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Effects of Blue Green Algae Extracts on Proliferation of Human Adult Stem Cell in Vitro

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Introduction

Sechium edule (Jacq.) Sw. (Cucurbitaceae), which is also known as chayote, is a neotropical constitute species that is endemic to Mexico. This species includes 12 varieties, some of which have high nutritional value. S. edule is used in traditional medicine for the treatment of different illnesses due to its hypotensive and vasodilator activities (Cambar et al. 1980; Cambar 1985). Its roots, stems and leaves are used as antimicrobial agents, and the leaves, when mixed with the seeds, tin human activity as antioxidants (Ordoñez et al. 2003, 2006). The fruit has been reported to have anti-inflammatory and cardiotonic activities (Salama et al. 1986) and is thus used to care for arteriosclerosis (Gordon et al. 2000). The fruit can also function equally a depressor of the central nervous system and an antiepileptic agent (Firdous et al. 2012).

Sechium edule fruit excerpt contains non-phenolic alkaloids, saponins, sterols, triterpenoids (Salama et al. 1986) and glycosylated flavonoids (Siciliano et al. 2004) that confer its anti-inflammatory (Salama et al. 1987), antihypertensive (Gordon et al. 2000), antimicrobial (Ordoñez et al. 2003), antioxidant (Ordoñez et al. 2006), antitumor (Cadena-Iñiguez et al. 2013; Aguiñiga-Sánchez et al. 2015), nephroprotective (Firdous et al. 2012), and hepatoprotective backdrop (Firdous et al. 2013); however, even though Due south. edule has been used for the handling of various diseases, about studies have not considered the diverseness of the varieties of this species. This consideration is important considering Cadena-Iñiguez et al. (2013) reported that 8 of the 12 varieties accept differential activities beyond different tumor jail cell lines and genotypes. 1 variety, nigrum spinosum, exhibits greater antiproliferative activity, particularly on the murine macrophage leukemic P388 cell line.

Hematopoietic tissue is responsible for the generation of both leukemic and normal hematopoietic cells (Orkin & Zon 2008). Leukemic cells are highly sensitive to the presence of antitumor agents because these agents reduce the number of cells undergoing cell partition, an event that can be quantified equally the mitotic index. As a side upshot, the cytotoxic upshot through the body (Yi et al. 2010; Alakoc & Eroglu 2011) results in a reduction in the number of generated hematopoietic cells, which compromises the patient'due south well-beingness (Culakova et al. 2014). Therefore, the utilize of selective antineoplastic agents that can eliminate tumor cells without harming normal cells would be desirable (Setzer & Setzer 2003; Alexander-Bryant et al. 2013). An inedible hybrid of South. edule known as H387 07 has these features (Aguiñiga-Sánchez et al. 2015). However, whether the edible S. edule var. nigrum spinosum tin can selectively kill leukemia cells without harming normal hematopoietic cells remains unknown. This study compared the in vitro and in vivo cytotoxic effects of fruit extract from South. edule var. nigrum spinosum and analyzed the secondary metabolites of the extract to provide insights that could help explain the observed biological effects.

Materials and methods

Plant cloth and excerpt preparations

Fruits of Southward. edule var. nigrum spinosum at horticultural maturity, specifically 18 ± ii days after anthesis, were collected by an Interdisciplinary Research Grouping of Sechium edule in Mexico, A.C. (GISeM) in the Bank of Germplasm of CRUO-UACh, Veracruz, Mexico, in 2011 and authenticated past the National Seed Inspection and Certification Service (SNICS; Ministry building of Agriculture, Livestock, Rural Development, Fisheries and Nutrient, Mexico) by Cadena-Iñiguez J. A voucher specimen (Accession 354-06 nigrum spinosum) remains in the Bank of Germplasm of CRUO-UACh (19°49′N, 97°21′East, Veracruz, Mexico). Immediately later collection, the fruits were cleaned, cut into small-scale pieces, dried at 45 °C to a moisture level of 10%, and ground to a standardized particle size of 2 mm (mesh No. 10). The plant material was so stored under aseptic weather at room temperature until use.

Extraction procedure

15 hundred grams of the stored plant material was extracted in batches with methanol (99.8%, ACS, Merck, Darmstadt, Germany) for 48 h at room temperature (20 ± 2 °C). The resultant alcoholic excerpt was filtered with Whatman cellulose filter paper (Sigma-Aldrich St. Louis, MO) for 30 passages, and the solvent was renewed until the macerated product did not show a colour. The solvent was and then evaporated at fifty °C under reduced pressure (Buchi Rotavapor R-114, Flawil, Switzerland) until a crude extract, without organic solvent, was obtained.

Additionally, to clarify whether the presence of sugars could change the extraction yields of compounds establish at lower concentration, we performed continuously selective extraction with lower-, medium- and college-polarity organic solvents, specifically hexane, dichloromethane and methanol, respectively. The solvents were then evaporated until an extract fraction without organic solvent was obtained.

Identification of compounds past HPLC

Preliminary tests were performed by sparse-layer chromatography (TLC) on silica gel plates (Merck G60 lxx–230 mesh, Germany) with a mobile phase of acetic acid and methanol (95:5) and cucurbitacins B, D, Eastward and I equally the standards. After, the content and type of the metabolites in the extract were identified by loftier-functioning liquid chromatography (HPLC). For the cucurbitacins, a symmetry shield column RP18 (4.6 × 250 mm, Waters Corporation, Milford, MA) was used, and an isocratic assay was conducted using water:methanol:acetonitrile (fifty:30:20) with the following experimental parameters: flow, 1 mL/min; pressure, 179 bar; temperature, 20 °C; injection volume, twenty μL; wavelength, 235 nm; and analysis time, 50 min. For the flavonoids, the analyses were performed on a Hypersil ODS (125 × xl mm) Hewlett Packard Cavalcade with a gradient of (A) HiiO at pH 2.5 with TFA (trifluoroacetic acid) and (B) ACN (acetonitrile) and the post-obit parameters: flow, 1 mL/min; temperature, 30 °C; variable injection volume; and analysis fourth dimension, 25 min. The standards used were rutin, phloridzin, myricetin, quercetin, naringenin, phloretin, apigenin and galangin. For phenolic acrid, a Nucleosil column (125 × 4.0 mm) from Macherey-Nagel was used with a slope of (A) HiiO at pH 2.5 with TFA (trifluoroacetic acid) and (B) ACN (acetonitrile). The other experimental parameters included the following: catamenia, 1 mL/min; temperature, 30 °C; variable injection volume; and assay time, 25 min. Caffeic, gallic, chlorogenic, vanillic, p-hydroxybenzoic, p-coumaric, ferulic and syringic acids were used equally the standards.

In vitro toxicity assays

Cell civilization

The murine macrophage-similar leukaemia P388 jail cell line was purchased from the American Blazon Civilization Drove (ATCC: The Global Bioresource Center, Manassas, VA) and maintained in Iscove'southward Modified Dulbecco's Medium (IMDM) culture medium (GIBCO-BRL Invitrogen Grand Island, NY) supplemented with 10% deactivated foetal bovine serum (FBS) (Invitrogen GIBCO-BRL HyClone, Carlsbad, CA) in drinking glass Petri dishes (Pyrex, US) at 37 °C in an incubator (Thermo Forma, Marietta, OH) with an temper of five% CO2 and 95% humidity. The cells (1 × 10five jail cell/mL) were seeded and split up every 48 h one time they reached xc% confluence. Total bone marrow cells from CD-ane mice were extracted from the femur and flushed with IMDM supplemented with 10% FBS. Mononuclear os marrow cells (MNCBMs) were isolated from the total cells through gradient separation with Ficoll-Pacque (Amersham Biosciences AB, Uppsala, Sweden) at a density of i.077 thousand/mL and were done twice with PBS. MNCBMs were cultured for 120 h in IMDM supplemented with 15% (v/5) FBS, 5% (v/v) equus caballus Equus ferus Linnaeus (Equidae) serum (Gibco-BRL, Carlsbad, CA) and 5 ng/mL recombinant mouse interleukin-3 (rmIL3; R&D System, Minneapolis, MN). The cells were cultured in a humidified temper with five% CO2 at 37 °C for a maximum elapsing of 120 h.

Proliferation, cell viability and Annexin-V in vitro assays

P388 cells and MNCBMs were grown in 96-well plates (Corning Costar, St. Louis, MO) at initial densities of two × 10four and 1 × xv, respectively, with or without the addition of different concentrations of the extract. To evaluate prison cell proliferation after growth, the cultures were and then fixed with 1.ane% glutaraldehyde and stained with crystal violet in 0.1% formic acid (Sigma-Aldrich St. Louis, MO). The dye was solubilized in 10% acerb acrid, and the optical density at 570 nm was determined using a plate reader (Tecan Spectra, Grödig, Austria). The data were plotted, and the IC50 value was adamant by linear regression. Trypan blue (Sigma, St. Louis, MO) exclusion assays were used to decide the number of viable cells in each treated civilization equally well as the IC50. The prison cell viability was determined by direct counting in a Neubauer chamber, and the non-stained cells were considered viable. The results are shown as the hateful cell viability percentages ± standard deviations (SDs) from triplicate cultures. Finally, to place the induction of apoptosis in the early stages, the cells were washed and labeled with an Annexin V-FITC kit (BD Biosciences, Franklin Lakes, NJ). After incubation, the cells were analyzed using a BD FACSAria Ii flow cytometer (BD Biosciences, The states).

In vivo toxicity assays

Experimental animals

Both female and male ten- to 12-week-old syngeneic CD-1 mice were maintained in the animal facility of FES-Zaragoza with an advertisement libitum sterile standard powdered rodent diet. All experimental protocols were canonical by the Ethics Committee of FES-Zaragoza and were performed in accordance with the inquiry methodologies and evaluation protocols for traditional medicine, the 'Guide for the Care and Use of Laboratory Animals, Eighth Edition' published by the National Institutes of Health and the national regulations for the care and utilise of experimental animals (NOM-062-ZOO-1999).

Determination of acute toxicity (LD50)

The method used for the determination of acute toxicity was described by Lorke in 1983. Ten groups of five mice each (both male and female) were used, and a single dose of the extract (8, 16, 40, 160, 400, 800, 1600, 2900, 5000 and 0 mg/kg extract in PBS/methanol, ane:100 v/v vehicle) was intraperitoneally administered. The mice were monitored every iii, half dozen, 9 and 12 h for the first 24 h post-treatment for signs of toxicity and death and were then monitored in one case a day for seven days. The LD50 was then determined based on the obtained data.

Organic disorders and blood samples

8 days afterward handling (and prior to cede), the mice were weighed, and the value was recorded. The organs, such equally the spleen, thymus and liver, were carefully removed and weighed, and the organ index was calculated as the ratio of the organ weight to the total body weight. Peripheral claret was collected by cardiac puncture with and without EDTA every bit an anticoagulant. Whole blood was used for the analysis of total leukocytes (WCBs), full cerise claret cells (RCBs), hemoglobin (Hb), hematocrit (Hct), mean corpuscular hemoglobin (HCMC), hateful corpuscular volume (MCV), platelets (PLTs) and differential counts of lymphocytes, granulocytes and monocytes with an automated hematology MICROS 60 musical instrument (HORIBA Medical, Kyoto, Nihon). Serum was used to determine the claret biochemistry parameters, such as the concentrations of glucose, cholesterol, triglycerides, creatinine, urea, uric acid, aminotransaminase (AST), alanine aminotransaminase (ALT) and bilirubin using a clinical automatic biochemical analyzer (Hitachi 912, Boehringer Mannheim, Germany).

Mitotic index

To evaluate the in vivo toxicity of the excerpt, the number of cells undergoing cell division (mitosis) in the os marrow (Antunes et al. 2000) was determined. Briefly, 24 h after the final inoculation of the extract, 300 μL of 0.1% colchicine (Sigma, St. Louis, MO) was intraperitoneally injected, and two h subsequently injection, both femurs were obtained using a hypotonic solution with 0.047 M KCl. These samples were then incubated for 20 min at 37 °C, and the cells were stock-still with a solution of methanol:acetic acid (3:1) and stained with Giemsa dye (Sigma, St. Louis, MO). One thousand cells were counted to obtain the percentage of dividing cells.

Statistical analysis

All analyses were performed using Microsoft Excel (Redmond, WA) and IBM SPSS Statistics (version xviii; Armonk, NY). All values are expressed every bit means ± SDs. Analysis of one-way variance was performed following Dunnett's comparison test. p ≤ 0.05 was considered statistically meaning.

Results

Effects of the Southward. edule var. nigrum spinosum extract on the P388 leukemia cell line and normal mononuclear bone marrow cells

We recently showed that S. edule var. nigrum spinosum reduces the proliferation of the macrophage leukemic P388 jail cell line (Cadena-Iñiguez et al. 2013), but it remains unclear whether it as well affects MNCBMs both in vitro and in vivo. The current results showed that the S. edule var. nigrum spinosum extract inhibited the proliferation of both leukemic P388 cells and normal cells, although the inhibition of proliferation was more than marked in the leukemic cells (Figure ane). This observation was confirmed by the ICfifty values of 927 and 1911 μg/mL obtained for the leukemic P388 cell line and MNCBMs, respectively. The extract at the IC50 concentration obtained for the leukemic line reduced the viability and induced the apoptosis of leukemic P388 cells but not the normal mononuclear bone marrow cells. Cytarabine damages both cell types every bit (), as was previously shown for this antineoplastic agent (Stentoft 1990; Verstappen et al. 2003).

Figure 1. In vitro effects of the South. edule var. nigrum spinosum extract on the proliferation of the P388 leukemia cell line and mononuclear bone marrow cells of healthy mice (MNCBMs). The cells were exposed to the S. edule var. nigrum spinosum excerpt, and the cellular proliferation was evaluated using a crystal violet assay. The data are presented as the means ± SDs (n = 4) and are representative of 4 contained experiments. The significance of the differences were determined by Tukey'southward test (p ≤ 0.05).

Table 1. Per centum of viability and apoptosis of P388 and bone marrow mononuclear cells from mice.

We previously quantified the concentrations of sugars in the extract as 3.178 ± 0.3326 one thousand of sugar/100 g of extract (Riviello 2015); however, nosotros analyzed the extraction yield of the compounds institute at lower concentrations (terpenes and flavonoids) in selective extract fractions and the biological activity of these individual fractions.

The selective extract fractions had yields of 0.52% in the hexane fraction (terpenes), one.20% in the dichloromethane (terpene) fraction and 54.82% in the methanol fraction (flavonoids). The comparison of the biological activities of the crude extract and the three selective fractions for the proliferation of MNCBMs revealed that the crude excerpt exerted a significant antiproliferative issue at concentrations college than 1180 μg/mL, whereas the fractions of hexane and methanol exerted effects at concentrations college than 2370 μg/mL, and the dichloromethane fraction does non announced to have biological activity (data not shown). As a consequence, the biological activity of the crude extract is higher than those of the individual fractions.

Toxicity of the Southward. edule var. nigrum spinosum extract in salubrious mice

Inspired by these findings and the fact that this variety of chayote eliminates leukemic cells by apoptosis without harming normal bone marrow cells (Aguiñiga-Sánchez et al. 2015), we evaluated the in vivo acute toxicity of the S. edule var. nigrum spinosum extract in accordance with Lorke (1983). The results indicated that the LDl was higher than 5000 mg/kg, which indicated that the S. edule var. nigrum spinosum excerpt is safe according to Lorke (1983).

Literature data suggest that repeated doses should be administered if the dose is less than the LD50 (Akhila et al. 2007); therefore, 800 mg/kg was administered i.p. every 48 h for seven days. The data indicated that this dose does not induce changes in the spleen, liver or thymus index (), and alterations in these parameters indicate toxicity. The evaluation of the biochemical parameters of the claret plasma () revealed that the South. edule var. nigrum spinosum extract only reduced the glucose levels, as previously published (Diré et al. 2006). Cytarabine, in contrast, increased the levels of uric acid and creatinine which indicatives kidney harm (Jesse 1982), and increased the levels of AST and total bilirubin which indicatis liver toxicity (Kumar et al. 2005; Karadeniz et al. 2011).

Table 2. Spleen, liver and thymic indexes of healthy mice treated i.p. with the Southward.east. nigrum spinosum extract at a dose of 800 mg/kg.

Tabular array 3. Blood biochemistry of good for you mice treated with the S.e. nigrum spinosum extract at a dose of 800 mg/kg.

The aforementioned treatment revealed that the Due south. edule var. nigrum spinosum extract increased the WBC without altering the other parameters, whereas cytarabine significantly reduced the hateful corpuscular volume (MCV) and increased the platelet levels (). These toxic effects were previously reported for cytarabine (Gobbi et al. 2009); thus, this dataset indicated that the S. edule var. nigrum spinosum extract does not induce systemic damage.

Table four. Blood count of healthy mice treated with the Due south.e. nigrum spinosum extract at a dose of 800 mg/kg.

Toxicity of the S. edule var. nigrum spinosum excerpt on mononuclear normal os marrow cells

Based on the increased number of WBCs in mice treated with the South. edule var. nigrum spinosum excerpt, nosotros evaluated the types of jail cell populations that were enhanced. The data indicated that the S. edule var. nigrum spinosum extract increased the levels of lymphocytes, monocytes and granulocytes, whereas cytarabine only increased the levels of monocytes and granulocytes (Effigy 2). As the S. edule var. nigrum spinosum extract and cytarabine increased the levels of some only non all WBCs, nosotros evaluated whether these increases are related to increased prison cell sectionalisation in the bone marrow, an organ that is highly sensitive to cytotoxic agents (Scatena et al. 2010). We constitute that only cytarabine exhibited agin effects, whereas the S. edule var. nigrum spinosum extract only increased the mitotic index in the bone marrow cells (Figure iii). It is known that cytarabine inhibits cell division in the bone marrow (Wei et al. 2013), and this finding was confirmed in this study.

Figure ii. Differential peripheral claret counts of salubrious mice treated with the South. edule var. nigrum spinosum extract at a dose of 800 mg/kg. n = 20 mice per treatment. The data are presented as the means ± SDs; *indicates a significant difference (Tukey's exam, p ≤ 0.05).

Figure 3. Mitotic index (MI) of bone marrow cells from healthy mice treated with the S. edule var. nigrum spinosum extract at a dose of 800 mg/kg. n = 20 mice per treatment. The data are presented as the ways ± SDs; *indicates a meaning difference (Tukey's test, p ≤ 0.05).

Identification of the chemical components of the S. edule var. nigrum spinosum excerpt

Chromatographic studies were performed to identify the secondary metabolites in the S. edule var. nigrum spinosum extract (Figure 4). Terpenes and flavonoids were the major components identified by colorimetric methods, and no alkaloids were identified in this written report. In add-on, an HPLC analysis indicated that the S. edule var. nigrum spinosum excerpt contains cucurbitacins B and D and traces of cucurbitacin I (one.008, 0.127 and 0.013 mg/thousand of extract, respectively), which are findings that have not been reported in the literature for this species and diverseness. Additionally, nosotros found some phenolic acids, such as gallic, chlorogenic, vanillic, p-hydroxybenzoic, caffeic and p-coumaric acids (0.072, 0.823, 0.032, 0.020, 0.091 and 0.032 mg/g of extract, respectively). Flavonoids are represented in the S. edule var. nigrum spinosum extract by phloridzin, naringenin, phloretin and apigenin (0.005, one.556, 0.018 and 0.292 mg/thou of extract, respectively).

Effigy 4. Representative high-functioning liquid chromatography (HPLC) chromatogram of the S. edule var. nigrum spinosum extract: (A) phenolic acids, (B) cucurbitacins C and D, and (C) flavonoids.

Discussion

Sechium edule var. nigrum spinosum is consumed by humans as nutrient and is also used as an antitumour agent (Alonso-Castro et al. 2011). In this written report, we show that the S. edule var. nigrum spinosum excerpt is used for treating leukemic cells, which is consistent with previous observations that showed normal cells are undamaged by the H387 07 chayote hybrid (Aguiñiga-Sánchez et al. 2015), even though S. edule var. nigrum spinosum has less potent biological activity than H387 07. In addition, these data betoken that this edible multifariousness presents biological selectivity.

The increased mitotic index of the bone marrow in response to the S. edule var. nigrum spinosum extract explains the increment in lymphocytes, monocytes and granulocytes in the peripheral claret. In this sense, a nutraceutical formulation of blueberry excerpt, green tea extract, carnosine and vitamin D3, which is known equally NT-020, promotes the proliferation of human hematopoietic stem cells in vitro and protects stem cells from oxidative stress when given chronically to mice in vivo (Shytle et al. 2010). In this written report, we show that the S. edule var. nigrum spinosum excerpt of an edible vegetable promotes blood cell generation and can thus be considered a nutraceutical agent.

Our study of the secondary metabolites in the S. edule var. nigrum spinosum extract identified terpenes and flavonoids as the major components. These data correlate with the presence of saponins and sterols/triterpene in fruits collected from Colombia, where the presence of alkaloids has as well been reported (Salama et al. 1987; Cadena-Iñiguez et al. 2007). We did not detect alkaloids in this study, and this difference might exist due to both the diverseness of the plant that was used and the growing conditions (Loraine & Mendoza-Espinoza 2010).

In addition, we institute cucurbitacins B, D and I in the S. edule var. nigrum spinosum excerpt. These compounds belong to a group of terpenes, which are metabolites recognized equally inhibitors of the proliferation of tumour and normal cells (Nelson & Falk 1992; Setzer & Setzer 2003; Shao et al. 2013; Li et al. 2015). Additionally, we plant gallic, chlorogenic, vanillic, p-hydroxybenzoic, caffeic and p-coumaric acids, but only gallic and caffeic acids have been reported in S. edule leaves (Ordoñez et al. 2006).

Cucurbitacins B, D and I are extremely toxic (Setzer & Setzer 2003), but their enrichment with the other phytochemicals found in the extract of Due south. edule var. nigrum spinosum together maintains the event of the extract on tumor cells merely allows the protection of normal cells in vitro. It is too likely that this machinery of action confers the nontoxic in vivo characteristics.

Interestingly, naringenin and apigenin accept been found in the stems and leaves simply not in the fruits. 1 possible caption for this difference in the phytochemical content in fruits might result from the variety of fruit used, which was unfortunately not reported by Siciliano et al. (2004). All of the phytochemicals plant in the S. edule var. nigrum spinosum fruit have been previously mentioned equally possible agents with cytotoxic action in leukaemia, lymphoma, and solid tumours (Nelson & Falk 1992; Wang et al. 2008; Shao et al. 2013; Spilioti et al. 2014).

Finally, nosotros want to emphasize that caffeic acrid is cytotoxic to SNU638 gastric cancer cells, AGS cells and HCT 116 colorectal carcinoma simply not to normal cells (Wang et al. 2005; Kim et al. 2011; Watanabe et al. 2011). Furthermore, information technology has been plant that gallic acid, caffeic acid, naringenin and phloridzin have antioxidant and anti-inflammatory activities (Hale et al. 2008; Wybranowski et al. 2014), which can help prevent jail cell impairment (Pietta 2000) and might explain their cytoprotective activities on normal cells. In the future, it would be interesting to analyze the metabolic pathways that are activated in neoplasm cell lines relative to normal cells in order to explain the observed cytotoxicity or cytoprotection and thus, strengthen the therapeutic potential of the S. edule var. nigrum spinosum extract. These data indicate that consuming this product provides qualities beyond its nutritional value as food, such as antiproliferative and nutraceutical activities (based on the identified metabolites), and this characteristic provides an added value to a nontraditional vegetable that is widely consumed, particularly among socially disadvantaged people.

Conclusions

The Due south. edule var. nigrum spinosum excerpt is not cytotoxic to mononuclear bone marrow cells in vitro and in vivo when it is administered intraperitoneally at doses of 800 mg/kg every 48 h for 7 days. The extract contains metabolites, such every bit flavonoids, phenolic acids and cucurbitacins, compounds that can eliminate neoplasm cells while protecting normal bone marrow cells. Therefore, the S. edule var. nigrum spinosum excerpt is an emerging natural agent that can be used for the treatment of various diseases without any harmful side furnishings.

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Source: https://www.tandfonline.com/doi/full/10.1080/13880209.2017.1316746

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