, anti-oxidant, anti-depressant, anti-apoptotic, and anti-viral [12], [19], [20], Kumar et al. demonstrated an anti-inflammatory activity for 1 by suppressing NF-kappa B and Ikappa B kinase activation [40]. These reported activities together with our findings render the piperine congeners a promising compound class endowed with a beneficial pharmacological profile to combat abnormal proliferation, migration and inflammation of VSMCs as key elements in the development of atherosclerosis and restenosis.

Among the investigated piperamides, compounds 4, 12, 14, and 20 were identified as potent VSMC proliferation blockers with IC₅₀ values of 3.38, 11.8, 6.00, and 7.85 µM, respectively. Whereas 4 and 14 showed some cytotoxicity at higher concentrations (30 µM), compounds 12 and 20 were identified as non-cytotoxic and selective inhibitors of VSMC proliferation. The obtained insights into the piperamidesʼ SAR yields knowledge that enables further development of more potent and cell type-specific VSMC growth inhibitors, based on the natural alkaloid scaffold piperine.

Materials and Methods

Isolation of amide alkaloids (1–10) from Piper nigrum fruits

General experimental procedures: NMR experiments were performed by using Bruker TXI600 NMR spectrometers, operating at 600 MHz. The samples were measured in MeOD (calibrated to the residual nondeuterated solvent signals). MS analysis was performed on a Bruker Esquire 3000 plus ion-trap mass spectrometer equipped with electrospray ionization (ESI) in the positive and negative modes: spray voltage, 4.5 kV; sheath gas, N₂, 30 psi; dry gas, N₂, 6 L/min, 350 °C; scanning range, m/z 50–1000. Column chromatography was performed using Merck silica gel 60 (40–63 µm) and Sephadex® LH-20 (20–100 µm; Pharmacia). Fast centrifugal partition chromatography was carried out on a FCPC A200 instrument from Kromaton (column volume: 200 mL). Fractions obtained from all chromatographic steps were analysed by TLC (mobile phase: toluene/diethyl ether/dioxane (6.25 : 2.15 : 1.6); stationary phase: Merck silica gel 60 PF₂₅₄, detected with staining reagents vanillin/H₂SO₄ at vis, UV₂₅₄, UV₃₆₆). HPLC was performed on a Shimadzu UFLC-XR instrument with a photodiode array (PDA) detector. LC-parameters: stationary phase: Phenomenex HyperClone, 150 × 4.60 mm, 5 µm; temperature: 35 °C; mobile phase: water (A); methanol (B); flow rate 1.0 mL/min; UV detection wavelength: 205, 254, 270, 360 nm; injection volume: 10 µL; gradient: 85 % A, 15 % B; 5 min; 45 % A, 55 % B; 30 min; 32 % A, 68 % B; 41 min; 2 % A, 98 % B; 50 min; 2 % A, 98 % B. Semi-preparative HPLC was performed on a Dionex (Thermo Scientific) UltiMate 3000 instrument with a photodiode array detector (DAD). LC-parameters: stationary phase: Phenomenex Gemini-NX 250 × 10.00 mm, 5 µm; temperature: 35 °C; mobile phase: water (A); methanol (B); flow rate 2.0 mL/min; UV detection wavelength: 270 nm. All chemicals and solvents used were analytical grade.

Plant material: Black pepper (dried unripe fruits of P. nigrum) was obtained from Dr. Kottas – Heilkräuter (Charge KLA80342). A voucher specimen (JR-20121114-A11) is deposited in the Herbarium of the Department of Pharmacognosy, University of Vienna, Austria.

General Procedure: 500 g dried ground material were defatted with 500 mL of n-hexane (ultrasonic bath, 1 h) and subsequently macerated with dichloromethane (at RT, for 72 h) followed by maceration with MeOH (at RT, three times for 72 h each). After removal of the solvent under vacuum, a combined CH₂Cl₂/MeOH (48.21 g) extract was obtained and subjected to silica gel column chromatography (Merck silica gel 60 PF₂₅₄, 37 cm × 3.5 cm) using a gradient system of CH₂Cl₂ and MeOH (CH₂Cl₂ 1000 mL; CH₂Cl₂/MeOH, v/v, 98 : 2; 96 : 4; 90 : 10; 50 : 50; MeOH; 500 mL each) to give 10 fractions (S1F1-S1F10). From fraction S1F4 (elution volume 890–1170 mL) 1.6 g 1 could be gained by crystallization from methanol. Fraction S1F5 (2.2 g, elution volume 1171–1561 mL) was further separated by means of Sephadex LH-20 column chromatography (mobile phase: MeOH, 81 cm × 2.5 cm) to give 8 fractions (S2F1-S2F8). Fractions S2F4 and S2F5 (elution volume 235–269 mL) were combined and subjected to silica gel column chromatography (Merck silica gel 60 PF₂₅₄, 87 cm × 2 cm) using a gradient system of CH₂Cl₂/EtOAc/MeOH (CH₂Cl₂ 1200 mL; CH₂Cl₂/EtOAc, v/v, 99 : 1; 98 : 2; 97 : 3; 96 : 4; 95 : 5; 90 : 10; 80 : 20, 500 mL each; 50 : 50, 350 mL; EtOAc 500 mL; MeOH 500 mL) yielding in 10 fractions (S3F0-S3F9). Fraction S3F7 (100.71 mg, elution volume 3483–3782 mL) was purified via Sephadex LH-20 (mobile phase: CH₂Cl₂, 98 cm × 2 cm) to yield 11 fractions (S4F0-S4F10) and 2.0 mg of 9 (S4F8, elution volume 165–177 mL). Fraction S3F6 (49.98 mg, elution volume 3288–3482 mL) was subjected to Sephadex LH-20 column chromatography (mobile phase: CH₂Cl₂, 98 cm × 2 cm) to gain 10 fractions (S5F0–S5F9) and 4.43 mg of 10 (S5F6, elution volume, 135–156 mL). Fraction S3F5 (elution volume 3118–3287 mL) was chromatographed over Sephadex LH-20 (mobile phase: CH₂Cl₂, 98 cm × 2 cm) giving 8 fractions (S6F0–F7). Fraction S6F2 (12.17 mg, elution volume 88–103 mL) was further separated using semi-preparative HPLC resulting in 2.08 mg of 7 (elution volume: 64–69 mL; purified via Sephadex LH-20 with MeOH). Fractions S4F3 (42.04 mg, elution volume 99–111 mL) and S4F4 (28.25 mg, elution volume 112–130 mL) were combined and subjected to Sephadex LH-20 column chromatography (mobile phase: MeOH, 83 cm × 2.5 cm) to give 7 fractions (S7F0–F6). From fraction S7F4 (57.46 mg, elution volume 240–257 mL) 21.00 mg of 2 were obtained by crystallization form methanol.

3.9 g of fraction S1F4 (elution volume 890–1170 mL) were fractionated by fast centrifugal partition chromatography using a biphasic equilibrated solvent mixture, consisting of n-hexane/ethyl acetate/MeOH/H₂O (2 : 6 : 3 : 2, v/v/v/v). The instrument was operated in descending mode (lower phase as mobile phase) and equilibrated at 800 rpm. The sample was dissolved in a mixture of mobile and stationary phase (1 : 1, v/v) and separation was carried out at a flow rate of 1 mL/min and a rotation speed of 1200 rpm in a counter clockwise direction to result in 14 fractions (S8F1–F14). Fraction S8F5 (126.96 mg, elution volume 256–269 mL) was separated using semi-preparative HPLC gradient (MeOH/H₂O, v/v, 73 : 27 to 80 : 20 in 12 min; 80 : 20 to 85 : 15 in 28 min; 85 : 15 to 98 : 2 in 1 min) yielding in 11 fractions (S9F1–F11). Fraction S9F4 (9.53 mg, elution volume 43.0–45.2 mL) was identified as compound 3, fraction S9F5 (3.99 mg, elution volume 47.5–49.5 mL) as compound 6, fraction S9F7 (3.51 mg, elution volume 51.7–54.7 mL) as compound 5, fraction S9F8 (2.74 mg, elution volume 54.8–57.8 mL) as compound 8, and S9F10 (3.46 mg, elution volume 61.0–64.0 mL) as compound 4. All pure compounds obtained from semi-preparative HPLC were purified over Sephadex LH-20. The physical and spectroscopic data of compounds 1 to 10 agreed with those published previously for piperine (1) [30], piperylin (2) [27], piperoleine A (3) [30], pipertipine (4) [25], pipernonaline (5) [27], dehydropipernonaline (6) [26], (2E,4E,8E)-9-(benzo[d][1,3]dioxol-5-yl)-1-(pyrrolidin-1-yl)nona-2,4,8-trien-1-one (7) [27], chabamide (8) [31], N-trans feruloylpiperidine (9) [32], and feruperine (10) [32]. Copies of the original NMR spectra are obtainable from the corresponding author. The purity of all compounds was checked using TLC and LC-MS and revealed to be > 95 % in all cases.

Synthesis of piperine analogs

We have previously reported the synthesis of compounds 14 [41] and 15–25 [33] including procedures in full experimental detail ([Fig. 5]). Purity of all synthesized compounds was > 95 % as determined by HPLC or NMR.

General experimental procedures for the synthesis of compounds 11–13: Unless otherwise noted, chemicals were purchased from commercial suppliers and used without further purification. Flash column chromatography was performed on silica gel 60 from Merck (40–63 mm), whereas most separations were performed by using a Büchi SepacoreTM MPLC system with a 9 g column. Evaporation of solvents was carried out either on standard rotary evaporators or on a Christ RVC 2–25 plus centrifugal evaporator with attached cooling system (Christ 2–4 LOplus). For TLC aluminum-backed silica gel was used. Melting points were determined by using a Kofler-type Leica Galen III micro hot stage microscope (Aigner-Unilab Laborfachhandel GmbH) and are uncorrected. For compounds unknown in the literature high-resolution MS was performed by E. Rosenberg at Vienna University of Technology, Institute for Chemical Technologies and Analytics; all samples were analyzed by LC-IT-TOF-MS in positive or negative ion detection mode with the recording of MS and MS/MS spectra. NMR-spectra were recorded on a Bruker AC 200 (200 MHz) or a Bruker Avance 400 (400 MHz) spectrometer and chemical shifts are reported in ppm. For assignment of ¹³C multiplicities standard ¹³C, DEPT or APT spectra were recorded. GC–MS runs were performed on a Thermo Finnigan Focus GC/DSQ II with a standard capillary column BGB 5 (ID = 30 m, 0.32 mm; Fisher Scientific GmbH) using standardized temperature programs: “short method” (2 min at 100 °C, 18 °C/min until 280 °C, 3 min at 280 °C) and “long method” (2 min at 100 °C, 18 °C/min until 280 °C, 10 min at 280 °C).

General procedure: Palladium(II)acetate (4 mol%, 0.2 mmol, 2.2 mg), tri-o-tolylphosphine (8 mol%, 0.4 mmol, 6 mg), arylbromide (0.5 mmol) and (piperidin-1-yl)pentadienone (0.5 mmol) were placed in a screw-cap vial and set under argon. Acetonitrile (1 ml) was added followed by triethylamine (1.5 equiv., 0.75 mmol, 0.1 ml). The reaction mixture was heated to 70 °C for 48 h and monitored with TLC and GC/MS. After letting the reaction cool to room temperature, the crude reaction mixture was adsorbed on silica and directly subjected to flash column chromatography (eluent LP/EA 20 %).

(2E,4E)-5-Phenyl-1-(piperidin-1-yl)penta-2,4-dien-1-one (11): Reaction stopped after 16 hours; 39 % yield (47 mg, 0.19 mmol), pale yellow solid, m. p. 85–87 °C, R_f 0.4 (LP/EA 2 : 1), ¹H NMR (CDCl₃, 200 MHz): δ = 1.55–1.70 (m, 6H, CH₂), 3.52–3.62 (m, 4H, CH₂), 6.77–6.98 (m, 2H), 7.22–7.38 (m, 3H), 7.42–7.49 (m, 3H); ¹³C NMR (CDCl₃, 50 MHz): δ 24.6 (t), 25.6 (t), 26.7 (t), 43.2 (t), 46.9 (t), 120.9 (d), 126.9 (d), 127.0 (d), 128.5 (d), 128.7 (d), 136.4 (s), 138.5 (d), 142.3 (d), 165.3 (s), HR ESIMS m/z 242.1544 [M+H]⁺ (calculated for C₁₆H₁₉NO 242.1539, diff 2.06 ppm).

(2E,4E)-5-(4-Fluorophenyl)-1-(piperidin-1-yl)penta-2,4-dien-1-one (12): 58 % yield (75 mg, 0.29 mmol), pale yellow solid, m. p. 77 °C, R_f 0.1 (LP/EA 20 %), ¹H NMR (CDCl₃, 200 MHz): δ = 1.48–1.67 (m, 6H), 3.49–3.59 (m, 4H), 6.44 (d, J = 14.7 Hz, 1H), 6.75 (s, 1H), 6.78 (d, J = 3.0 Hz, 1H), 6.98 (t, J = 8.6 Hz, 2H), 7.31–7.44 (m, 3H); ¹³C NMR (CDCl₃, 50 MHz): δ 24.6 (t), 25.6 (t), 26.7 (t), 43.3 (t), 46.9 (t), 115.7 (d, ²  J_C–F  = 21.8 Hz), 120.9 (d), 126.76 (d, ⁵  J_C–F  = 2.5 Hz), 128.5 (d, ³  J_C–F  = 8.1 Hz), 132.7 (d, ⁴  J_C–F  = 2.9 Hz), 137.1 (d), 142.1 (d), 162.8 (d, ¹  J_C–F  = 249.3 Hz), 165.3 (s), HR ESIMS m/z 260.1450 [M + H]⁺ (calculated for C₁₆H₁₈FNO 260.1445, diff 1.92 ppm).

(2E,4E)-5-(4-Methoxyphenyl)-1-(piperidin-1-yl)penta-2,4-dien-1-one (13): 59 % yield (80 mg, 0.29 mmol), pale yellow solid, m. p. 93 °C, R_f 0.06 (LP/EA 20 %), ¹H NMR (CDCl₃, 200 MHz): δ 1.48–1.70 (m, 6H), 3.52–3.62 (m, 4H), 3.80 (s, 3H), 6.43 (d, J = 14.6 Hz, 1H), 6.77–6.80 (m, 2H), 6.84–6.95 (m, 4H), 7.34–7.42 (m, 2H), 7.46 (dd, J = 6.6, 3.7 Hz, 1H); ¹³C NMR (CDCl₃, 50 MHz): δ = 24.7 (t), 25.6 (t), 26.7 (t), 43.2 (t), 46.9 (t), 55.3 (q), 114.0 (d), 119.6 (d), 125.0 (d), 128.3 (d), 129.3 (s), 138.2 (d), 142.8 (d), 160.0 (s), 165.5 (s), HR ESIMS m/z 272.1655 [M + H]⁺ (calculated for C₁₇H₂₁NO₂ 272.1645, diff 3.76 ppm).

Bioactivity evaluation

Chemicals and reagents: Rat aortic VSMCs were purchased from Lonza. HUVECs (HUVECtert) were provided by Dr. Hannes Stockinger (Medical University of Vienna) [42], [43]. Paclitaxel (taxol) and digitonin were obtained from Sigma-Aldrich with a purity of ≥ 95 % and ~ 50 %, respectively.

Cell culture: VSMCs were grown in Dulbeccoʼs modified essential medium – F12 (1 : 1) supplemented with 20 % fetal calf serum and gentamycin. For all tests, VSMCs were seeded in 96-well plates at 5 × 10³ cells/well and incubated for 24 h (37 °C, 5 % CO₂). Then the cells were serum-starved for another 24 h to render them quiescent. HUVECtert were cultivated in EBM supplemented with 10 % fetal calf serum, 100 U/mL penicillin, 100 µg/mL streptomycin, 1 % amphotericin B and EBM™ SingleQuots®, containing recombinant human epidermal growth factor, hydrocortisone, gentamicin sulphate, amphotericin B and 0.4 % bovine brain extract.

Resazurin conversion assay: Quiescent VSMCs were pre-treated for 30 min with compound or vehicle (0.1 % DMSO) as indicated, and subsequently stimulated with PDGF-BB (20 ng/mL) for 48 h. To measure the number of metabolically active VSMCs by resazurin conversion [44], [45], [46], cells were washed with phosphate-buffered saline and incubated for 2 h in serum-free medium containing 10 µg/mL resazurin. Total metabolic activity was measured by monitoring the increase in fluorescence at a wavelength of 590 nm using an excitation wavelength of 535 nm in a 96-well plate reader (Tecan GENios Pro). HUVECtert were seeded in 96-well plates at 5 × 10³ cells/well and incubated for 24 h (37 °C, 5 % CO₂). Then, HUVECtert [42], [43] were treated with compound or vehicle (0.1 % DMSO) as indicated and incubated for another 48 h. Afterwards, cells were washed with phosphate-buffered saline and incubated in culture medium containing 10 µg/mL resazurin for 2 h. The detection step was performed as described above. Results were expressed in RU compared to the fluorescence of PDGF/vehicle-treated control cells, which was set to 1. Paclitaxel (taxol) was used as a reference compound, and inhibited the proliferation of VSMC and HUVECtert with IC₅₀ = 108 nM and 4 nM, respectively [46].

5-bromo-2′-deoxyuridine (BrdU) incorporation assay: Quiescent VSMCs were pre-treated for 30 min with compound, or vehicle (0.1 % DMSO) as indicated and stimulated with PDGF (20 ng/mL) for 2 h. Then BrdU was added to estimate de novo DNA synthesis in VSMCs [47], [48], [49]. 22 h later, the BrdU incorporation was quantified according to the manufacturerʼs instructions (Roche Diagnostics).

Assessment of cytotoxicity: Quiescent VSMCs were pre-treated for 30 min with compound, or vehicle (0.1 % DMSO) as indicated, and subsequently stimulated for 24 h with PDGF-BB (20 ng/mL). Afterwards the supernatant of the treated cells was assessed for soluble cytosolic protein LDH activity, as an indicator for loss of cell membrane integrity and cell death [43], [50]. For estimation of the total LDH, identically treated samples were incubated for 45 min in the presence of 1 % Triton X-100. The released and the total LDH enzyme activity was measured for 30 min in the dark in the presence of 4.5 mg/mL lactate, 0.56 mg/mL NAD⁺, 1.69 U/mL diaphorase, 0.004 % (w/v) bovine serum albumin, 0.15 % (w/v) sucrose, and 0.5 mM 2-p-iodophenyl-3-nitrophenyl tetrazolium chloride. The enzyme reaction was stopped by adding 1.78 mg/mL oxymate and the absorbance was measured at 490 nm. Potential effects on cell viability were estimated as percentage of extracellular LDH enzyme activity. The cytotoxic natural product digitonin (100 µg/mL) was used as a positive control.

Statistical analysis: Statistics were performed by ANOVA/Bonferroni test, using GraphPad PRISM software, version 4.03. The number of experiments is given in the figure legends, and a probability value < 0.05 was considered significant.

Acknowledgements

This work was supported the European Union Seventh Framework Program (EU-FP7) Marie Curie Fellowship (R. L.) and the University of Vienna “Back to Research Grant” (R. L.), as well as by the Austrian Science Fund (FWF): P24587 (C. E. M.), S10704 (NFN-project “Drugs from Nature Targeting Inflammation”), P23317-B11 and P25971-B23.

Conflict of Interest

The authors declare no conflict of interest.

^* Dedicated to Professor Dr. Dr. h. c. mult. Adolf Nahrstedt on the occasion of his 75th birthday.

^# These authors contributed equally to this work.

• References

• 1 Lusis AJ. Atherosclerosis. Nature 2000; 407: 233-241
  CrossrefPubMedSearch in Google Scholar
• 2 Rudijanto A. The role of vascular smooth muscle cells on the pathogenesis of atherosclerosis. Acta Med Indones 2007; 39: 86-93
  PubMedSearch in Google Scholar
• 3 Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993; 362: 801-809
  CrossrefPubMedSearch in Google Scholar
• 4 Katz G, Harchandani B, Shah B. Drug-eluting stents: the past, present, and future. Curr Atheroscler Rep 2015; 17: 11
  CrossrefPubMedSearch in Google Scholar
• 5 Windecker S, Remondino A, Eberli FR, Juni P, Raber L, Wenaweser P, Togni M, Billinger M, Tuller D, Seiler C, Roffi M, Corti R, Sutsch G, Maier W, Luscher T, Hess OM, Egger M, Meier B. Sirolimus-eluting and paclitaxel-eluting stents for coronary revascularization. N Engl J Med 2005; 353: 653-662
  CrossrefPubMedSearch in Google Scholar
• 6 Raja SG. Drug-eluting stents and the future of coronary artery bypass surgery: facts and fiction. Ann Thorac Surg 2006; 81: 1162-1171
  CrossrefPubMedSearch in Google Scholar
• 7 Degertekin M, Serruys PW, Foley DP, Tanabe K, Regar E, Vos J, Smits PC, van der Giessen WJ, van den Brand M, de Feyter P, Popma JJ. Persistent inhibition of neointimal hyperplasia after sirolimus-eluting stent implantation: long-term (up to 2 years) clinical, angiographic, and intravascular ultrasound follow-up. Circulation 2002; 106: 1610-1613
  CrossrefPubMedSearch in Google Scholar
• 8 Iakovou I, Schmidt T, Bonizzoni E, Ge L, Sangiorgi GM, Stankovic G, Airoldi F, Chieffo A, Montorfano M, Carlino M, Michev I, Corvaja N, Briguori C, Gerckens U, Grube E, Colombo A. Incidence, predictors, and outcome of thrombosis after successful implantation of drug-eluting stents. Jama 2005; 293: 2126-2130
  CrossrefPubMedSearch in Google Scholar
• 9 Mehilli J, Byrne RA, Tiroch K, Pinieck S, Schulz S, Kufner S, Massberg S, Laugwitz KL, Schömig A, Kastrati A. Randomized trial of paclitaxel- versus sirolimus-eluting stents for treatment of coronary restenosis in sirolimus-eluting stents: the ISAR-DESIRE 2 (intracoronary stenting and angiographic results: drug eluting stents for in-stent restenosis 2) study. J Am Coll Cardiol 2010; 55: 2710-2716
  CrossrefPubMedSearch in Google Scholar
• 10 Jagella T, Grosch W. Flavour and off-flavour compounds of black and white pepper (Piper nigrum L.) I. Evaluation of potent odorants of black pepper by dilution and concentration techniques. Eur Food Res Technol 1999; 209: 16-21
  CrossrefPubMedSearch in Google Scholar
• 11 Meghwal M, Goswami TK. Piper nigrum and Piperine: an update. Phytother Res 2013; 27: 1121-1130
  CrossrefPubMedSearch in Google Scholar
• 12 Vasavirama K, Upender M. Piperine: a valuable alkaloid from Piper species. Int J Pharm Pharm Sci 2014; 6: 34-38
  PubMedSearch in Google Scholar
• 13 Labruyère B. Determination of pungent constituents of Piper nigrum . J Agr Food Chem 1966; 14: 469-472
  CrossrefPubMedSearch in Google Scholar
• 14 Srinivasan K. Black pepper and its pungent principle-piperine: A review of diverse physiological effects. Crit Rev Food Sci Nutr 2007; 47: 735-748
  CrossrefPubMedSearch in Google Scholar
• 15 Dawid C, Henze A, Frank O, Glabasnia A, Rupp M, Büning K, Orlikowski D, Bader M, Hofmann T. Structural and sensory characterization of key pungent and tingling compounds from black pepper (Piper nigrum L.). J Agric Food Chem 2012; 60: 2884-2895
  CrossrefPubMedSearch in Google Scholar
• 16 Meghwal M, Goshwami T. Chemical composition, nutritional, medicinal and functional properties of black pepper: A review. Open Access Sci Rep 2012; 1: 1-5
  PubMedSearch in Google Scholar
• 17 OʼMahony R, Al-Khtheeri H, Weerasekera D, Fernando N, Vaira D, Holton J, Basset C. Bactericidal and anti-adhesive properties of culinary and medicinal plants against Helicobacter pylori . World J Gastroenterol 2005; 11: 7499-7507
  CrossrefPubMedSearch in Google Scholar
• 18 Agbor GA, Vinson JA, Oben JE, Ngogang JY. Comparative analysis of the in vitro antioxidant activity of white and black pepper. Nutr Res 2006; 26: 659-663
  CrossrefPubMedSearch in Google Scholar
• 19 Singh VK, Singh P, Patel AMA, Yadav KKM. Piperine: delightful surprise to the biological world, made by plant “pepper” and a great bioavailability enhancer for our drugs and supplements. World J Pharm Res 2014; 3: 2084-2098
  PubMedSearch in Google Scholar
• 20 Jiang ZY, Liu WF, Zhang XM, Luo J, Ma YB, Chen JJ. Anti-HBV active constituents from Piper longum . Bioorg Med Chem Lett 2013; 23: 2123-2127
  CrossrefPubMedSearch in Google Scholar
• 21 Zaugg J, Baburin I, Strommer B, Kim HJ, Hering S, Hamburger M. HPLC-based activity profiling: discovery of piperine as a positive GABA(A) receptor modulator targeting a benzodiazepine-independent binding site. J Nat Prod 2010; 73: 185-191
  CrossrefPubMedSearch in Google Scholar
• 22 Lee KP, Lee K, Park WH, Kim H, Hong H. Piperine inhibits platelet-derived growth factor-BB-induced proliferation and migration in vascular smooth muscle cells. J Med Food 2015; 18: 208-215
  CrossrefPubMedSearch in Google Scholar
• 23 Subehan. Usia T, Kadota S, Tezuka Y. Alkamides from Piper nigrum L. and their inhibitory activity against human liver microsomal cytochrome P450 2D6 (CYP2D6). Nat Prod Commun 2006; 1: 1-7
  PubMedSearch in Google Scholar
• 24 Okumura Y, Narukawa M, Iwasaki Y, Ishikawa A, Matsuda H, Yoshikawa M, Watanabe T. Activation of TRPV1 and TRPA1 by black pepper components. Biosci Biotechnol Biochem 2010; 74: 1068-1072
  CrossrefPubMedSearch in Google Scholar
• 25 Siddiqui BS, Gulzar T, Begum S. Amides from the seeds of Piper nigrum Linn. and their insecticidal activity. Heterocycles 2002; 57: 1653-1658
  CrossrefPubMedSearch in Google Scholar
• 26 Lee SW, Rho MC, Park HR, Choi JH, Kang JY, Lee JW, Kim K, Lee HS, Kim YK. Inhibition of diacylglycerol acyltransferase by alkamides isolated from the fruits of Piper longum and Piper nigrum . J Agric Food Chem 2006; 54: 9759-9763
  CrossrefPubMedSearch in Google Scholar
• 27 Wei K, Li W, Koike K, Pei Y, Chen Y, Nikaido T. New amide alkaloids from the roots of Piper nigrum . J Nat Prod 2004; 67: 1005-1009
  CrossrefPubMedSearch in Google Scholar
• 28 Wei K, Li W, Koike K, Chen Y, Nikaido T. Nigramides AS, dimeric amide alkaloids from the roots of Piper nigrum . J Org Chem 2005; 70: 1164-1176
  CrossrefPubMedSearch in Google Scholar
• 29 Inatani R, Nakatani N, Fuwa H. Structure and synthesis of new phenolic amides from Piper nigrum L. Agric Biol Chem 1981; 45: 667-673
  CrossrefPubMedSearch in Google Scholar
• 30 Strunz GM, Finlay H. Concise, efficient new synthesis of pipercide an insecticidal unsaturated amide from Piper nigrum and related compounds. Tetrahedron 1994; 50: 11113-11122
  CrossrefPubMedSearch in Google Scholar
• 31 Rukachaisirikul T, Prabpai S, Champung P, Suksamram A. Chabamide, a novel piperine dimer from stems of Piper chaba . Planta Med 2002; 68: 853-855
  Thieme ConnectPubMedSearch in Google Scholar
• 32 Correa EA, Hogestatt ED, Sterner O, Echeverri F, Zygmunt PM. In vitro TRPV1 activity of piperine derived amides. Bioorg Med Chem Lett 2010; 18: 3299-3306
  CrossrefPubMedSearch in Google Scholar
• 33 Schoffmann A, Wimmer L, Goldmann D, Khom S, Hintersteiner J, Baburin I, Schwarz T, Hintersteininger M, Pakfeifer P, Oufir M, Hamburger M, Erker T, Ecker GF, Mihovilovic MD, Hering S. Efficient modulation of gamma-aminobutyric acid type A receptors by piperine derivatives. J Med Chem 2014; 57: 5602-5619
  CrossrefPubMedSearch in Google Scholar
• 34 Khom S, Strommer B, Schoffmann A, Hintersteiner J, Baburin I, Erker T, Schwarz T, Schwarzer C, Zaugg J, Hamburger M, Hering S. GABA_A receptor modulation by piperine and a non-TRPV1 activating derivative. Biochem Pharmacol 2013; 85: 1827-1836
  CrossrefPubMedSearch in Google Scholar
• 35 Sahu PK, Sharma A, Rayees S, Kour G, Singhi A, Khullar M, Magotra A, Paswan SK, Gupta M, Ahmed I, Roy S, Tikoo MK, Sharma SC, Singh S, Singh G. Pharmacokinetic study of Piperine in wistar rats after oral and intravenous administration. Int J Drug Delivery 2014; 6: 82-87
  PubMedSearch in Google Scholar
• 36 Bhardwaj RK, Glaeser H, Becquemont L, Klotz U, Gupta SK, Fromm MF. Piperine, a major constituent of black pepper, inhibits human P-glycoprotein and CYP3A4. J Pharmacol Exp Ther 2002; 302: 645-650
  CrossrefPubMedSearch in Google Scholar
• 37 Khatri S, Ahmed FJ, Rai P. Formulation and evaluation of floating gastroretentive capsules of acyclovir with piperine as a bioenhancer. Pharma Innovation 2015; 3: 78-81
  PubMedSearch in Google Scholar
• 38 Motiwala MN, Rangari VD. The effect of piperine on oral bioavailability and pharmacokinetics of paclitaxel in rats. Int J Pharm Phytopharm Res 2014; 3: 399-403
  PubMedSearch in Google Scholar
• 39 Wadhwa S, Singhal S, Rawat S. Bioavailability enhancement by piperine: a review. Asian J Biomed Pharm Sci 2014; 4: 1-8
  PubMedSearch in Google Scholar
• 40 Kumar S, Singhal V, Roshan R, Sharma A, Rembhotkar GW, Ghosh B. Piperine inhibits TNF-alpha induced adhesion of neutrophils to endothelial monolayer through suppression of NF-kappaB and IkappaB kinase activation. Eur J Pharmacol 2007; 575: 177-186
  CrossrefPubMedSearch in Google Scholar
• 41 Wimmer L, Schonbauer D, Pakfeifer P, Schoffmann A, Khom S, Hering S, Mihovilovic MD. Developing piperine towards TRPV1 and GABA_A receptor ligands – synthesis of piperine analogs via Heck-coupling of conjugated dienes. Org Biomol Chem 2015; 13: 990-994
  CrossrefPubMedSearch in Google Scholar
• 42 Schiller HB, Szekeres A, Binder BR, Stockinger H, Leksa V. Mannose 6-phosphate/insulin-like growth factor 2 receptor limits cell invasion by controlling alphaVbeta3 integrin expression and proteolytic processing of urokinase-type plasminogen activator receptor. Mol Biol Cell 2009; 20: 745-756
  PubMedSearch in Google Scholar
• 43 Fakhrudin N, Waltenberger B, Cabaravdic M, Atanasov AG, Malainer C, Schachner D, Heiss EH, Liu R, Noha SM, Grzywacz AM, Mihaly-Bison J, Awad EM, Schuster D, Breuss JM, Rollinger JM, Bochkov V, Stuppner H, Dirsch VM. Identification of plumericin as a potent new inhibitor of the NF-kappaB pathway with anti-inflammatory activity in vitro and in vivo . Br J Pharmacol 2014; 171: 1676-1686
  CrossrefPubMedSearch in Google Scholar
• 44 Kurin E, Atanasov AG, Donath O, Heiss EH, Dirsch VM, Nagy M. Synergy study of the inhibitory potential of red wine polyphenols on vascular smooth muscle cell proliferation. Planta Med 2012; 78: 772-778
  Thieme ConnectPubMedSearch in Google Scholar
• 45 Joa H, Vogl S, Atanasov AG, Zehl M, Nakel T, Fakhrudin N, Heiss EH, Picker P, Urban E, Wawrosch C, Saukel J, Reznicek G, Kopp B, Dirsch VM. Identification of ostruthin from Peucedanum ostruthium rhizomes as an inhibitor of vascular smooth muscle cell proliferation. J Nat Prod 2011; 74: 1513-1516
  CrossrefPubMedSearch in Google Scholar
• 46 Liu R, Heiss EH, Sider N, Schinkovitz A, Groblacher B, Guo D, Bucar F, Bauer R, Dirsch VM, Atanasov AG. Identification and characterization of [6]-shogaol from ginger as inhibitor of vascular smooth muscle cell proliferation. Mol Nutr Food Res 2015; 59: 843-852
  CrossrefPubMedSearch in Google Scholar
• 47 Schwaiberger AV, Heiss EH, Cabaravdic M, Oberan T, Zaujec J, Schachner D, Uhrin P, Atanasov AG, Breuss JM, Binder BR, Dirsch VM. Indirubin-3′-monoxime blocks vascular smooth muscle cell proliferation by inhibition of signal transducer and activator of transcription 3 signaling and reduces neointima formation in vivo . Arterioscler Thromb Vasc Biol 2010; 30: 2475-2481
  CrossrefPubMedSearch in Google Scholar
• 48 Blažević T, Schwaiberger AV, Schreiner CE, Schachner D, Schaible AM, Grojer CS, Atanasov AG, Werz O, Dirsch VM, Heiss EH. 12/15-lipoxygenase contributes to platelet-derived growth factor-induced activation of signal transducer and activator of transcription 3. J Biol Chem 2013; 288: 35592-35603
  CrossrefPubMedSearch in Google Scholar
• 49 Liu R, Heiss EH, Guo D, Dirsch VM, Atanasov AG. Capsaicin from chili (Capsicum spp.) inhibits vascular smooth muscle cell proliferation. F1000Res 2015; 4: 26
  PubMedSearch in Google Scholar
• 50 Sepp A, Binns RM, Lechler RI. Improved protocol for colorimetric detection of complement-mediated cytotoxicity based on the measurement of cytoplasmic lactate dehydrogenase activity. J Immunol Methods 1996; 196: 175-180
  CrossrefPubMedSearch in Google Scholar

Correspondence

• References

• 1 Lusis AJ. Atherosclerosis. Nature 2000; 407: 233-241
  CrossrefPubMedSearch in Google Scholar
• 2 Rudijanto A. The role of vascular smooth muscle cells on the pathogenesis of atherosclerosis. Acta Med Indones 2007; 39: 86-93
  PubMedSearch in Google Scholar
• 3 Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993; 362: 801-809
  CrossrefPubMedSearch in Google Scholar
• 4 Katz G, Harchandani B, Shah B. Drug-eluting stents: the past, present, and future. Curr Atheroscler Rep 2015; 17: 11
  CrossrefPubMedSearch in Google Scholar
• 5 Windecker S, Remondino A, Eberli FR, Juni P, Raber L, Wenaweser P, Togni M, Billinger M, Tuller D, Seiler C, Roffi M, Corti R, Sutsch G, Maier W, Luscher T, Hess OM, Egger M, Meier B. Sirolimus-eluting and paclitaxel-eluting stents for coronary revascularization. N Engl J Med 2005; 353: 653-662
  CrossrefPubMedSearch in Google Scholar
• 6 Raja SG. Drug-eluting stents and the future of coronary artery bypass surgery: facts and fiction. Ann Thorac Surg 2006; 81: 1162-1171
  CrossrefPubMedSearch in Google Scholar
• 7 Degertekin M, Serruys PW, Foley DP, Tanabe K, Regar E, Vos J, Smits PC, van der Giessen WJ, van den Brand M, de Feyter P, Popma JJ. Persistent inhibition of neointimal hyperplasia after sirolimus-eluting stent implantation: long-term (up to 2 years) clinical, angiographic, and intravascular ultrasound follow-up. Circulation 2002; 106: 1610-1613
  CrossrefPubMedSearch in Google Scholar
• 8 Iakovou I, Schmidt T, Bonizzoni E, Ge L, Sangiorgi GM, Stankovic G, Airoldi F, Chieffo A, Montorfano M, Carlino M, Michev I, Corvaja N, Briguori C, Gerckens U, Grube E, Colombo A. Incidence, predictors, and outcome of thrombosis after successful implantation of drug-eluting stents. Jama 2005; 293: 2126-2130
  CrossrefPubMedSearch in Google Scholar
• 9 Mehilli J, Byrne RA, Tiroch K, Pinieck S, Schulz S, Kufner S, Massberg S, Laugwitz KL, Schömig A, Kastrati A. Randomized trial of paclitaxel- versus sirolimus-eluting stents for treatment of coronary restenosis in sirolimus-eluting stents: the ISAR-DESIRE 2 (intracoronary stenting and angiographic results: drug eluting stents for in-stent restenosis 2) study. J Am Coll Cardiol 2010; 55: 2710-2716
  CrossrefPubMedSearch in Google Scholar
• 10 Jagella T, Grosch W. Flavour and off-flavour compounds of black and white pepper (Piper nigrum L.) I. Evaluation of potent odorants of black pepper by dilution and concentration techniques. Eur Food Res Technol 1999; 209: 16-21
  CrossrefPubMedSearch in Google Scholar
• 11 Meghwal M, Goswami TK. Piper nigrum and Piperine: an update. Phytother Res 2013; 27: 1121-1130
  CrossrefPubMedSearch in Google Scholar
• 12 Vasavirama K, Upender M. Piperine: a valuable alkaloid from Piper species. Int J Pharm Pharm Sci 2014; 6: 34-38
  PubMedSearch in Google Scholar
• 13 Labruyère B. Determination of pungent constituents of Piper nigrum . J Agr Food Chem 1966; 14: 469-472
  CrossrefPubMedSearch in Google Scholar
• 14 Srinivasan K. Black pepper and its pungent principle-piperine: A review of diverse physiological effects. Crit Rev Food Sci Nutr 2007; 47: 735-748
  CrossrefPubMedSearch in Google Scholar
• 15 Dawid C, Henze A, Frank O, Glabasnia A, Rupp M, Büning K, Orlikowski D, Bader M, Hofmann T. Structural and sensory characterization of key pungent and tingling compounds from black pepper (Piper nigrum L.). J Agric Food Chem 2012; 60: 2884-2895
  CrossrefPubMedSearch in Google Scholar
• 16 Meghwal M, Goshwami T. Chemical composition, nutritional, medicinal and functional properties of black pepper: A review. Open Access Sci Rep 2012; 1: 1-5
  PubMedSearch in Google Scholar
• 17 OʼMahony R, Al-Khtheeri H, Weerasekera D, Fernando N, Vaira D, Holton J, Basset C. Bactericidal and anti-adhesive properties of culinary and medicinal plants against Helicobacter pylori . World J Gastroenterol 2005; 11: 7499-7507
  CrossrefPubMedSearch in Google Scholar
• 18 Agbor GA, Vinson JA, Oben JE, Ngogang JY. Comparative analysis of the in vitro antioxidant activity of white and black pepper. Nutr Res 2006; 26: 659-663
  CrossrefPubMedSearch in Google Scholar
• 19 Singh VK, Singh P, Patel AMA, Yadav KKM. Piperine: delightful surprise to the biological world, made by plant “pepper” and a great bioavailability enhancer for our drugs and supplements. World J Pharm Res 2014; 3: 2084-2098
  PubMedSearch in Google Scholar
• 20 Jiang ZY, Liu WF, Zhang XM, Luo J, Ma YB, Chen JJ. Anti-HBV active constituents from Piper longum . Bioorg Med Chem Lett 2013; 23: 2123-2127
  CrossrefPubMedSearch in Google Scholar
• 21 Zaugg J, Baburin I, Strommer B, Kim HJ, Hering S, Hamburger M. HPLC-based activity profiling: discovery of piperine as a positive GABA(A) receptor modulator targeting a benzodiazepine-independent binding site. J Nat Prod 2010; 73: 185-191
  CrossrefPubMedSearch in Google Scholar
• 22 Lee KP, Lee K, Park WH, Kim H, Hong H. Piperine inhibits platelet-derived growth factor-BB-induced proliferation and migration in vascular smooth muscle cells. J Med Food 2015; 18: 208-215
  CrossrefPubMedSearch in Google Scholar
• 23 Subehan. Usia T, Kadota S, Tezuka Y. Alkamides from Piper nigrum L. and their inhibitory activity against human liver microsomal cytochrome P450 2D6 (CYP2D6). Nat Prod Commun 2006; 1: 1-7
  PubMedSearch in Google Scholar
• 24 Okumura Y, Narukawa M, Iwasaki Y, Ishikawa A, Matsuda H, Yoshikawa M, Watanabe T. Activation of TRPV1 and TRPA1 by black pepper components. Biosci Biotechnol Biochem 2010; 74: 1068-1072
  CrossrefPubMedSearch in Google Scholar
• 25 Siddiqui BS, Gulzar T, Begum S. Amides from the seeds of Piper nigrum Linn. and their insecticidal activity. Heterocycles 2002; 57: 1653-1658
  CrossrefPubMedSearch in Google Scholar
• 26 Lee SW, Rho MC, Park HR, Choi JH, Kang JY, Lee JW, Kim K, Lee HS, Kim YK. Inhibition of diacylglycerol acyltransferase by alkamides isolated from the fruits of Piper longum and Piper nigrum . J Agric Food Chem 2006; 54: 9759-9763
  CrossrefPubMedSearch in Google Scholar
• 27 Wei K, Li W, Koike K, Pei Y, Chen Y, Nikaido T. New amide alkaloids from the roots of Piper nigrum . J Nat Prod 2004; 67: 1005-1009
  CrossrefPubMedSearch in Google Scholar
• 28 Wei K, Li W, Koike K, Chen Y, Nikaido T. Nigramides AS, dimeric amide alkaloids from the roots of Piper nigrum . J Org Chem 2005; 70: 1164-1176
  CrossrefPubMedSearch in Google Scholar
• 29 Inatani R, Nakatani N, Fuwa H. Structure and synthesis of new phenolic amides from Piper nigrum L. Agric Biol Chem 1981; 45: 667-673
  CrossrefPubMedSearch in Google Scholar
• 30 Strunz GM, Finlay H. Concise, efficient new synthesis of pipercide an insecticidal unsaturated amide from Piper nigrum and related compounds. Tetrahedron 1994; 50: 11113-11122
  CrossrefPubMedSearch in Google Scholar
• 31 Rukachaisirikul T, Prabpai S, Champung P, Suksamram A. Chabamide, a novel piperine dimer from stems of Piper chaba . Planta Med 2002; 68: 853-855
  Thieme ConnectPubMedSearch in Google Scholar
• 32 Correa EA, Hogestatt ED, Sterner O, Echeverri F, Zygmunt PM. In vitro TRPV1 activity of piperine derived amides. Bioorg Med Chem Lett 2010; 18: 3299-3306
  CrossrefPubMedSearch in Google Scholar
• 33 Schoffmann A, Wimmer L, Goldmann D, Khom S, Hintersteiner J, Baburin I, Schwarz T, Hintersteininger M, Pakfeifer P, Oufir M, Hamburger M, Erker T, Ecker GF, Mihovilovic MD, Hering S. Efficient modulation of gamma-aminobutyric acid type A receptors by piperine derivatives. J Med Chem 2014; 57: 5602-5619
  CrossrefPubMedSearch in Google Scholar
• 34 Khom S, Strommer B, Schoffmann A, Hintersteiner J, Baburin I, Erker T, Schwarz T, Schwarzer C, Zaugg J, Hamburger M, Hering S. GABA_A receptor modulation by piperine and a non-TRPV1 activating derivative. Biochem Pharmacol 2013; 85: 1827-1836
  CrossrefPubMedSearch in Google Scholar
• 35 Sahu PK, Sharma A, Rayees S, Kour G, Singhi A, Khullar M, Magotra A, Paswan SK, Gupta M, Ahmed I, Roy S, Tikoo MK, Sharma SC, Singh S, Singh G. Pharmacokinetic study of Piperine in wistar rats after oral and intravenous administration. Int J Drug Delivery 2014; 6: 82-87
  PubMedSearch in Google Scholar
• 36 Bhardwaj RK, Glaeser H, Becquemont L, Klotz U, Gupta SK, Fromm MF. Piperine, a major constituent of black pepper, inhibits human P-glycoprotein and CYP3A4. J Pharmacol Exp Ther 2002; 302: 645-650
  CrossrefPubMedSearch in Google Scholar
• 37 Khatri S, Ahmed FJ, Rai P. Formulation and evaluation of floating gastroretentive capsules of acyclovir with piperine as a bioenhancer. Pharma Innovation 2015; 3: 78-81
  PubMedSearch in Google Scholar
• 38 Motiwala MN, Rangari VD. The effect of piperine on oral bioavailability and pharmacokinetics of paclitaxel in rats. Int J Pharm Phytopharm Res 2014; 3: 399-403
  PubMedSearch in Google Scholar
• 39 Wadhwa S, Singhal S, Rawat S. Bioavailability enhancement by piperine: a review. Asian J Biomed Pharm Sci 2014; 4: 1-8
  PubMedSearch in Google Scholar
• 40 Kumar S, Singhal V, Roshan R, Sharma A, Rembhotkar GW, Ghosh B. Piperine inhibits TNF-alpha induced adhesion of neutrophils to endothelial monolayer through suppression of NF-kappaB and IkappaB kinase activation. Eur J Pharmacol 2007; 575: 177-186
  CrossrefPubMedSearch in Google Scholar
• 41 Wimmer L, Schonbauer D, Pakfeifer P, Schoffmann A, Khom S, Hering S, Mihovilovic MD. Developing piperine towards TRPV1 and GABA_A receptor ligands – synthesis of piperine analogs via Heck-coupling of conjugated dienes. Org Biomol Chem 2015; 13: 990-994
  CrossrefPubMedSearch in Google Scholar
• 42 Schiller HB, Szekeres A, Binder BR, Stockinger H, Leksa V. Mannose 6-phosphate/insulin-like growth factor 2 receptor limits cell invasion by controlling alphaVbeta3 integrin expression and proteolytic processing of urokinase-type plasminogen activator receptor. Mol Biol Cell 2009; 20: 745-756
  PubMedSearch in Google Scholar
• 43 Fakhrudin N, Waltenberger B, Cabaravdic M, Atanasov AG, Malainer C, Schachner D, Heiss EH, Liu R, Noha SM, Grzywacz AM, Mihaly-Bison J, Awad EM, Schuster D, Breuss JM, Rollinger JM, Bochkov V, Stuppner H, Dirsch VM. Identification of plumericin as a potent new inhibitor of the NF-kappaB pathway with anti-inflammatory activity in vitro and in vivo . Br J Pharmacol 2014; 171: 1676-1686
  CrossrefPubMedSearch in Google Scholar
• 44 Kurin E, Atanasov AG, Donath O, Heiss EH, Dirsch VM, Nagy M. Synergy study of the inhibitory potential of red wine polyphenols on vascular smooth muscle cell proliferation. Planta Med 2012; 78: 772-778
  Thieme ConnectPubMedSearch in Google Scholar
• 45 Joa H, Vogl S, Atanasov AG, Zehl M, Nakel T, Fakhrudin N, Heiss EH, Picker P, Urban E, Wawrosch C, Saukel J, Reznicek G, Kopp B, Dirsch VM. Identification of ostruthin from Peucedanum ostruthium rhizomes as an inhibitor of vascular smooth muscle cell proliferation. J Nat Prod 2011; 74: 1513-1516
  CrossrefPubMedSearch in Google Scholar
• 46 Liu R, Heiss EH, Sider N, Schinkovitz A, Groblacher B, Guo D, Bucar F, Bauer R, Dirsch VM, Atanasov AG. Identification and characterization of [6]-shogaol from ginger as inhibitor of vascular smooth muscle cell proliferation. Mol Nutr Food Res 2015; 59: 843-852
  CrossrefPubMedSearch in Google Scholar
• 47 Schwaiberger AV, Heiss EH, Cabaravdic M, Oberan T, Zaujec J, Schachner D, Uhrin P, Atanasov AG, Breuss JM, Binder BR, Dirsch VM. Indirubin-3′-monoxime blocks vascular smooth muscle cell proliferation by inhibition of signal transducer and activator of transcription 3 signaling and reduces neointima formation in vivo . Arterioscler Thromb Vasc Biol 2010; 30: 2475-2481
  CrossrefPubMedSearch in Google Scholar
• 48 Blažević T, Schwaiberger AV, Schreiner CE, Schachner D, Schaible AM, Grojer CS, Atanasov AG, Werz O, Dirsch VM, Heiss EH. 12/15-lipoxygenase contributes to platelet-derived growth factor-induced activation of signal transducer and activator of transcription 3. J Biol Chem 2013; 288: 35592-35603
  CrossrefPubMedSearch in Google Scholar
• 49 Liu R, Heiss EH, Guo D, Dirsch VM, Atanasov AG. Capsaicin from chili (Capsicum spp.) inhibits vascular smooth muscle cell proliferation. F1000Res 2015; 4: 26
  PubMedSearch in Google Scholar
• 50 Sepp A, Binns RM, Lechler RI. Improved protocol for colorimetric detection of complement-mediated cytotoxicity based on the measurement of cytoplasmic lactate dehydrogenase activity. J Immunol Methods 1996; 196: 175-180
  CrossrefPubMedSearch in Google Scholar