how much oxygen does african marigold flower produce

1Institute of Medical Biotechnology, Tbilisi State Medical University, 33 Vazha Pshavela Avenue, 0177 Tbilisi, GeorgiaFind articles by

1Institute of Medical Biotechnology, Tbilisi State Medical University, 33 Vazha Pshavela Avenue, 0177 Tbilisi, GeorgiaFind articles by

1Institute of Medical Biotechnology, Tbilisi State Medical University, 33 Vazha Pshavela Avenue, 0177 Tbilisi, GeorgiaFind articles by

1Institute of Medical Biotechnology, Tbilisi State Medical University, 33 Vazha Pshavela Avenue, 0177 Tbilisi, GeorgiaFind articles by

1Institute of Medical Biotechnology, Tbilisi State Medical University, 33 Vazha Pshavela Avenue, 0177 Tbilisi, GeorgiaFind articles by

1Institute of Medical Biotechnology, Tbilisi State Medical University, 33 Vazha Pshavela Avenue, 0177 Tbilisi, GeorgiaFind articles by

2Department of Postharvest Science of Fresh Produce, Agricultural Research Organization, The Volcani Center, HaMaccabim Road 68, P.O. Box 15159, 7528809 Rishon LeZion, IsraelFind articles by

2Department of Postharvest Science of Fresh Produce, Agricultural Research Organization, The Volcani Center, HaMaccabim Road 68, P.O. Box 15159, 7528809 Rishon LeZion, IsraelFind articles by

The flowers of French marigold (Tagetes patula L.) are widely used in folk medicine, in particular for treating inflammation-related disorders. However, cellular mechanisms of this activity demand further investigation. In the present work, we studied the potential of T. patula compounds to alleviate the oxidative stress in hydrogen peroxide-challenged human lymphoblastoid Jurkat T-cells. Crude extracts of marigold flowers and purified fractions containing flavonoids patuletin, quercetagetin, and quercetin and their derivatives, as well as the carotenoid lutein, were brought in contact with Jurkat cells challenged with 25 or 50 μM H2O2. Hydrogen peroxide caused oxidative stress in the cells, manifested as generation of superoxide and peroxyl radicals, reduced viability, arrested cell cycle, and enhanced apoptosis. The stress was alleviated by marigold ingredients that demonstrated high radical-scavenging capacity and enhanced the activity of antioxidant enzymes involved in neutralization of reactive oxygen species. Flavonoid fraction rich in quercetin and quercetagetin showed the highest cytoprotective activity, while patuletin in high dose exerted a cytotoxic effect associated with its anticancer potential. T. patula compounds enhanced the production of anti-inflammatory and antioxidant interleukin-10 (IL-10) in Jurkat cells. Both direct radical-scavenging capacity and stimulation of protective cellular mechanisms can underlay the anti-inflammatory properties of marigold flowers.

The genus Tagetes (Asteraceae) is native to Americas but some of its members (in particular T. erecta and T. patula) commonly known as marigolds were naturalized in the Old World (India, North Africa, and Europe) as early as in 16th century [1]. Moreover, some researchers suggest that both species reached India anciently through pre-Columbian transoceanic voyages [2]. Marigold was introduced to Georgia from India, and its ground dried petals became one of the most popular local spices [3]. Both T. erecta and T. patula are grown in Georgia as spice and dye plants [4] recognized for their health-beneficial properties [5].

Tagetes is a multipurpose plant having ornamental, ritual, medicinal, anthelmintic, insecticidal, colorant, food, and forage applications [6, 7]. Healing properties of Tagetes species have been implemented by folk medicine for centuries [8]. In particular, flowers and entire herb of T. patula (French marigold) are used for preparing ethnobotanical remedies against rheumatism, stomach and intestinal problems, kidney and hepatic disorders, fever, and pneumonia [6, 9]. The infusion of T. patula flowers is also implemented as eyewash [6]. The efficacy of orally administered methanolic extracts of T. patula florets against acute and chronic inflammation was confirmed in experiments with animal models [10]. Similar results were obtained for T. erecta (African marigold) extracts [11]. Furthermore, a double-blind placebo-controlled clinical trial showed effectiveness of marigold therapy using T. patula preparations in treating human inflammation-associated disorders such as bunion [12]. The anti-inflammatory effect of T. patula extracts could be reproduced in animal model by oral administration of its flavonoid constituents, patuletin and patulitrin [13]. Lipophilic ingredients of marigold flowers, the carotenoid lutein and essential oil compounds, were also reported to possess anti-inflammatory properties [14, 15]. In our previous study, both hydrophilic and lipophilic fractions from T. patula petals showed the highest radical-scavenging capacities among all Georgian spices tested [16].

However, the cellular mechanisms by which the marigold extracts exert their anti-inflammatory effects are not fully understood and demand further investigation. Methanol extracts of T. patula flowers as well as isolated patuletin were reported to scavenge peroxyl and superoxide radicals in chemical systems and in human neutrophils and at the same time to exert cytotoxic and growth inhibitory effects towards a range of human cancer cell lines, in particular HeLa cells [17]. On the other hand, ethanolic and ethyl acetate extracts of marigold flowers showed no cytotoxicity towards H460 lung cancer and the Caco-2 colon cancer cell lines in an MTT assay [18]. Furthermore, the MTT assay revealed a cytoprotective effect of patuletin on the human lung carcinoma GLC4 cell line challenged by cytotoxic sesquiterpene lactone helenalin [19]. Activation of antioxidant enzymes rather than direct free radical scavenging was suggested as a possible mechanism underlying this phenomenon. Mesaik et al. [20] demonstrated that immunomodulatory and antiarthritic potential of patuletin was associated with inhibited production of the proinflammatory cytokine TNF-α with no cytotoxic property. Chew et al. [21] reported that marigold-derived dietary lutein enhanced phytohemagglutinin-induced lymphocyte proliferation in mice but had no effect on interleukin-2 production or lymphocyte cytotoxicity.

Human lymphoblastoid T-cell Jurkat line is a popular model for the study of immune signaling and inflammation [22]. Jurkat cells can imitate both healthy and inflammatory T-cells in their response to oxidative metabolites, such as hydrogen peroxide [23]. Although H2O2 plays an important role in antigen-dependent lymphocyte activation [24], excessive production of H2O2 induces oxidative stress and impairs T-cell activity, leading to chronic inflammation and cell death. In the presence of oxygen in aqueous medium, hydrogen peroxide can produce additional cytotoxic reactive oxygen species (ROS), such as superoxide and peroxyl radicals [25]. To control the level of ROS, cells employ antioxidant enzymes, for example, catalase that decomposes hydrogen peroxide, and superoxide dismutase neutralizing superoxide radicals, as well as low-molecular antioxidants. The latter group includes internally produced glutathione and dietary antioxidants such as ascorbic acid and phenolic compounds. The function of the antioxidant system is maintained by additional enzymes such as glutathione reductase that restores the antioxidant capacity of oxidized glutathione. Signaling for regulation of oxidative stress and inflammatory responses involve cytokines such as anti-inflammatory and antioxidant interleukin-10 (IL-10) [26].

The oxidative stress can interfere with normal progression of cell growth and division arranged in a cell cycle. In eukaryotes, a normal cell cycle consists of four main stages: G1, during which a cell is metabolically active and continuously grows; S phase, during which DNA replication takes place; G2, during which the growth of cell continues and the cell prepares for division; and the M (mitosis) phase, during which the cell divides into two daughter cells, each with a full copy of DNA. After the M phase, the cells can enter G1 or G0, a quiescent phase. When the cell detects any defects (e.g., oxidative DNA damage) which necessitate halting the cell cycle in G1, cell cycle arrest occurs. Efforts to correct these problems may slow growth and induce cell death [27].

The response of Jurkat cells to H2O2 is dose-dependent. Reversible oxidative changes that could be repaired by cellular antioxidant systems occurred at a H2O2 concentration of 20 μM, while the signs of apoptosis (programmed cell death) were noted at 50 μM H2O2 [28]. Both apoptosis and necrosis (a nonprogrammed cell death caused by damage) were observed in the Jurkat cells exposed to 100 μM H2O2 [29], whereas the necrosis prevailed at 500 μM H2O2 [30].

The balance between prooxidant and antioxidant repair mechanisms determining cellular survival and function can be affected by dietary bioactive compounds possessing radical-scavenging and anti-inflammatory activity. Therefore, in the present work, we investigated the effects of anti-inflammatory T. patula flower extract and of its purified fractions on the behavior of the H2O2-challenged Jurkat cells.

3. Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis

The samples were dissolved in HPLC-grade methanol and filtered through a Millex-HV Durapore (PVDF) membrane (0.22 μm) before being injected into the LC-MS instrument. Mass spectral analyses were carried out using the Ultraperformance LC-Quadruple Time of Flight (UPLC-QTOF) instrument (Waters Premier QTOF, Milford MA, USA), with the UPLC column connected online to a PDA detector and then to an MS detector equipped with an electrospray ion (ESI) source (used in ESI-positive mode). Separation was performed on a 2.1 × 50 mm i.d., 1.7 μm UPLC BEH C18 column (Waters Acquity).

The chromatographic and MS parameters were as follows: the mobile phase consisted of 0.1% formic acid in water (phase A) and 0.1% formic acid in acetonitrile (phase B). The linear gradient program was as follows: 100% to 95% A over 0.1 min, 95% to 5% A over 9.7 min, held at 5% A over 3.2 min, and then returned to the initial conditions (95% A) in 4.2 min. The flow rate was 0.3 mL min−1 and the column was kept at 35°C. Masses of the eluted compounds were detected with a QTOF Premier MS instrument. The UPLC-MS runs were carried out at the following settings: capillary voltage of 2.8 kV, cone voltage of 30 eV, and collision energy of 5 eV. Argon was used as the collision gas. The m/z range was 70 to 1,000 D. The MS system was calibrated using sodium formate and Leu-enkephalin was used as the lock mass. The MassLynx software version 4.1 (Waters) was used to control the instrument and calculate accurate masses. The compounds were identified using the molecular formulae calculated on the basis of accurate mass and isotopic pattern information, and UV/visible spectra, in comparison with authentic standards of quercetin, quercetagetin, and quercetagetin-7-glucoside (Extrasynthese, Genay, France).

4. Cell Culture and Experimental Design

The human T-cell leukemia lymphoblastoid Jurkat cells (DSMZ ACC 282) were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany). The cells were grown in suspension culture at 37°C under 5% humidified CO2 in bioactive medium RPMI 1640 (Gibco, Grand Island, NY, USA) containing inactivated embryonic bovine serum (Sigma, St Louis MO, USA), L-glutamine (4 mM), penicillin (100 U mL−1), and streptomycin (100 U mL−1). The experiments were carried out at cell densities of 0.3 to 0.6 × 106 cells mL−1. In order to imitate the oxidative stress conditions, H2O2 (Sigma) was added to the Jurkat culture to reach the concentrations of 25 and 50 μM, corresponding to low and intermediate stress severity, respectively [28]. In the unstressed control treatment, water was added to the samples instead of H2O2. The crude marigold extracts and isolated fractions were added to the cultures at a rate of 2 mg mL−1, if not specified differently in the text.

Why YOU Should Plant Marigolds. EVERYWHERE.

FAQ

Are African marigolds good for pollinators?

Do African marigolds do well in pots?

Do African marigolds like full sun?

What are the benefits of marigolds for the environment?

How big do African marigolds get?

African marigolds are taller and more tolerant of hot, dry conditions than French marigolds. They also have larger flowers that can be up to 6 inches (15 cm.) in diameter. If deadheaded regularly, African marigold plants will usually produce many large blooms. They grow best in full sun and actually seem to prefer poor soil.

How long do African marigold plants take to grow?

African marigold plants propagate easily from seed started indoors four to six weeks before last frost date or sown directly in the garden after all danger of frost has passed. Seeds usually germinate in 4 to 14 days. African marigold plants can also be purchased at most garden centers in the spring.

How much water do African marigolds need?

The answer to this question depends on several factors, such as the climate, soil type, and stage of growth. In general, African marigolds should be watered deeply once a week during the growing season. This means that you should water the plants until the soil is moist to a depth of 6 inches.

Are African marigolds annuals?

Also called American marigolds or Aztec marigolds, African marigolds are annuals that bloom from early summer until frost. African marigolds are taller and more tolerant of hot, dry conditions than French marigolds. They also have larger flowers that can be up to 6 inches (15 cm.) in diameter.