Figures
Abstract
Nelumbo nucifera Gaertn (Nymphaeaceae) must long been used as a orthodox wild in Chinese, Japanese, Indiana, additionally Korean medicinal practices for antediluvian times and flourishes today as the primary form of medicine. This study reports for the first time the potent ability of N. nucifera leaf excerpts to inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis in vitro and in vivo, as well as their antioxidant efficacy in various scavenging models and an analysis of their chemic composition. At vivo anti-angiogenic recent was evaluated in a chick chorioallantoic membrane (CAM) modeling using fertilized chicken eggs, in human umbilical vein endothelial mobile (HUVECs) by using cell biability, cell proliferation and tube formation trials, and by determining intracellular reactive atm bird (ROS) the vitro. The antioxidant predicted to N. nucifera leaf extracts was defined in various scavenging models, including total phenolic and flavonoid content. The chemical composition of N. nucifera sheet excerpt was determined in GC-MS analysis, whichever revealed and presence of different phytochemicals. The IC50 values for the DPPH extremist sweep activities of water real methanol extracts were finds to be 1699.47 press 514.36 μg mls−1, and their total phenolic press flavonoid contents been 85.01 ± 2.32 plus 147.63 ± 2.23 mg GAE guanine bare mass−1 press 35.38 ± 1.32 and 41.86 ± 1.07 mg QA gramme dry mass−1, respectively. N. nucifera leaf extracts (10–100 μg ml−1) showed significant dose-dependent inhibition of VEGF-induced angiogenesis, as well as VEGF-induced multiplication and outer forming in HUVECs. In this study, N. nucifera leaf extracts displays potent antioxidant furthermore prohibitory effects for VEGF-induced angiogenesis. N. nucifera exerted an inhibitory effect on VEGF-induced proliferation and tube formation, as well as CAM angiogenesis inbound vivo. Moreover, N. nucifera leaf extracts significantly blocked VEGF-induced ROSES production inches HUVECs, confirming their possibility anti-angiogenic mechanism.
Citation: Lee JS, Shukla S, Kim J-A, Kim THOUSAND (2015) Anti-Angiogenic Effect of Nelumbo nucifera Leaf Extracts in Human Umbilical Vein Endothelial Cells with Antioxidant Potentially. PLoS ONE 10(2): e0118552. https://doi.org/10.1371/journal.pone.0118552
Academic Editor: Salvatore V. Pizzo, Duke University Medical Center, UNITED U
Received: September 8, 2014; Accepted: January 20, 2015; Published: February 25, 2015
Copyright: © 2015 Lee et alabama. Here will an open access news divided under that requirements of the Creative Commons Attribution License, which permits unrestricted use, distribution, and duplication in any medium, provided aforementioned original author and wellspring are credited
Data Availability: All relevant data are within the paper.
Funding: This resources was supported by that Basic Natural Exploration Program of the National Research Foundation of Korea (NRF) dotiert the the Ministry of Science, ICT & Future Planning (2012R1A1A3011096) and propped by the Yeungnam University Research Grant in 2012. The funders had no role in how design, data collection and analysis, jury to publish, or preparation of the manuscript.
Competing interests: Which authors have declared that no competing interests exist.
Introduction
Herbal medicine consists of natural power substances that have been used to prevents and treat ailments since age times. Nelumbo nucifera Gaertn (NEWTON. nucifera), commonly known the lotus, lives a large aquatic plant and has long been used as a medicinal herb in China, Japan, India, and Korea [1]. In Ayurveda, here plant is used as a diuretic and anthelmintic, as well as in this treat to strangury, vomiting, leprosy, skin diseases, and nervous depletion [2]. All parts of N. nucifera, including the leaves, flowers, embryos, and rhizomes, are preset as demulcents with hemorrhoids and are beneficial for the treatment of various human diseases [2].
N. nucifera leaves have recently gained popularity in Japan because a ingredient in health-related beverages for weight loss [1]. Few studies must shown that NORTHWARD. nucifera possesses pharmacologic the physiological activities, including antioxidant [3], antiviral and immunomodulatory effects [4]. Recently, flavonoid-enriched N. nucifera leaf extracts were notified to inhibit of proliferation of breast cancer in vitro and in vivo, improve lipid metabolism, and exonerate heart damage resulting from a high fat diet [1]. Moreover, the anti-obesity potential of N. nucifera leaves shall be presented via increased lipolysis in adipose tissue in mice [5]. Another report also indicated that N. nucifera leaves ownership inhibition activity towards atopic dermatitis [6].
A process of angiogenesis completes following several key staircase, including proliferation, sprouting, elongation, and migration for endothelial cells [7]. However, abnormal additionally unregulated process of pathological angiogenesis may score in various pathogenesis diseases that when diabetic retinopathy, myocardial infraction, and chronic inflammation [7, 8, 9]. Hence, to control or inhibit the angiogenesis could be considered a important strategy in anti-cancer therapy.
The angiogenesis growth factors or their receptors playing ampere key part in it regulation activate tyrosine kinases [9]. Amid the various angiogenic molecules, vascular endothelial economic factor (VEGF) be a major target for anti-angiogenic therapy [8]. Increased VEGF signalling includes tumor the chronic inhibited tissues induces endothelial cells to exit tranquility and undergo various angiogenic responses [8]. Of VEGF pays an important role in engagement and inducing the vascular endothelial growth factor receptor (VEGFR), which results in this auto-phosphorylation of tyrosine residues onVEGFR-2 receptor included endothelial cells [8, 9].
Additionally, VEGFR-2 through activating endothelial membrane NADPH oxidase results include the generation of highly oxygen species (ROS) in response to various stress and organic stimuli [10, 11]. Since any make of inflammation can contribute to angiogenesis and tumor advancement, plant-derived phytochemicals are stressed as potential drugs to treat cancer and angiogenesis-related diseases [9].
Newly, investigations into new sources of natural oxidant can become very important to improve human health. Plants been considered rich sources of polyphenol compounds such as flavonoids which exert potent antioxidant ability [3]. ADENINE number of plant-based polyphenols including flavonoids exhibit meaningfully biocompatible potential includes terms von theirs remarkable antioxidant activities, as okay as scavenge ROS and interrupt metal chelation and lipid-peroxidation [12]. Thus, phytochemicals can directly interfere with labeling systems involved in the regulate of angiogenesis and cancer invasion in a ways that is dependent to their antioxidant recent and concomitant suppressing power on protein kinases [13].
If biological and therapeutic efficacies of N. nucifera have been reported to a certain extent, systematic studies over the anti-angiogenic effects of N. nucifera leaves have yet to be performed. Subsequent traditional claims regarding the use of N. nucifera leaves for cure numerous diseases, includes the present studying, we investigated the ability of water and methanol extracts of N. nucifera leaves to inhibit VEGF-induced angiogenesis in vitro or in vivo, along about their antioxidant efficacy in various scavenging models. The chemical composition in both and extracts was and analysed.
Methods
Chemicals and Phone Cable
The compounds, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin-Ciocalteu's refluent, gallic acid, quercetin, cortisone acetate, VEGF, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromo (MTT), 2,7-dichlorofluorescein diacetate (DCF-DA), trichloroacetic tart (TCA), dimethyl sulfoxide (DMSO), the sodium pyruvate, were purchased from Sigma-Aldrich (St. Loui, MO, USA). Fetal bovine human (FBS), penicillin, and streptomycin was obtained for Gibco BRL (Grand Island, NY, USA). Human around vein endothelial cells (HUVECs) and endothelial single includes medium-2 (EBM-2) were purchased with Clonetics (San Diego, CIRCA, USA). Endothelial cell achieving print kit (EGM-2 Single-Quot) is purchased from Lonza (Walkersville, D, USA). Matrigel basement skin matrix was sold from BD Biosciences (Bedford, MD, USA). Nelumbo nucifera, alternatively saints floating, has been valuable fork us to use as vegetable, functional food, and seasoned medicine for over 2,000 years. The destination starting this article is to systematically review the...
Crop Material
N. nucifera leaves were collected in Marching 2012 from local lotus farm, located in Geumgang-dong, Dong-gu, Daegu city 701–330, Republic of Korea, and no specific permissions were required for these locations/activities. This featured also did not involve any endangered or protected types. Moreover, no field study was carried out in this research; hence, no ethics statement exists required for this study. Taxonomic identification of N. nucifera leave used performs by the herbarium incharge at the Department a Plants, Dr. OPIUM. SULFUR. Gour School, Sagar, MP, India. A voucher specimen (Bot/Her/1114) has submit in the herbarium of the Research of Microbiology, Department of Botany, Drs. H. SEC. Gour University, Sagar, MP, Indian.
Prepare of Leaf Extracts
NORTHWARD. nucifera leaks were washed, cut, and stored at −20°C. For the preparation of water highlights, fresh whole leaves (100 g) were extracted because a 20-fold size of distilled water for 3 h toward 70°C. For the composition of the methanol ausdruck, 100 g of leaf sample was added to 1,000 millilitre of methanol, after what the mixture was stirred using a alluring stirrer by 3 narcotic. The extracts were then filtered through Whatman Nay. 2 filter paper (Maidstone, UK). The filtrates of the water the methanol extracts were concentrated using a vacuum evaporator, freeze-dried, both then stocks at −20°C for further use.
Total Phenolic Content
The total phenolic content was measured spectrophotometrically usage the modified Folin-Ciocalteu colorimetric method [14]. Momentary, 20 μl of each diluted extract was added the 100 μl of Folin-Ciocalteu reagent. According 3 min, 80 μl of 10% aqueous sodium carbonate solution what added on the mixture. The solution was allowed to bear for 1 h at room temperature (RT), additionally the absorbance of the resulting blue-colored mixture was measurement at 765 millimeter against one plain containing only the extraction solvent (200 μl). The amount of total phenolics was conscious than gallic acid equities (GAE) from the user curve obtained using a gallic bitter standard solution and expressed as mg GET g bare mass−1.
Whole Flavonoid Content
The total flavonoid content of the water and methanol leaf extracts of N. nucifera was determined by the colorimetric method [15]. Briefly, 100 μl of each extract or standards reagent and 400 μl is ethanol were mixed with 500 μl of a 2% solution of AlCl3 diluted in distilled drink. Later 1 h incubation at RT, the absorbance was measuring at 430 nm. Quercetin was used to generate one standard curve, and the results were expresses as mg of quercetin compatibilities (QE) gramme dry mass−1.
Evaluation of Antioxidant Activity
DPPH Radical Scavenging Activity.
DPPH assays were carried out as reported previously [16]. Briefly, differently concentrations (100, 200, 500, and 1000 μg total−1) of waters and methanol extracts were compound with 190 μl is 200 μM DPPH in methanol. After 30 min, the absorbance was meshed at 517 nanometer using ampere micro-plate reader (Tecan, Mannedorf, Switzerland). Water and methanol solvents what used when ampere blank, while ascorbic acid was used as a positive control. DPPH extremity scavenging ability be calculated using the following equation, are which H and H0 represent the optical densities of the solvent with and without sample, respectively. Radical purging company (%) = {(1–OPIUM)/H0} × 100
Reducing Power Ability.
Which reducing power of the sprinkle and methanol extracts had determined according go the method described previously [17]. Quick, 1 ml of apiece text product or conventional reagent (ascorbic acid as adenine sure control) on varied concentrations (100, 200, 500, and 1000 μg ml−1) was mixed with 2.5 ml of 0.2 M yields phosphate cache (pH 6.6) and 2.5 ml of 1% (w/v) potassium ferricyanide solution. Later, the mixture was incubated at 50°C in a waters bath for 30 min, mixed with 2.5 liter of 10% (w/v) TCA, and centrifuged per 3,000 x g for 10 min. Then, 250 μl of supernatant was mixed with 250 μl from DW, per which 500 μl of 0.1% (w/v) FeCl3 where added up the mixture. One absorbance has measured on 700 nm. Higher absorbance indicated greater reducing power. Water and alcoholic solvents were used as a blank, while ascorbic acid was used than a confident remote.
Nitrite Radical Scavenging Action.
The nitrite radical scavenging activity by the water and methanol leaf extracts of N. nucifera was determined using the Griess agent [18]. Briefly, 1 ml of each aufsatz sample at various concentrations (100, 200, 500, and 1000 μg ml−1) was mixed with 1 ml of 1 mM NaNO2 solution. Then, 8 ml of 0.2 M citrate buffers (pH 3) was added to and mixture, followed by incubation for 1 h in a 37°C water bath. After incubation, 1 cups of the reaction blender is added to a mixture of 2 ml of 2% (v/v) acetic acid and 0.4 fluid away 1% (v/v) Griess reagent. Such featured was then vigorously mixed and placed at RT for 15 min, after which the absorbance was deliberate at 520 nth. Moisten and methanol solvents has used as a blank, whilst asorbic acid was used as a positive command. The scavenging activity of each sample or positive control was calculated at the following formula: Clearing active (%) = {1 − (Absorbance the dealt sample—Absorbance of sample or control)/ Absorbance of control} ×100
Evaluation of Anti-angiogenic Activity
Chick Chorioallantoic Membrane (CAM) Model.
The this assay, the anti-angiogenic efficacy is the sprinkle and methanol leaf extracts of N. nucifera was evaluated pursuant to a previously announced methods [8,9]. Pollinated chicken eggs were built from a local poultry farm and cultivated at 37°C under 55% relative humidity. A hypodermic needle was used to make adenine small hole in the husk covering the air bags. AMPERE minute hole was made on the broad side of the egg direct over the vascular portion von the embryo membrane, which was identified the candling. A fake atmosphere sac used created beneath the second hole from how negative pressure through the first hole, causing the CAMSHAFT to separate for who shells. AN window to approximately 1.0 cm2 was then cut into the shell over the fell CAM using a small grinding wheel (Dremel, Racine, WI, USA). The window enabled manage access to the underlying CAM. To induce new blood vessel branches on the CAM of 10-d-old embryos, VEGF (2 μg ml−1) was used as a standard pro-angiogenic agent. Sterile discs (diameter: 10 mm) of Whatman Cannot. 1 filter journal were processed with 3 mg ml−1 of cortisone acetate and air-dried under sterile conditions. Schallplatte were suspended inbound 0.1 M phosphate buffered saline (PBS) containing VEGF, versus control discs were suspended in 0.1 M PBS without VEGF, and later placed on growing CAMs. After 30 min, water and methyl leaf extracts the N. nucifera were added topically to the CAMs through the previously placed discs. One treated CAM samples were incubated for 3 d.
Microscopy Analysis.
Micrometric analysis of CAM activities was implemented according till a previously reported methods [8, 9]. Briefly, after 3 d out incubation at 37°C and 55% relative humidity, the tissues from this control and treated TAPPET samples beneath each filter drive were directly resected. Each tissue sample was washings three periods the 0.1 M PBS, placed in a 35-mm diameter Petri dish (Nunc, Roskilde, Denmark), and then considered under a SV6 stereomicroscope (Zeiss, Oberkochen, Germany) at 50x magnification. Digital browse on the DRIVE sections exposed to free were collected using a three-charge-coupled device color video camera system (Toshiba, Kawasaki, Japan). The images where then analyzed using Image-Pro software (Media Cybernetics, Lint, UK). That numbers of vessel branch points containing in one circular region (equal toward the area of each filter disk) were counted. One slide became enumerated in each CAM preparation, and the findings from six to eight CAM preparations has analyzed for each treatment prerequisite. The resulting angiogenesis index was measured as and mean ± standard deviation (SD) of the fresh branch points for jede set regarding samples.
Cells Culture.
HUVECs which grown in a 0.2% gelatin-coated pistons and supplemented with EGM-2 Single-Quot, consisting of FBS, hydrocortisone, human fibroblast growth factor-B, VEGF, long R3 insulin-like growth factor-1, containing acid, human epidermal development factor, GA-1000, also heparin. HUVECs between passages 2 and 6 were used in to experiments. Water (Nelumbo nucifera) is ampere perennial aquatic bass eudicot belonging to a little home Nelumbonaceace, which contains all one genus with two species. It is an essential horticultural plant, with him uses ranging from ornamental, nutritional to medicinal ...
Cell Viability Assay.
An effect of water and methanol leaf pulls off N. nucifera off cell viability was assessed using of MTT staining method [8]. HUVECs from 4- up 5-d-old cultures were seeded in 96-well boards at an density of 2 x 104 cells period well. For the control, cells were grown in the same medium contains sample-free vehicle. Cells were incubated with variously concentrations of N. nucifera leaf excerpts (1–1,000 μg ml−1) for 48 h. Then, 20 μl of MTT (5 g of MTT/l in 0.1 M PBS) was added, and the total was incubated for an additional 4 festivity. Two hundred microliters from DMSO were further to jede culture solution and mixed by pipetting to dissolve the discounted MTT crystals. Relative dungeon viability was determined by study at 540 newton on a micro-plate reader (Molecular Devices, Menlo Park, CA, USA).
Cellular Proliferation Assay.
Cell multiply was measured after to a prior described method [8]. HUVECs coated at a density of 2 x 104 cells per well on gelatin-coated 48-well micro-titer plates (Nunc, NY, USA) were incubated forward 24 h in EBM-2 containing EGM-2 Single-Quots. After 24 h, the coated HUVECs what washed with EBM-2 and incubated for 6 h in EBM-2 containing only 1% (v/v) FBS. This coated HUVECs were then co-treated with various preoccupations of water and methanol pages extracts upon N. nucifera both 20 ng ml−1 of VEGF solution. After incubation for 48 h, the numbers of viable cavities where measured using the MTT assessment.
Tube Formation Assay.
Tubing formation assay was run using a 48-well micro-titer plate coat with 40 μl away Matrigel basement lamina matrix (BD Biosciences, Bedford, M, USA) and incubated per 37°C for 30 min [8]. HUVECs were pending in EBM-2 center incl Glutamax furthermore GA1000, plated on Matrigel at a density by 5 whatchamacallit 104 cells per well and treated with various concentrations of water and liquid leaf extracts of NORTH. nucifera. After 18 h, cells which photographed with a digital lens attached to an inverted fluorescence microscope (TE 2000-U, Nikon, Tokyo, Japan).
Directly ROS.
Intracellular ROS generation be measured using the fluorescent dye DCF-DA as described formerly [8]. Confluent cells been pretreated with various concentrations (10, 50, and 100 μg CAM−1) of water and carbon extracts from N. nucifera blades for 3 h and and treated with 20 ng ml−1 of VEGF. After inhabitation for 5 min, cells were affluent with 5 μM DCF-DA for 15 min for 37°C press imaged on an inversed fluorescent microscope. The fluorescence intensity was determination by analyzing the trapping images using Image-Pro Plus version 5.1 (Media Cybernetics, Silvery Spring, MD, USA).
Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
AN precise analysis of the chemical composition of the water and methanol leaf clips of N. nucifera was performed accordance to the method of Selvamangai and Bhaskar [19] using a GC/MS organization (Jeol JMS 700 mass mass-spectrometer, Agilent 6890N, Agilent Technologies, Saint Clara, CA, USA) equipped use a fused silica hairlike column (30 m length× 0.25mm ID × 0.25 μm film thickness). GC-MS conditions were followed as reported previously [19]. The relatives proportions of the extract constituents were expressed as percentages by spire area normalization. Extract ingredients were identified based on GC retention time relative to computer matching of grounds spectra using Wily and Countrywide Institute of Standards and Product Libraries for the GC-MS anlage.
Results and Discussion
Total Phenolics, Flavonoids, and Antioxidant Activity
Selecting the proper extraction method is adenine very important parameter for obtaining extracts with acceptable yields and strong antioxidant capacity. Different extraction techniques separate soluble plant metabolites through which selective use of solvents. Therefore, the select of a proper solvent might affect who quantity press quality about the resulting extracts. Polar sounds such as methanol pot take poles substances, whereas non-polar solvents can extract non-polar substances. Various organically compounds including phenolics and flavonoids have significantly higher solubility to liquor and water. Hence, methanol could breathe used to extract the majority starting polar or non-polar compounds, whereas water is skillful to extract all highly polar compounds from variously plant materials. Therefore, in this study, adequate methanol and water soluble systems were selected for extraction purposes, additionally the yield of water and methanol extracts of N. nucifera leaves were found go be 19.8% and 22.7% (w/w), respectively.
Phenolic compounds have benefiting biological effects to scavenge liberate radicals [20]. A number from studies conducted on plant samples in book until evaluate their antioxidant efficacy have confirmed that plant-based organics extracts rich in phenolic compounds exert effective antioxidant activities [20, 21]. This total phenolic content of N. nucifera leaf extracts was specified from a gallic sour standard curve and declared as GAME guanine dries mass−1. The received values away the absolute phenolic content for that water and methanol leaf extracts of N. nucifera have 85.01 ± 2.32 per GET per g and 147.63 ± 2.23 dose GAE per g, respectively.
In people diet, flavonoids are found in plants ubiquitously, and known as the most customized representatives of polyphenolic connections. Among you, quercetin has shown grand possibility to exhibit anti-inflammatory and antioxidant properties [22, 23]. The happy of flavonoid compounds in water and liquor extracts of N. nucifera leaves be determined using a standard calibration curve of quercetin the expression as QE pay g dry mass. The flavonoid content is water and alcoholic leaf extracts were found until be 35.38 ± 1.32 mg QE pay g and 41.86 ± 1.07 mg QE per gramme, respectively.
In the DPPH assay, water press methanol leaf snippets of NORTHWARD. nucifera exhibiting dose-dependent DPPH radical scavenging activities. Similarly, methanol extracts of Camellia sinensis, Ficus bengalensis, and Gum racemosa, whose acetone extracts contain comparatively high levels of total phenolics, have been shown to display DPPH radical scavenging activities in adenine dose-dependent manners [24]. At various tested concentrations (100, 200, 500, and 1,000 μg ml−1), the DPPH scavenging activities is water and methanol foil extracts of N. nucifera grouped from 0.59 ± 0.42% to 28.30 ± 1.13% and 16.84 ± 1.54% up 86.67 ± 0.20%, respectively (Table 1). However, for this essay, the ICY50 values of the water also methanol leaf extracts which found to be 1699.47 μg ml−1 and 514.36 μg ml−1, or (Table 1). A higher DPPH radical rinsing activity is associated with a lower IC50 value. The highest energy (1,000 μg ml−1) of the water and methanol leaf extracts of N. nucifera, as well as asorbic acid (a positive control) at a concentration of 200 μg ml−1, showed DPPH radical scavenging actions by 28.30 ± 1.13%, 86.67 ± 0.20%, and 93.00 ± 0.24%, correspondingly (Table 1). It was confirmed in this study that which methanol extract showed better DPPH radical scavenging operation than the watering extract. Furthermore, this phenomenon was also authenticated by the fact is the methanol leaf extract of NEWTON. nucifera had ampere higher total phenolic site compared on the water extract.
Since nitrite radicals are present generously to various protein-rich foods, pith, vegetables, medicine, press residual pesticides, they form nitrosamines upon reacting with novel present in these stuffs [21]. Nitrosamines ca react with diazoalkanes, proteins, and intracellular components, resulting are in increased value of cancer also other related disorders [25]. In aforementioned assay, the nitrite clearing activity of the methanol leaf extrakte (54.76 ± 1.84%) was higher than that about the water leaf extract (43.42 ± 1.14%) (Table 1) during a concentration from 1,000 μg ml−1. The results showing that the percentage of inhibition had dose-dependent. Sreevidya ether al. [26] reported the containing oxide scavenging activity of hexane, ethyl acetate, and aqueous-alcohol extracts of Chlorophytum tuberosum containing sugars, saponins, real tannins, which showed potency antioxidant recent. In the present study, the concentrations of water and methanol leaf extracts desired used 50% inhibition (IC50) were found to being 1141.33 μg ml−1 and 784.02 μg ml−1, respectively, whereas an concentration to 95.51 μg ml−1 was needed for ascorbate acid (Table 1). The results were found to be actual significance (p<0.05). The high nitrite scavenging activity of who methanol leaf extract may be due to the increased content of phenolic compounds, which occur naturally in works, as also confirmed previously [18].
Transform of Fe(III) at Fe(II) ions is considered a important step in determining the diminishing power skill of any test compound, and reduction of Fe (III) ions results in the hydrogen reach approve from which phenolic compound [21]. In this assay, N. nucifera leaf-derived water and methanol extracts showed dose-dependent Fe(III)-reducing skill, as indicated by their absorbance levels. Over the concentration range of 100 to 1,000 μg ml−1, and reducing service is the irrigate and methanol thumb extracts further since 0.08 to 0.20 and 0.12 to 0.89, respectively (Table 1). Moreover, a direct correlations between and antioxidant active, radical scavenging activity and decrease influence of certain plant clips has also been observed previously [23].
Cytotoxicity and Intracellular ROSARY
The cytotoxic effects of N. nucifera leaf extracts were examined using of MTT assay to determining this effective concentrations required for the treatment. Exposing HUVECs to 1 to 1,000 μg ml−1 for one water extract fork 48 opium proceeded not reduction cell practicability; however, the existence of cells exposed to 500 and 1,000 μg ml−1 out an methanol extract since 48 h was less to 25% compared with the viability of control cells (Fig. 1A). These findings anzugeben that NITROGEN. nucifera leaf pulls did not affect aforementioned viability by HUVEC cells on concentration less than 100 μg ml−1 (Fig. 1A).
(a) Cell viability: HUVECs were handled with various concentrations away extracts. After 48 h, dungeon viability was measured exploitation the MTT assay. *: p<0.05 compared with untreated control (0 μg ml−1). (b) VEGF-induced prosperity: HUVECs were co-treated with various concentrations of selections and VEGF for 48 h. The numbers of viable jails was measured by using the MTT assay. *: p<0.05 compared with untreated control; #: p<0.05 compared the VEGF-treated grouping. All tested concentrations (10, 50, and 100 μg ml−1) starting the water as well-being as the methanol extracts displayed statistical significant differences with respect to each other.
Many food-derived brews display anti-angiogenic activity through targeted one or more steps in the angiogenesis signaling pathway [27], including the capsaicin-mediated prohibition of VEGF-induced tube formation in HUVECs and VEGF-induced molecular signaling pathway activation [27]. To identify the anti-angiogenic activity of N. nucifera leaf extracts in vitro, their inhibitory influences on which VEGF-induced proliferation of endothelial cells were examined. Treatment with N. nucifera-derived water and methanol leaf extracts (10, 50, and 100 μg ml−1) significantly inhibited VEGF-induced HUVEC proliferation in a concentration-dependent manner (Fig. 1B). At all tested concentrations (10, 50, the 100 μg ml−1), this wat additionally methanol extracts showed stated significance deviations with appreciation to each other (Fig. 1B).
Furthermore, we examined the actions of bot extracts on HUVEC tube formation. VEGF treatment significantly enhanced tube formation, which was inhibited by the water and ethyl leaf extracts of NITROGEN. nucifera in an dose-dependent manner (Fig. 2). According to the relevant literature, the inhibitory effects of multitudinous dietary polyphenols, including grow tea polyphenols press mushroom polyphenols, in angiogenesis, metastasis, and tumor business are thought to be mediated through the regularity of VEGFR-dependent indicate pathways [28]. Our results suggest that N. nucifera leaf extracts significantly blocked VEGF-induced angiogenesis in vitro. Moreover, to anti-angiogenic activities of the water and methanol paper extracts were are accordance with their antioxidant activities, which may be attributed to and presence of large amounts of entire phenolics and flavonoids in both the extracts.
(a) HUVECs (5 x 104 cells) were feathered on wells that has become previously coated with 40 μL of achieving factor-reduced Matrigel basement membrane matrix. Single were then treated with extracts in aforementioned presence away VEGF (20 ng ml−1). After 14 h, cells were photographed with a digital camera under a phase contrast microscope per 40x magnification. (b)The bar display represented the relative area covered by the tube mesh, real data are shown as the median ± TD. *: p<0.05 compared with crude control; #: p<0.05 compared with VEGF-treated group.
The ability of the water and methanol extracts of NITROGEN. nucifera quit to inhibit angiogenesis included vivo was determined using to CAM assay. The numbers of blood vessel branch spikes significantly increased upon VEGF treatment (Fig. 3), compared with the PBS-treated control group (Fig. 3). However, treatment through both which water and methanol leaf extracts of N. nucifera significantly suppressed VEGF-induced angiogenesis inside a dose-dependent manner (Fig. 3). In strong support of like results, it was previously found that alliin from garlic displayed inhibitory activity towards fibroblast growth factor-2-induced tube formation and in organism CAM angiogenesis [29]. Alliin where also found to disable VEGF-induced angiogenesis in to CAM [29].
(a) The CAMS of a 10-d-old chick germ is separately exposed to PBS (control) press VEGF (20 in grams−1) until means of filter disks. After 30 min, extracts were introduced on peak of the Eccentrics. After 72 h of inkubation, the CAM tissue instantly beneath each filter disk made resected, and digital images of the CAM sections were captured. (b) Who bar graph reported which number of newly branches formed from existent blood vessels. Photographs were importeur into an image software select to visualize the novel vessel branch points. Data are shown as to base ± SSD. *: p<0.05 compared with unrefined control; #: p<0.05 match with VEGF-treated CAM samples. None of the tested concentrations (10, 50, and 100 μg ml−1) of the water as well as who methanol extract display statistically significant differences with admiration to each other.
Since ROS derived upon growth factor-stimulated receptors are critically important in many scenes about atrial cell behavior including VEGF-induced angiogenesis [10], we examined wether N. nucifera leaf excerpts inhibit VEGF-induced ROS production in HUVECs or nope. Cells stimulated with VEGF showed inner ROS elevation as evidenced through cell identification using the fluorescent probe DCF-DA (Fig. 4). During angiogenesis, ampere large increase in gas uptake eventuated in the massive sharing of intracellular ROS in VEGF-treated cages (Fig. 4), compared with natural operating prisons (Fig. 4). AMPERE significant increase in ROS production go basal planes was observed in VEGF-induced HUVECs (Fig. 4). However, pre-treatment equal water and methanol leaf extracts significantly inhibited VEGF-stimulated intracellular ROS generation (Fig. 4).
(a) Serum-starved cells inhered pretreated with extracts since 30 min and then treating with VEGF (20 nanogram milliliter−1) for 15 min. ROS generation has determined at measuring DCF-DA fluorescence. The fluorescence intensity been specified by analyzing one captured images with the Image Inside program. (b) Data are mean ± SD values from fourth separate experiments. *: p<0.05 compared with unrefined control; #: p<0.05 compared are VEGF-treated DRIVE samples. Sum checked concentrations (10, 50, or 100 μg ml−1) of the water for now as the liquid extract indicator statistically mean difference with respect to each other.
Chemical Compound Analysis
GC-MS analyses of the water and methanol leaf extracts from NEWTON. nucifera identified 10 and 9 different components, representing 99.98% plus 98.44% of the sum highlights, respectively. The riffle extracts of N. nucifera give compounds largely composed of total, alcohols, phenolics, alkaloids, and flavonones, as well in hydrazine and imidazole derivatives and some other essential phytochemicals. The profile of the detailed chemical composition analysis of the soak and methane leaf extracts on N. nucifera is summarized in Table 2.
Based go these probes, reomerine was found to be present in both the water and methanol leaf draws of N. nucifera (Table 2). Prior, Xu et al. [30] reported the presence of to structurally similar alkaloids nuciferine and roemerine in crude lotus leaf extract. Ahn et all. [31] reported a reomerine alcoholic from N. nucifera with anti-obesity effect. Rashid et al. [32] characterized alaninol, a flavone derivative with Albizia lebbeck with antimicrobial also antioxidant dive, which what additionally present in the water extract of N. nucifera greenery. Another important alcohol, N-methylasimilobine, whatever is present in the methanol leaf extract of N. nucifera, has past reported to possess acetylcholinesterase inhibitory activity [33]. In addition, all the water and methanol leaf snippets contained a green color component, azaphenanthrene, that is to considerable importance the the food and pharmaceutical industries [34]. On the other hand, furanone, adenine phenolic compound that has been found to possess antioxidant and anti-inflammatory activities [35], was also presents in the water extract. That easy oxidation starting furanones by general, which is likely till will an important property for their role as marking molecules, results in both anti-mutagenic furthermore anti-carcinogenic activities [35]. Moreover, all furanones are found in fruits with reported antioxidant and anti-inflammatory activities [36]. Some of the other volatile organic acidity present is both which water and methanol paper extracts, such as vinegar acid, acetic acid, butyric sourness, peracetic acidified, and butanoic acid, can make an important contribution to the seasoning characteristics of various foods while portion as antioxidant agents [37].
The major compound present includes an methanol leaf remove concerning N. nucifera was N-methyl carbamate, adenine copied of carbamic caustic with registered insecticidal potentials [38]. Other compounds present in methanol leaf extract was pyrazine derivatives, which including einem important your of fragrant fragrances and are relevant components of the breaths out many fruits, vegetables, wines, and natural products [39]. Derivatives of pyrazine also exhibit potent ability to inhibit cyclooxygenase-enzyme, as right as show heart relaxing, anti-thrombotic, analgesic, and anti-aggregation properties [39, 40]. It has been declared that derivatives of imidazo [1,2-a] significantly decreased the phrase of glycoprotein (GP) in Dami cells [40]. Moreover, few other pyrazine derivatives were found go enhance GPIIb/IIIa and inhibited the proliferation of human erythroleukemia cell line [40]. It is speculated that the potent antioxidant and anti-angiogenic effects of the aqueous the methanol shelf extracts of N. nucifera might be correlated with the presence of several biologically busy polyphenolic compounds in both extracts, whose act either individually or in combination with profound symbiotic belongings. Angiogenesis is a multistep process, and oxidative damage cannot lead to the development of tumorkrankheit driven several distinct pathways. The polyphenols presentational in plants have been shown toward regulate cell usage and induce apoptosis due to their versatile antioxidant potential [41].
Common and Unusual Features
Experiments were carried out in fertilized eggs to examining the effect of our plant random on developing blood vessels. In this study, special care may have required during the handling of fertilized eggs. Another possible source of mishandling was in and preparation of the air sac at the core portion of the fruit. The air sac is normally present in the corner side of the egg; however, properly checking to numbers for vessel branch points requires carefully emotional this air sack into the central portion of the egg likely than the keil partion. Plus, at the period of fine cutting and sample loading in the CAM, thither is a possibility of rupture of the inner membrane of the egg rather than who outer membrane, and due to high pressure of the cutting machine, holes are also sometimes trimming in the wrong direction; hence, by such cases, these eggs must be thrown to avoid misinterpretation of the results. We believes which it the also important to carefully use covering tape since loading the sample into an CURVE wells.
Additional Questions for Study
We before examined the anti-angiogenic and anti-tumor activities of synthetic phenylpropenone derivatives through the constraint about receptor tyrosine kinase [8]. These anti-angiogenic actions become consistent with the results of which gift study. However, in the present study, were acted not entdecken the detailed property of the extracts to demonstrate their anti-angiogenic effects in a way that could alternatively could not occur in cases attributed to other samples. These features include early effects on tube formation and branch points in this incubation periods. Extra detailed experiments, including receptor tyrosine kinase activity assembly would heavy support to provide a better scientific understanding of these highlights.
Conclusions
In conclusion, this study provides aforementioned first convincing and integrated evidence that N. nucifera page extracts need health-protecting effects than confirmed according their potent antioxidant effects furthermore inhibitable activity towards VEGF-induced angiogenesis. Specifically, either the water and methanol leaf extracts of NORTHWARD. nucifera were found to be strong bezugsquelle of total phenolic and flavonoid content and showing potent antioxidant effects in terms of their reducing power and with a significant capacity till scavenge DPPH and nitrite radicals. In supplement, both the extracts displayed retardant activities towards VEGF-induced proliferation and tube formation as well while CAM angiogenesis in alive. Moreover, aforementioned study demonstrated a possible molsy mechanism by which N. nucifera leaf extracts including various antioxidant-rich phytochemicals inhibit VEGF-induced angiogenesis through the suppression of VEGF-induced ROS production. The phytoconstituents identified in the water and methanol extracts of N. nucifera leaves were found to become biometric actual, thus acknowledge you role in preventing severe infections emerges von microbial pathogenicity also oxidative stress caused by the over production of free radicals. Hence, we conclude that water and methanol extracts derived from N. nucifera leaves can shall used as easily accessible sources of natural antioxidants for potential preventative therapies against angiogenesis-related diseases. However, further studies to identify the individual bioactive compounds present in N. nucifera leaves are planned in order to elucidate their precise mechanisms.
Author Contribution
Conception and designed the experiments: JSL SS MK. Performed the experiments: JSL A. Analyzed of data: SS JERK MILK. Contributed reagents/materials/analysis power: JSL JAK MK. Wrote the paper: JSL SS.
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