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Cyclocurcumin, an Antivasoconstrictive Constituent of Curcuma longa (Turmeric) Keunyoung Kim,† Jung-Jun Kim,† Yeryeon Jung,† Ji-Yoon Noh,† Ahmed Shah Syed,‡ Chul Young Kim,‡ Moo-Yeol Lee,§ Kyung-Min Lim,⊥ Ok-Nam Bae,*,‡ and Jin-Ho Chung*,† †
College of Pharmacy, Seoul National University, Seoul 08826, Korea College of Pharmacy, Hanyang University, Ansan 15588, Korea § College of Pharmacy, Dongguk University, Goyang 10326, Korea ⊥ College of Pharmacy, Ewha Womans University, Seoul 03760, Korea ‡
S Supporting Information *
ABSTRACT: Despite the increasing attention on the therapeutic potential of Curcuma longa (turmeric), the biological activities of curcuminoids other than curcumin are not well understood. Here, we investigated antivasoconstrictive activities of C. longa extract and its ingredients using freshly isolated rat aortic rings. C. longa extract significantly suppressed agonist-stimulated vasoconstriction, and cyclocurcumin was found to be the most potent (IC50 against phenylephrine-induced vasoconstriction: 14.9 ± 1.0 μM) among the 10 tested ingredients including four curcuminoids. Cyclocurcumin significantly inhibited contraction of vascular smooth muscle, which was mediated by the suppression of myosin-light-chain phosphorylation and calcium influx via the Ltype calcium channel. The inhibitory effect of cyclocurcumin was observed to be reversible and without cytotoxicity. Taken together, we demonstrated that cyclocurcumin, a bioactive ingredient in C. longa, may have a therapeutic potential as a novel antivasoconstrictive natural product.
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curcuminoids may reduce circulating levels of C-reactive protein (CRP), a key biomarker of CVDs. Potential therapeutic activity of curcuminoids on pulmonary hypertension has also been suggested.15 However, the effects of curcuminoids on vascular tone, an important therapeutic target for CVDs and CVD-related mortality, have yet to be established. To investigate the antivasoconstrictive effects of curcuminoids, the effects of C. longa extract on vascular contraction induced by phenylephrine (PE), an adrenergic contractile agonist, were examined in a freshly isolated rat aortic ring system (Figure 1A). C. longa extract (0.1%, 1 mg/mL) significantly inhibited PE-induced vasocontraction. To identify the active components for the antivasoconstrictive effects of C. longa, 10 major ingredients of C. longa were examined for the inhibition of vasocontraction (25 μM: Figure 1B). As a result, demethoxycurcumin and cyclocurcumin significantly inhibited PE-induced vasocontraction. Especially, cyclocurcumin exhibited a strong potency and almost abolished PE-induced vasoconstriction at 25 μM. Cyclocurcumin inhibited PEinduced vasocontraction in a concentration-dependent manner (IC50 = 14.9 ± 1.0 μM), of which potency was superior to that
urcuminoids, the family of curcumin and curcumin derivatives, are major components of Curcuma longa (turmeric; C. longa), which has long been used as a yellow curry spice and a traditional herbal medicine.1,2 Curcumin is believed to account for the diverse therapeutic effects of C. longa since it represents up to 80% of all the curcuminoids in this plant.3 Accordingly, intensive efforts have been made to elucidate the pharmacological activities of curcumin including antioxidant, anti-inflammatory, neuroprotective, and chemopreventive effects.2,4,5 In addition to curcumin, other curcuminoids have been isolated from C. longa,6 which include bisdemethoxycurcumin, demethoxycurcumin, and cyclocurcumin, but there exist few published studies on their biological activities. Of note, recent studies illuminated that these minor curcuminoids have distinct pharmacological effects and, more importantly, may act synergistically with curcumin,7,8 suggesting that their biological activities should be investigated in more detail. Accumulating evidence suggests that curcumin or C. longa is effective against cardiovascular diseases (CVDs)2,9 and CVDrelated mortality. Against this backdrop, beneficial effects of curcuminoids on CVDs have been studied that include cholesterol-lowering activity,10,11 modulation of vasoactive factors,12 and alleviation of myocardial damage.13 Sahebkar14 has published meta-analysis of human studies suggesting that © 2017 American Chemical Society and American Society of Pharmacognosy
Received: May 2, 2016 Published: January 9, 2017 196
DOI: 10.1021/acs.jnatprod.6b00331 J. Nat. Prod. 2017, 80, 196−200
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Figure 1. Effects of C. longa extract and curcuminoids on agonist-induced vasoconstriction. (A and B) Effect of C. longa extract (C.L.; A) or major ingredients in C. longa (25 μM; B) on phenylephrine (PE)-induced vasoconstriction examined in freshly isolated rat aortic rings. (C) Concentrationdependent effect of cyclocurcumin (left panel) or curcumin (right panel) on PE-induced vasoconstriction. (D) Serotonin (5-HT) was used to induce vasoconstriction after treatment of cyclocurcumin. Values are mean ± SEM of at least three independent experiments. Emodin (25 μM) was used as a positive control for antivasoconstrictive activity in isolated rat aortic rings as previously reported.18 *Significant differences from the control (p < 0.05).
of curcumin (IC50 > 100 μM) (Figure 1C), yielding a Schild plot-based pA2 value of 5.2 ± 0.1. The amount of cyclocurcumin was analyzed to be 1.84 mg in 1 g of crude extract, while curcumin was 122.92 mg/g, as determined by HPLC quantification (Figure S1 in the Supporting Information). The antivasoconstrictive effect of cyclocurcumin was reproduced with other types of vascular agonists (serotonin; 5HT) (Figure 1D), suggesting that cyclocurcumin may target contractile machinery rather than specific agonist−receptor interaction. Vascular tone is regulated by the balance between smooth muscle-dependent contraction and endothelium-dependent relaxation.16 To elucidate the mechanisms underlying antivasoconstrictive effects of cyclocurcumin, an endotheliumdenuded aortic ring system was employed. The inhibitory activity of cyclocurcumin was maintained in aortic rings without endothelium, suggesting that it targets contractile pathways in vascular smooth muscle (Figure 2A). Inhibition of vascular smooth muscle contraction by cyclocurcumin was also demonstrated in primary vascular smooth muscle cells as shown by reduced generation of phospho-myosin light chain (pMLC), a key molecular activation event in the vascular contraction (Figure 2B). Phosphorylation of MLC and vasocontraction are initiated by an agonist-induced increase in intracellular calcium level. The effect of cyclocurcumin on intracellular calcium increase was examined in primary vascular smooth muscle cells. Cyclocurcumin inhibited influx of intracellular calcium in a dose-dependent manner (Figure 2C). Agonist-induced calcium influx is mainly accomplished by opening of L-type calcium channels.17 Cyclocurcumin inhibited L-type calcium channel-mediated vasoconstriction in a
concentration-dependent manner, as observed in aortic rings stimulated by 60 mM K+ (IC50 = 18.5 ± 4.3 μM; Figure 2D) or by the specific agonist Bay K8644 (IC50 = 13.5 ± 1.1 μM; Figure 2E). Lastly, washout experiments demonstrated that the anticontractile effect of cyclocurcumin was reversible, which was in contrast to the irreversible inhibition manifested by a vascular toxicant, monomethylarsonous acid (MMA3+) (Figure 3A). Nonspecific tissue damage was not observed in cyclocurcuminexposed aortic rings as measured by a TUNEL assay or leakage of lactate dehydrogenase (LDH) (Figure 3B and C). In this study, we demonstrated that cyclocurcumin, a previously unnoticed curcuminoid in C. longa, can have anticontractile effects through modulating L-type calcium channel-mediated calcium influx in vascular smooth muscle. We believe that our study may give new insights into the possible contribution of cyclocurcumin in biological benefits of C. longa, supporting the need to re-evaluate the therapeutic potential of curcuminoids and cyclocurcumin. Further studies including in vivo demonstration of antihypertensive effects and elucidation of the metabolic/pharmacokinetic profile of cyclocurcumin and other curcuminoids would be necessary to fully illustrate the therapeutic benefits and mode of action for cardiovascular protection manifested by C. longa.
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EXPERIMENTAL SECTION
Chemicals and Reagents. Phenylephrine, serotonin creatinine sulfate, NADH, pyruvate, (−)-(S)-Bay K8644, and DMSO were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Cyclocurcmin (95.6% pure as determined by HPLC, Karl Fischer (water content), GC (residual solvent) including mass spectroscopy, and NMR profiles by the manufacturer) was obtained from 197
DOI: 10.1021/acs.jnatprod.6b00331 J. Nat. Prod. 2017, 80, 196−200
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Figure 2. Effect of cyclocurcumin on Ca2+ mobilization and smooth muscle contraction. (A) After cyclocurcumin was used to treat aortic rings without an endothelium, concentration-dependent effects on 5-HT-induced vasoconstriction were examined. Emodin (25 μM) was used as a positive control. (B) 5-HT-enhanced phosphorylation of myosin light chain (MLC) in primary cultured rat vascular smooth muscle cells was determined by Western blot analysis. (C) Inhibition of 5-HT-induced calcium increase by cyclocurcumin was observed in primary vascular smooth muscle cells using fluo-2 in confocal microscopy. (D and E) Effect of cyclocurcumin on L-type calcium channel-mediated vasoconstriction was measured by using 60 mM K+ (D) or the specific agonist Bay K8644 (E; 10−6 M). Values are mean ± SEM of at least three independent experiments. *Significant differences from the corresponding control (p < 0.05). #Significant differences from the 5-HT-treated cells (p < 0.05). Chromadex Inc. (Irvine, CA, USA). Curcumin (98% pure as determined by HPLC including mass spectroscopy and melting point by the manufacturer) was purchased from Cayman Chemical (Ann Arbor, MI, USA). Other ingredients in C. longa were provided from the National Center for Standardization of Herbal Medicines in Korea, with a purity of >98%. Fura-2/AM was obtained from Molecular Probes (Eugene, OR, USA), and reagents and media used in cell culture were purchased from Gibco Co. (Carlsbad, CA, USA). Collagenase and elastase were obtained from Worthington Biochemical Corp. (Lakewood, NJ, USA). Myosin light chain antibody, pMLC antibody, and horseradish peroxidase-conjugated anti-rabbit secondary antibody were from Cell Signaling Technology Inc. (Danvers, MA, USA). Halt protease and phosphatase inhibitor cocktail and RIPA buffer were from Thermo Scientific (Rockford, IL, USA). All other reagents used were of the highest purity available. Plant Material. The plant was authenticated, and a voucher specimen (CU2009-06) has been deposited at the herbarium of Chosun University (Gwangju, Korea). Dry powders of Curcuma longa were extracted with 70% ethanol at 70−80 °C for 3 h. The extraction was repeated three times. After filtration and concentration under reduced pressure, extracts were lyophilized, and the resultant powder was stored at −20 °C. For experiments extracts were dissolved in DMSO. Quantification of major compounds of crude extract was performed by HPLC analysis (Supporting Information). Animals. The entire animal protocol was approved by the Ethics Committee of Animal Service Center at Seoul National University. Male Sprague−Dawley rats (SamTako, Seoul, Korea) weighing 250− 300 g were used. Before the experiments, animals were acclimated for 1 week in the laboratory animal facility maintained at constant temperature and humidity with a 12 h light/dark cycle. Food and water were provided ad libitum. Isolated rat aortic rings were used to examine vasoconstriction, and tissue integrity was observed with TUNEL staining and histological analysis. Possible nonspecific cytotoxicity was evaluated by measuring the leakage of lactate
dehydrogenase. Primary rat vascular smooth muscle cells (VSMCs) were isolated and used to elucidate molecular mechanisms underlying antivasoconstrictive cyclocurcumin that include phosphorylation of myosin light chain and intracellular calcium influx. Measurement of Vasoconstriction in Isolated Rat Aortic Rings. After rats were humanely decapitated to exsanguinate, the thoracic aorta was carefully isolated and cut into ring segments in lengths of 3−4 mm on ice. Aortic rings without endothelium were prepared by gently rubbing the intimal surface of the aortic rings with a cotton swab. The rings were then mounted on organ baths filled with Krebs-Ringer solution (115.5 mM NaCl, 4.6 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, 25 mM NaHCO3, and 11.1 mM glucose, pH 7.4) continuously saturated with a 95% O2/5% CO2 gas mixture and maintained at 37 °C. The change in tension was measured with Grass FT03 force transducers (Grass Instrument Co., Quincy, MA, USA) and recorded using the AcqKnowledge III (BIOPAC Systems Inc., Goleta, CA, USA). To investigate the effect on vasoconstriction, the aortic rings were treated with herb extracts or components, and vasoconstriction was initiated by the cumulative addition of PE, serotonin, or 60 mM K+. To investigate the effect on Bay K8644-induced vasoconstriction, vasoconstriction was initiated by Bay K8644 in 15 mM K+ buffer solution (105 mM NaCl, 15.0 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, 25.0 mM NaHCO3, 0.026 mM EDTA, and 11.1 mM glucose, pH 7.4). The extent of contraction was calculated as percent of pre-estimated basal contraction response induced by 90 mM K+. TUNEL Staining of Aortic Rings and Histopathological Assessment. Aortic rings placed in minimum essential media (MEM) containing 100 U/mL penicillin and 100 μg/mL streptomycin were treated with cyclocurcumin, MMA3+, or vehicle and incubated in a 95% air/5% CO2 incubator for 24 h at 37 °C. After incubation, aortic rings were fixed in buffered formalin solution (10%) and embedded in paraffin. A terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay was performed using a commercial kit 198
DOI: 10.1021/acs.jnatprod.6b00331 J. Nat. Prod. 2017, 80, 196−200
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Figure 3. Reversible effect of cyclocurcumin on agonist-induced vasoconstriction. (A) After treatment with cyclocurcumin, 5-HT-induced contraction was measured at “I”. 5-HT-induced contraction was measured again at “II”, 4 h after a washout of cyclocurcumin-containing buffer in an organ bath. Reversible effect of cyclocurcumin is summarized in the right panel. (B and C) Nonspecific cytotoxicity in aortic ring was monitored via histological examination following TUNEL staining (B) or determination of lactate dehydrogenase (LDH) leakage (C). Monomethylarsonous acid (MMA3+) or lysophosphatidiylcholine (LPC) was used as a positive control. Values are mean ± SEM of at least three independent experiments. *Significant differences from the corresponding control (p < 0.05). #Significant differences from cyclocurcumin-treated group before washout (p < 0.05). chopped finely and VSMCs were liberated from the tissue using collagenase and elastase. VSMCs grown in T-25 flasks were treated with cyclocurcumin for 30 min, and 10−5 M serotonin was added for 10 min. VSMCs were harvested and lysed in RIPA buffer containing protease/phosphatase inhibitor cocktail on ice, and cell debris was removed by centrifugation at 14000g for 10 min. The protein extract was subjected to Western blot using SDS-PAGE, and antibodies specific against MLC, pMLC. The bands were detected and analyzed with a Chemidoc XRS system (Bio-Rad, Hercules, CA, USA). Measurement of Intracellular Calcium Level in Primary Rat Smooth Muscle Cells. Intracellular calcium level was measured with the fluorometric method employing fura-2 and digital imaging. After the endothelium and adventitia were removed, aortic rings were chopped finely, and smooth muscle cells were liberated from the tissue using collagenase and elastase. Vascular smooth muscle cells grown on coverslips were treated with cyclocurcumin for 30 min. To load fura-2, cells were incubated in physiological salt solution (PSS; 140 mM NaCl, 5 mM KCl, 5 mM NaHCO3, 1.8 mM CaCl2, 1.4 mM MgCl2, 1.2 mM NaH2PO4, 11.5 mM glucose, and 10 mM HEPES, pH 7.4) containing 1 μM fura-2/AM and 1% bovine serum albumin for 60 min. Coverslips were mounted in a superfusion chamber on the microscope stage and were superfused with PSS (2 mL/min). All experiments were performed at 33 °C. Cells were imaged with a Nikon Eclipse Ti− U inverted microscope equipped with an S Fluor 40× (NA 1.30, oil) objective lens (Nikon, Melville, NY, USA) and an Evolve EMCCD camera (Photometrics, Tucson, AZ, USA). Illumination was provided by a Sutter DG-4 filter changer (Sutter Instruments, Novato, CA, USA). Excitation and emission wavelengths used for fura-2 were 340/ 380 and 535 nm, respectively. Images were acquired and analyzed with a Meta Imaging System (Molecular Devices, West Chester, PA, USA). Statistical Analysis. The means and standard errors of means were calculated for all treatment groups. The data were subjected to one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test or Student’s t test using SPSS software (Chicago, IL, USA) to determine which means were significantly different from the
according to the manufacturer’s instructions (Chemicon International, Temecula, CA, USA). The embedded tissue was sectioned with 4 μm thickness and placed on an adhesive slide. The section was deparaffinized by washing with xylene following serial rehydration with aqueous ethanol (100%, 95%, 80%, and 70%). Sections were then treated with 0.3% H2O2 to quench endogenous peroxidase activity followed by 20 μg/mL DNase-free Proteinase K to retrieve antigenic epitopes. Sections were next treated with terminal deoxynucletidyl transferase enzyme reagent for 1 h at 37 °C to label free 3′-OH termini with digoxigenin-dUTP. To detect incorporated digoxigenin-conjugated nucleotides, horseradish peroxidase-conjugated antidigoxigenin antibody and 3,3′-diaminobenzidine (DAB) were used. Sections were treated with antidigoxigenin-peroxidase for 30 min at room temperature followed by DAB development. Sections were counterstained with Mayer’s hematoxylin. Dehydrated sections were cleared in xylene and mounted. For assessment of TUNEL-positive cells, the number of total cell nuclei and positive cell nuclei was counted at four fields in each specimen, and the percentage of positive cell nuclei was calculated. Measurement of Lactate Dehydrogenase Leakage. To examine the nonspecific cytotoxicity of cyclocurcumin, the extent of LDH leakage from aortic rings was measured. After incubation with cyclocurcumin for 30 min and lysophosphatidylcholine for 24 h, aliquots were collected. A 50 μL portion of aliquot was added to 1 mL of Tris-EDTA-NADH buffer (56 mM tris(hydroxymethyl)aminomethane, 5.6 mM EDTA, 0.17 mM β-NADH, pH 7.4) and then incubated for 10 min at 37 °C. After incubation, 100 μL of prewarmed 14 mM pyruvate solution at 37 °C was added. The reduction in absorbance at 340 nm by the conversion of NADH to NAD+ was measured for 1 min to evaluate LDH activity in the aliquots. Detection of pMLC by Western Blot. The phosphorylation of MLC was observed in primary rat vascular smooth muscle cells. After the endothelium and adventitia were removed, thoracic aorta was 199
DOI: 10.1021/acs.jnatprod.6b00331 J. Nat. Prod. 2017, 80, 196−200
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control. In all cases, a p value of
