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    • 专利摘要:
    Extracts of floral biorresiduos of saffron after the separation of the spice, to be used as active principles of cosmetic products with antioxidant properties, once confirmed the antioxidant activity expected from them for its high content of phenols, flavonoids and anthocyanosides, and evaluated favorably said activity against cellular oxidative stress by experimentation in vitro and on cell culture of hepatic carcinoma. Its importance lies in that in addition to serving as the basis for the development of a new range of cosmetics with these properties, there is an advantage to the large amount of floral waste generated in the collection of saffron, for which so far no has found no practical use. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2646415A1
    申请号:ES201630788
    申请日:2016-06-09
    公开日:2017-12-13
    发明作者:Nuria Acero De Mesa；Dolores MUÑOZ-MINGARRO
    申请人:Bielsa Pons Eva Ma；Fundacion Universitaria San Pablo CEU；
    IPC主号:
    专利说明:

    Saffron bio-waste extracts as active ingredients of antioxidant cosmetic products.
    Extracts obtained from the floral residues of saffron after spice separation are presented and claimed to be used as active ingredients of cosmetic products with antioxidant properties. Its importance lies in that in addition to serving as a basis for the development of a new range of cosmetics with these
    10 properties, take advantage of the large amount of floral waste or bio-waste generated in the collection of saffron, for which so far no practical utility has been found.
    Knowing that saffron shows high antioxidant activity, the
    15 recent works on the antioxidant capacity of the bio-wastes of this plant in order to use them as a basis for obtaining extracts for the aforementioned purpose, these extracts, cold and hot polar extracts, have been confirmed, the expected antioxidant activity of the same for its high content of phenols, flavonoids and anthocyanosides, and this activity has been evaluated against cellular oxidative stress through
    20 experimentation in vitre and on cell culture of liver carcinoma, obtaining results that make them suitable for use as active ingredients of cosmetic products for body and facial rejuvenation.
    APPLICATION FIELD.
    The field in which the invention is framed is the development of cosmetics and products for hygiene and beauty treatments for people, finding application in the pharmaceutical and cosmetic industry in particular. 30 STATE OF THE TECHNIQUE.
    In the traditional production of saffron, the collection of the flowers of the plant is done by hand, and the separation of the red stigmas from the rest of the flower, which constitute the saffron spice, too. This practice makes the final product more expensive, but it also generates a large amount of floral waste, estimated at 63 kg of bio-waste for each kg of spice produced, for which interest has recently increased in seeking some application.
    The search for new natural compounds or complex phytoextracts with antioxidant activity, is particularly interesting at present for the food and cosmetic industry (Karimi el al., 2010). His study is of interest since antioxidants are molecules capable of preventing or retarding the oxidation of others.
    5 compounds, and have significant potential to reduce the development of some increasingly common diseases in the world population.
    In this sense and as demonstrated by a simple search on the Internet, it is known that saffron, such as the spice derived from the Crocus sativus flower, among other benefits for
    10 health, it has antioxidant properties, and can potentially be used as a base of cosmetic products (IMujer and Botanical), due to active components such as crocina and safranal, which are substances that "sequester" free radicals, delaying the aging of cells of the body and improving overall health.
    15 More specifically, studies on the chemical composition of C. saliva tepals reveal the presence of quinsenoside, goodieroside, 3-hydroxy-and-butyrolactone (Righi et al., 2015), as well as crocina and a high concentration of phenols (Montoro et al., 2012), especially kaeplerol and its glycosides (Zeka el al., 2015). The antioxidant activity of the tepals has been determined by the ABTS and DPPH assay, revealing a
    20 high antioxidant activity, sometimes higher than that of a-Tocopherol (Li et al., 2004).
    In view of these data it has already been proposed in different scientific papers that the floral remains of saffron have a high commercial potential as a natural source of antioxidants (Zeka el al., 2015; Righi el al., 2015; Hosseinzadeh and Younesi, 2002 ).
    25 Termentzi and Kokkalou (2008) attribute the antioxidant capacity and possible applications of this part of the plant to the presence of f1avonoids. On the other hand, but in the same line of antioxidation studies, Omidi et al. (2014) conclude that tepal extracts of C. sativus reduce the complications of hepatotoxic induced oxidative stress in rats.
    There are several publications that demonstrate a high content of phenols and other antioxidant compounds in saffron bio-waste, giving it high activity and presenting them as a possible functional food or ingredient. In addition to the antioxidant activity, other biological activities have been detected in extracts of bio-waste from saffron, such as: absence of cytotoxicity in fibroblasts (Serrano-Díaz et al., 2013); anti-inflammatory and antinociceptive effect (Serrano-Díaz el al., 2012; Hosseinzadech and Younesi,
    2002); tyrosinase inhibitory activity (Kubo and Kinst-Hori, 1999); or ability to reduce blood pressure (Fatehi et al., 2003).
    However, none of these publications proposes that bio-waste from
    5 saffron can have a possible cosmetic use, which is precisely the objective of thepresent invention, that of providing extracts of the floral residues of Crocussafivus that can serve as antioxidant active ingredients for the preparation ofCosmetics and hygiene and personal beauty treatment products.
    10 SUMMARY OF THE INVENTION.
    The bio-waste extracts of the saffron in question have been obtained from samples
    frozen and fresh from the floral waste of Crocus sativus, once separated
    Red stigmas of the flower during the traditional spice harvesting process, both
    15 by continuous hot extraction, as by cold alcohol extraction, used methanol as a solvent, and have been characterized by their content in phenols, flavonoids, and anthocyanosides.
    The phenol content, determined by the Folin-Ciocalteau method, has been found to be
    20 117.10 mg eq gallic acid Ig extract obtained in cold and 51, 39-42.5 mg eq gallic acid Ig extract obtained hot, as shown in the graph in figure 1 at the end of this report, which puts It is clear that the optimal extraction method to obtain the active ingredients with antioxidant activity of the proposed drug is cold extraction, since the concentration of phenolic compounds responsible for this activity is
    25 significantly higher when the extract is not heated.
    The content of flavonoids and anthocyanosides, determined by calorimetric assay for the cold extract, has been 20.12 ± 4.45 mg eq epicatechin / g extract, and 9.3013 ± 0.764 mg cyanidin 3-glucoside Ig extract, respectively, according to the graph in figure 2.
    Subsequently, the antioxidant activity of the extracts has been evaluated against cellular oxidative stress through in vitro experimentation, carried out by DPPH and Xantinal Xanthine oxidase assays, and on hepatic carcinoma cell culture, by MTT assay, diacetate assay. 2-7-diciorodihydrofluorescin (DCFH-DA) and test
    35 glutathione of Cayman.
    The result of the DPPH test for hot extracts at an extract concentration of 500 IJg / mL in methanol reveals a capacity to capture the DPPH-radical compared to gallic acid used as a reference substance, with a lesion of 5,664 ± 0.232 IJg / mL, 27%, and for extracts obtained cold to it
    5 concentration in methanol, 37%,
    The Xanthine / Xanthine oxidase assay reveals the ability to capture the superoxide radical 0 2 'compared to gallic acid with ICs of 7.01 ± 0.4731 IJg / mL, and the ability to inhibit Xanthine Oxidase compared to gallic acid at a
    10 concentration of 8.33 IJg / mL, shown in Figures 3 and 4 in percentages for different concentrations of hot extract, both from fresh and frozen samples,
    The results of these in vitro tests allow us to conclude that the extracts studied have a capacity to capture DPPH and O, which is low compared to the reference substance,
    Through the MTT cell culture test, it has been determined that the effect on the viability of Hep G2 cells under normal conditions and under oxidative stress
    20 induced by H20 2 (200 IJM) for different concentrations of hot obtained extract, both from fresh and frozen samples, is the graphs of Figures 5 and 6 in percentages of cell growth inhibition versus control with PBS (100%)
    25 By testing the 2-7-dichlorodihydrofluorescin (DCFH-DA) diacetate, it has also been possible to determine that the ability to reduce the concentration levels of intracellular ROS in Hep G2 culture for different concentrations of hot and fresh frozen extract obtained it is the one shown in the graphs of figures 7 and 8 in percentages of fluorescence at 90 minutes with respect to the control with PBS (100%), in
    The presence and absence of H20 Z (stressed and unstressed control), and in Figures 9 and 10 in fluorescence indices over time for different concentrations of cold-obtained extract,
    Finally, the Cayman glutathione assay, based on the enzymatic recycling of GSSG to 35 GSH in the presence of the enzyme glutathione reductase for the quantification of GSH, determines that the ability to increase levels of GSH concentration in culture of
    Hep G2 is the one shown in Figure 11 in percentages of GSH versus control in the absence of H20 2 (unstressed control).
    The results of these tests performed on cell cultures show
    5 that at the concentrations tested, the extracts lack relevant cytotoxic activityin Hep G2 cells, and that show significant antioxidant activity as protectorsCellular versus induced oxidative stress, reducing intracellular ROS levels andincreasing the concentration of GSH.
    10 All of this demonstrates that extracts obtained from saffron bio-wastes can be used as active ingredients of antioxidant cosmetic products, since, on the one hand, the extracts are capable of capturing free radicals, reducing the oxidative stress that could be caused by External factors such as pollution or stress cells, and, on the other hand, are able to prepare the cell for eventual stress
    15 oxidative, acting as a preventive of possible cellular damage.
    FIGURES AND GRAPHICS.
    At the end of the present specification the following figures are included with the graphs 20 of the results of the tests carried out, which will be explained and discussed in detail in the embodiment of the invention:
    Figure 1. Amount of phenols present in the extracts of Crocus sativus, (saffron plant) determined by the Folin-Ciocalteau method.
    Figure 2: Concentration of flavonoids and anthocyanosides in Crocus sativus extracts
    obtained in cold, determined by calorimetric test.
    Figure 3. Percentage of capture of the Radical 0 2-versus the different concentrations of the
    30 hot extracts, determined by the Xanthine Xanthine oxidase assay
    Figure 4. Percentages of inhibition of xanthine oxidase against the different
    concentrations of Crocus sativus and gallic acid extracts at a concentration of 8.33 IJg / mL, determined by the same test.
    Figure 5. Effect of frozen saffron extract on the viability of Hep G2 cells under normal conditions and under oxidative stress induced by HzOz (200 IJM), determined by MTT cell culture assay
    5 Figure 6. Effect of fresh saffron extract on the viability of Hep G2 cells, innormal conditions and low oxidative stress induced by H20 2 (200¡1M), throughprevious essay.
    Figure 7. Effect of Crocus sativus flower extracts on ROS concentration
    10 intracellular in Hep G2 culture, determined by the 2-7 dichlorodihydrofluorescin (DCFH-DA) diacetate assay
    Figure 8. Protective effect of Crocus sativus flower extracts on the concentration of intracellular ROS in Hep G2 culture, determined by DCFH-DA.
    Figure 9: Direct effect of different concentrations of saffron extract on the concentration of intracellular ROS over time, according to DCFH-DA.
    Figure 10: Protective effect of different concentrations of saffron extract on the concentration of intracellular ROS over time, according to DCFH-DA.
    Figure 11: Effect of saffron floral bio-waste extracts on reduced glutathione (GSH) levels, determined by the Cayman glutathione test.
    25 FORM OF REALIZATION.
    1. Obtaining the extracts of bio-waste from saffron.
    The plant samples were transferred by Don Antonio Bielsa Moliner, interested farmer
    30 in the recovery of the traditional cultivation of organic saffron in the municipality of Vinaceite, province of Teruel. After remaining in the fallow farm for twenty years, in June 2013, bulbs of native C. sativus from Teruelo were planted by hand. The flowers were collected at the beginning of November 2013, given that the flowering was somewhat late due to the weather. After obtaining the stigmata, part of the floral waste was transferred in
    35 refrigerator to the laboratory for extraction, constituting the fresh samples; another part was frozen at -18 oC, and transferred to the cold laboratory to avoid defrosting.

    1.1. Obtaining hot extracts.
    Continuous hot extraction of frozen and fresh samples was carried out for 180 minutes, with the help of a Soxhlet equipment (Buchi B-811). To do this
    5 used 2 g of sample and 80% methanol as solvent.The methanolic extracts obtained were evaporated to dryness in a rotary evaporator (Buchi R-114)and kept in a refrigerator at 4 oC until use.
    The humidity percentage of the sample was 85.21 ± 0.16% and the yields obtained of 10.15% and 40.83% for frozen and fresh samples respectively.
    1.2. Obtaining the cold extract.
    A cold alcoholic extraction of the frozen samples was carried out, preserving the light extract at all times to avoid possible alterations of its components.
    15 35 g of frozen saffron flowers and 300 mL of methanol with 0.1% commercial Hel (37%) were used as solvent. The mixture was introduced in an ultrasonic bath for ten minutes on 4 occasions: at the beginning of the extraction, at 3, 9 and 24 h.
    The methanolic extract obtained was evaporated to dryness in a rotary evaporator (Buchi R-114) at 22 oC 20 and stored in a refrigerator at 4 oC and protected from light.
    The moisture percentage of the sample was 85.58 ± 0.46%; and the extraction yield was 8.91% (plp).
    25 2. Experimental study and results. 2.1. Determination of the content in total phenols.
    The total phenolic content was determined by the Folin-Ciocalteau 30 method (Abdelwahab et al., 2014), where the phenolic groups are oxidized with phosphomolibdicotungstic acid, forming a quantifiable green-bluish complex at 750 nm.
    To perform the test, 5 µL of the extracts were placed at different concentrations in a 96-well plate. Then 80 IJL of 10% Folin-Ciocalteau were added and the plate was shaken so that the reagents were mixed effectively. After five minutes 160 IJL of sodium carbonate (Na2C03) was added at 7.5%. The mixture was stirred again and 30 minutes later the absorbance at 765 nm was measured in Versa Max plate reader
    (Molecular Devices). The total phenolic content was expressed as equivalents of gallic acid in mg / g of extract, obtained by interpolation in the calibration line of gallic acid. All trials were performed in triplicate.
    5 Results:
    The results obtained are shown in the graph of figure 1, which reveals that the phenolic content of the Crocus sativus extracts under study is much higher than that of the Iranian stigmas, despite not being particularly striking if we compare it with the
    10 content of other plant extracts (Acero and Muñoz-Mingarro, 2012). The content in this type of compounds was found to be significantly higher in the cold-obtained extracts. Phenolic compounds have been described on multiple occasions as responsible for the antioxidant effect of plant extracts (Wenzig et al., 2008), for this reason and since the stigmas of the flowers of C. sativus, have demonstrated a notorious antioxidant effect,
    15 the fact that the tepals have a content almost eight times higher than that of stigmata, makes us assume that this part of the flower could also be of interest as a natural antioxidant. 2.2. Determination of flavonoid and anthocyanoside content.
    Once the phenol content was analyzed, the concentration of flavonoids and anthocyanosides was studied.
    To determine the amount of total flavonoids present in the different extracts, a calorimetric test was used (Zhishen et al., 1999). A calibration line with epicatechin was performed as standard.
    The test protocol consists of using 200 I-IL of extract at different concentrations, 200 I-IL of Mili-Q water as a blank, or 200 I-IL of different concentrations of
    30 epicatechin (5 serial dilutions starting from 0.1 mg / mL), to which 800 I-IL of Mili-Q water and 60 I-IL of 5% sodium nitrite (NaN02) were added. After five minutes, 60 I-IL of aluminum trichloride (AICI3) at 10% was added, and the next minute 400 I-IL of sodium hydroxide (NaOH) 1 M. Immediately the appearance of color occurs, the absorbances being read against the blank at 510 nm in UV-1603 spectrophotometer (Shimadzu).
    35 All trials were performed in triplicate. After interpolating in the calibration line, the results were expressed as milligrams of epicatechin per gram of extract.
    The total anthocyanide content was determined by the differential pH method (Fuleki and Francis, 1968a, b). It is based on the spectroscopic measurement of the reversible structural transformation of the anthocyanin chromophore as a function of pH, providing different absorbance spectra.
    From the saffron extract (2 mg / mL), two solutions were prepared, in triplicate, of 500 ~ L of the extract in 7000 ~ L of 0.2 M KCI buffer at pH 1.0 or 1 M sodium acetate at pH 4, 5.
    10 The solutions were incubated for 15 min, protected from light. The absorbance of both solutions was measured at 510 nm and 700 nm.
    The absorbance calculation was performed according to equation 1:
    Abs = (A510 -A700) pH 1.0 - (A510 -A70Q) pH 4.5
    The anthocyanoside content was calculated using the Lambert-Beer equation (Abs = E * C * L) and was expressed as mg of equivalents of delfinidine-3-glucoside I 100g of saffron extract according to equation 2:
    20 C (mg / L) = Abs 1 (, "1)" Pm "dilution factor" 103
    Equation 2: Lambert-Beer equation adapted. e = concentration; Abs = absorbance; E = cyanidin-3-glycoside molar extinction coefficient: 29000L / mol * cm; L = cuvette width: 1cm; Pm = molecular weight of delfinidine-3-glycoside: 500.84g / mol
    25 Delfinidine-3-glycoside has been described as the majority anthocyanide in saffron bio-wastes (Serrano-Diaz et al., 2014), which is why it was used to calculate anthocyanoside equivalents.
    30 Results:
    The graph in Figure 2 shows the concentration of flavonoids and anthocyanosides in saffron extracts obtained in cold
    35 Flavonoid content is 20.12 ± 4.45 mg epicatechin / g extract, this data was not obtained for hot extracts.
    The anthocyanoside content was 9.3013 ± 0.764 mg / g of equivalent cyanidin 3-glycoside extract.
    As can be seen in the aforementioned graph, there is a high amount of
    5 anthocyanides in saffron floral bio-wastes. The color of the flower points to theexistence of this type of compounds, so it was analyzed. In fact, theextract obtained in cold had a color clearly different from that obtained in hot. This typeof compounds, also present in blackberries, strawberries, blueberries, etc., has a highantioxidant potential, however, degrade with relative ease, so in the
    10 hot extracts may have degraded due to temperature. 2.3. Determination of antioxidant activity in vitro. 2.3.1 DPPH test.
    DPPH radical capture capacity.
    The study of antioxidant capacity through the DPPH test (Koleva al. • 2002). it consists in studying the capacity of the extract, in this case of saffron, to reduce the
    20 radical DPPH. by donating a hydrogen giving rise to the DPPH-H form. Said reduction results in a discoloration passing the compound of blue color in its oxidized form, to yellow in the reduced one.
    For the development of the method three batteries of dilutions in methanol were prepared with each
    25 one of the extracts and a reference substance, in this case, gallic acid. The test was performed in 96-well plates. To each well 100lJL of each of the dilutions prepared were added. and 100 ~ L of 1 mM DPPH in methanol. The control was carried out with 100 ~ L of methanol and 100IJL of 1mM DPPH. All trials were performed in triplicate.
    30 The plate was allowed to incubate for 20 minutes, at room temperature and in the dark. Subsequently, absorbances at 517 nm were measured on a Versa Max (Molecular Devices) plate reader. With the data obtained, the% reduction was calculated as [(Ao-A1) / Aol x 100, where Ao is the absorbance of the control and A1 that of the extract / gallic acid at a certain concentration. Next, the lesion of the extracts and gallic acid was estimated, which
    35 indicates the concentration produced by the 50% reduction of the DPPH • radical.
    Results:
    As for the antioxidant activity, a greater antioxidant activity has been detected incold extracts; if in hot extracts at a concentration of 500IJg / mL we obtained a reduction of 27% of the radical, with those obtained cold the percentage5 reduction increased to 37% reduction at the same concentration. For him
    Gallic acid leso resulted in 5,664 ± 0.232 ~ glmL.
    In view of these results, saffron shows a reduced capacity to capture the DPPH radical when compared to the reference substance used.
    10 Given the complexity of oxidation-antioxidation processes and the chemical composition of plant extracts, it is clear that the use of a single method is not sufficient in the evaluation of the antioxidant properties of complex mixtures. In this sense, many authors confirm the need to use several methods to determine
    15 antioxidant activity (Koleva et al., 2002), so two antioxidant tests were carried out in cell culture, which are set out below. 2.3.2. Xanlina I Xanlina oxidase assay.
    20 As a consequence of the activity of the enzyme xanthine oxidase, hypoxanthine is transformed into xanthine and it is converted into uric acid. During this last step, a superoxide radical is also generated, capable of producing the reduction of NBT (nitrotetrazolium blue), resulting in crystals of formazan, colored product and therefore, quantimetrically quantifiable (Figure 4). Substances with antioxidant capacity are capable of
    25 capture this radical and avoid the reduction of the NBT.
    The objective of this test is to study whether our extracts have the capacity to inhibit the reduction of NBT, by capturing the superoxide radical. On the other hand, the ability of the extracts to inhibit the enzyme will be checked, by quantifying the
    30 uric acid production.
    Capture capacity of the O2 'radical The capture capacity of the superoxide radical by the hot extracts is quantified by decreasing the absorbance at 560 nm, since they will prevent
    35 the reduction of the NBT. In view of the results, we decided not to study the effect of cold extracts. To perform the test, they prepared:
    White: 100 ~ L of 50 mM phosphate buffer, 10 ~ L of 15 mM EDTA, 15 ~ L of 3 mM hypoxanthine and 25 ~ L of 0.6 mM NBT.
    Positive Control: 75 ~ L of 50 mM phosphate buffer, 10 ~ L of 15 mM EDTA, 15 ~ L of
    3 mM hypoxanthine, 25 IJL of 0.6 mM NBT and 25 IJL of xanthine oxidase. Samples: 62.51JL of 50 mM phosphate buffer, 10 IJL of 15 mM EDTA, 15IJL of hypoxanthine 3
    10 mM, 25 IJL of 0.6 mM NBT and 25 IJL of xanthine oxidase and 12.5 IJl of each extract and reference substance at each concentration.
    The test was carried out in a 96-well plate, following the protocol of Mc Cune and Johns (2002). All reagents were prepared in phosphate buffer (50 mM, KH2P04 / KOH,
    15 pH = 7.4), as well as dilutions of gallic acid and extracts. Absorbances were measured for 30 minutes, at 5 minute intervals, on the Versa Max (Molecular Divices) plate reader. All tests were performed in triplicate. The result was expressed as a percentage of inhibition of the reduction of N8T.
    20 The statistical analysis of the data was carried out with a repeated measures ANOVA, using the IBM SPSS Statistics 20 program. The peer comparison based on the estimated marginal averages was carried out by means of adjustment for multiple DMS comparisons (Minimum Difference Significant). 25 Xanthine oxidase inhibition ability.
    The ability to inhibit xanthine oxidase can be quantified by analyzing the production of uric acid at 295 nm. 30 To perform the test, the following were added:
    White: 750 ~ L of 50 mM phosphate buffer, 60 ~ L of 15 mM EDTA, 90 ~ L of 3 mM hypoxanthine.
    35 Positive Control: 600 ~ L of 50 mM phosphate buffer, 60 ~ L of 15 mM EDTA, 90 ~ L of 3 mM hypoxanthine and 150 IJL of xanthine oxidase.
    Samples: 525IJL of 50 mM phosphate buffer, 60 IJL of 15 mM EDTA, 90 IJL of 3 mM hypoxanthine IJL, 150 IJL of xanthine oxidase and 75 IJL of each extract and reference substance at each concentration.
    5 The trial was carried out following the protocol of Mc Cune and Johns (2002). All theReagents were prepared in phosphate buffer (50 mM, KH2P04 / KOH, pH = 7.4), as were thedilutions of gallic acid and extracts. Absorbances were measured inspectophotometer for 40 minutes, at 10 minute intervals. All the trials areThey performed in triplicate. The result was expressed as a percentage of production inhibition.
    10 uric acid
    The statistical analysis of the data was carried out with a repeated measures ANOVA, using the IBM SPSS Statistics 20 program. The pairwise comparison based on the estimated marginal averages was carried out by means of adjustment for comparisons.
    15 multiple DMS.
    Results:
    Both extracts are capable of capturing the radical O2., Avoiding the reduction of the NBT, with
    20 greater efficiency than that detected in the DPPH test. The results are represented in the graph of figure 3 by the percentage of capture of the Radical O2 • compared to the different concentrations of the extracts. The different letters indicate significant differences (ANOVA-DMS Test p <0.05).
    25 A dose-dependent capacity is observable, more evident in the case of frozen saffron than in fresh. However, at the same concentration no significant differences were detected between fresh and frozen saffron, the antioxidant capacity of frozen at slightly higher doses being slightly higher. As in the previous trial, 50% of radical capture was not reached, so it was impossible for us to calculate the
    30Iso of the extracts, which in the case of gallic acid was found to be 7.01 ± 0.4731 IJg / mL In addition to capturing the superoxide radical some of the extract components could inhibit xanthine oxidase, thereby reducing the production of uric acid.
    The graph in Figure 4 reflects the percentages of inhibition of xanthine oxidase against
    35 the different concentrations of C. sativus and gallic acid extracts at a concentration of 8.33 IJg / mL. The different letters indicate significant differences (ANOVATest DMS p <0.05).
    It is observed that neither of the two extracts is capable of considerably inhibiting the enzyme. The maximum inhibition achieved by the extracts is around 7.5% and is reached with the concentration of 83.33 IJg / mL. There are no significant differences between the two extracts of the plant at the same concentration. nor between nearby concentrations
    5 of the same extract. 2.4. Determination of antioxidant activity in cell culture. 2.4.1. Study of cell viability.
    10 Before carrying out the studies to evaluate the antioxidant capacity of saffron extracts in Hep G2 cell culture, its possible toxicity on this cell line was determined by the MTT test. This technique is based on the metabolic transformation of the 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium (MTT) bromide salt of
    15 yellow, in purple formazan crystals. The reaction takes place in the mitochondria of living cells, so greater absorbances at 550 nm, imply greater cell survival.
    The effect of the extracts on tumor cell growth in 20 normal conditions and under conditions of oxidative stress induced with hydrogen peroxide was analyzed.
    96-well plates were prepared, at the rate of 8,000 cells / well of the Hep G2 line, in EMEM culture medium supplemented with 1% non-essential amino acids, 2 mM 25 glutamine, 1% fetal bovine serum and 1 % of antibiotics (10,000 U of penicillin and 10 mg of streptomycin / mL). The plate was incubated for 24h and subsequently 50 IJL of each of the extract concentrations prepared in PBS were added. Five concentrations of each extract, between 0.5 mg / mL and 0.0625 mg / mL, were studied. A control with PBS and a negative one without cells were also performed. The plates were incubated
    30-20 h at 37 oC. After this time, the medium was aspirated and medium was added alone or with H202 (200 IJM). After three hours of incubation the medium was aspirated and 150 IJL of EMEM medium and 50 IJL of 0.1% MTT in PBS were added. The plates were re-incubated for 3 hours, the medium was then aspirated and adding 100 IJL of DMSO, with the aim of dissolving the formed formazan crystals (Denizot and Lang, 1986).
    The absorbance was measured on a Versa Max (Molecular Devices) plate reader at 550 nm. The results were expressed in percentages of cell growth inhibition versus control with PBS. Calculating from this data. if applicable, the concentration of the extract that inhibits 50% of cell growth (le50).
    The statistical analysis of the data was carried out with a repeated measures ANOVA,
    5 using the IBM SPSS Statistics 20 program. Peer comparison based onEstimated marginal averages, was carried out through adjustment for comparisonsBonferroni multiples. Determination of antioxidant activity in cell culture.
    Results:
    10 The graphs of Figures 5 and 6 show the effect of Croeus sativus extracts on the viability of Hep G2 cells, under normal conditions and under oxidative stress induced by H, O, (200 ~ M).
    15 Figure 5 shows the effect of frozen saffron extract on the viability of Hep G2 cells under normal conditions and under oxidative stress induced by H20 2 (200 ... 1M). The data are represented as% growth with respect to the control with PBS (100%).
    20 Figure 6 shows the effect of fresh saffron extract on the viability of Hep G2 cells, under normal conditions and under oxidative stress induced by H20 2 (200 IJM). The data are represented as% growth with respect to the control with PBS (100%).
    25 There are clear differences between the two extracts; While that from frozen saffron does not reduce the percentage of growth below 67%, the fresh bio-waste extract produces a more pronounced cytotoxicity. This fact allows us to calculate the leso for this extract that turned out to be 272 ± 27¡Ig / mL. It is also evident in the case of fresh saffron extract, a dose-dependent effect than in the other extract
    30 is not detected.
    According to The American National Caneer Institute (NCI), a crude extract exhibits considerable cytotoxic activity when its ICso is less than 20 IJg / ml (Boyd, 1997). Therefore, in view of the results, we can conclude that Crocus sativus extracts do not have a remarkable cytotoxic activity. This data allowed considering the studied extracts as possible protective agents against oxidative damage, lacking relevant cytotoxicity. These data agree with the low toxicity of six extracts of
    floral bio-residues of C. sativus, described by Serrano-Diaz et al. (2014) in cell lines of non-tumor fibroblasts.
    When treating the cells with H20 2, it is observed how there is a significant decrease in
    5 cell growth versus control with PBS. The objective of the study is to analyze whether extractsobtained are able to reverse or prevent damage caused by this agentoxidizing Neither extract is able to reverse this stress to concentrationof toxic tested, in fact the fresh extract even reduces, to more concentrationshigh, cell growth in the presence of hydrogen peroxide.
    10 Both when subjecting cells to oxidative stress, and under normal conditions, the same general trend is observed in each extract: the frozen extract does not significantly modify cell growth with respect to the control (treated or not with H20 2, in each case) ; while the fresh extract significantly reduces it, at
    15 higher concentrations. 2.4.2. Determination of reactive oxygen species (ROS).
    The ability of extracts to reduce intracellular ROS levels was studied.
    20 For this purpose, the direct effect of the extracts and their protective capacity against oxidative damage caused by a prooxidant agent, such as hydrogen peroxide (Wang and Joseph, 1999; Fernández-Gómez el al., 2005), were determined.
    To carry out the determination of reactive oxygen species, the
    25 diacetate 2-7-dichlorodihydrofluorescin (DCFH-DA) assay. This non-fluorescent compound crosses the cell membrane thanks to its low polarity and is deacetylated by intracellular esterases resulting in 2-7-dichlorodihydrofluorescin (DCFH), which is also not fluorescent. However, in the presence of reactive oxygen species, it passes into its oxidized form, 2-7-dichlorodihydrofluorescein (DCF), which emits fluorescence.
    It can be said that the emitted fluorescence is directly proportional to the concentration of ROS, and therefore, a higher antioxidant potential of the extract will be associated with low levels of flowering (Cardomy and Cotter, 2000).
    35 To perform this test, 96-well plates were prepared on which they were placed
    8,000 cells / well in a final volume of 150 IJL of EMEM medium supplemented with 2 mM glutamine, 1% fetal bovine serum, 1% non-essential amino acids and 1% antibiotics (10,000 U of penicillin and 10 mg / mL of streptomycin). The plates were kept in a 24 h incubator at 37 ° C and an atmosphere of 5% CO2.
    Then, to evaluate the direct effect of the extracts, the medium was aspirated, and
    5 added the 0.02 mM DCFH-DA (200 I-IUcillo). The plates were incubated 30 minutes indarkness in incubator. The handling of the plates from the moment it is addedDCFH-DA was performed in darkness. After aspiration and washing with PBS glucosate (1.8mg glucose / mL PBS) different concentrations of the extracts in PBS were addedGlucose, using PBS Glucose as a control.
    10 To study the protective effect against stress induced by H20 2, we pretreated the cells for 20 h with different doses of each extract. After that time, DCFH-DA was added and incubated 30 minutes. The medium was then aspirated, washed with glucose PBS and culture medium was added with H20 2 200 I-IM.
    15 Fluorescence was measured at the time of adding the extracts (direct effect) or the hydrogen peroxide (protection test) for 90 minutes at 15 minute intervals
    in Fluostar optima plate reader (BMG Labtech) with l. 485 nm excitation and 520 nm emission.
    The results were expressed as a percentage of fluorescence at 90 minutes, with respect to the control with PBS (100%). The statistical analysis of the data was carried out with a repeated measures ANOVA, using the IBM SPSS Statistics 20 program. The peer comparison based on the estimated marginal averages was carried out.
    25 by adjustment for multiple Bonferroni comparisons.
    Results:
    The graph in Figure 7 reflects the effect of C. sativus flower extracts on the
    30 concentration of intracellular ROS in Hep G2 culture. The fluorescence units were measured 90 minutes after adding the extract. the data is expressed as% of ROS with respect to the control. Different letters indicate significant differences (ANOVA-Test Bonferroni p <0.005). Ac: Frozen saffron extract obtained hot; Af: Fresh saffron extract obtained hot.
    As shown in Figure 7, the two highest concentrations of the two hot extracts significantly increase the concentration of intracellular ROSs with respect to the control.
    5 The results of the test performed to observe the possible protective effect ofExtracts of flowers of C. sativus against oxidative stress, that is, on the concentrationof intracellular ROS in Hep G2 culture, is shown in Figure 8. The units ofFluorescence were measured 90 minutes after adding the oxidizing agent (200 µM) after20 h pretreatment with the extract. Data are expressed as% of ROS with respect
    10 to control without H20 2 (100%). Different letters indicate significant differences (ANOVA-Test Bonferroni p <0.005). Ac: Frozen saffron extract obtained hot; Af: Fresh saffron extract obtained hot.
    It is observed in this other figure how in the presence of H20 2 an increase occurs
    15 significant levels of ROS in the stressed control (in red) versus the unstressed control (in green). The treatments with the highest concentrations are able to keep ROS levels close to the values observed in the control of unstressed cells, which demonstrates an antioxidant activity as cellular protectors against a possible oxidative stress.
    20 The graphs of Figures 9 and 10 show the effect of cold extracts of C. sativus on the concentration of intracellular ROS in Hep G2 culture over time. In Figure 9 the direct effect of different concentrations of the extract, and in Figure 10, the protective effect of different concentrations of the extract. The different letters indicate
    25 significant differences between treatments for each time (ANOVA-Bonferroni Test p <O, 005).
    With regard to the direct effect (figure 9), it is seen how the lowest levels of ROS occur in the control, while treatments with saffron flower extract 30 produce an increase in the levels of these radicals, not always significant with respect to control. At the highest concentration, ROS production soars significantly higher than control at all times. However, when the results of the protective effect are observed (Figure 10), it is seen how all treatments and control follow the same pattern, but the lowest concentration of ROS is obtained with the
    35 more concentrated treatment of the extract. Despite all this, there are no significant differences between the control and this treatment, so we cannot talk about a marked protective effect of the extract in terms of intracellular ROS levels.
    The results of the test performed to observe the possible protective effect ofExtracts against oxidative stress is shown in the aforementioned figure 10. In the presence ofH20 2, it is observed how there is a significant increase in ROS levels in theStressed control (in red) versus unstressed control (in green). The5 treatments with the highest concentrations, are able to maintain levelsof ROS close to the values observed in the control of unstressed cells, whichdemonstrates antioxidant activity as cell protectors against possible stressoxidative This effect may be due to the ability of phenolic compounds tosequester oxygen radicals in cell cultures (Pavlica and Gebhardt, 2010). The
    10 results indicate that ROS generated during oxidative stress are neutralized by the compounds present in the extracts. Therefore, the extracts show a relevant antioxidant activity although only as cellular protectors against possible oxidative stress, which could lead to multiple pathologies, such as cancer, inflammation or various skin disorders, among others (Bickers and Athar, 2006). 2.4.3. Study of the state Red-ox cellular.
    The cellular redox state was studied by determining the levels of intercellular GSH and GSSH. The concentration of these compounds allowed to see if the extract has
    20 antioxidant capacity by increasing their concentration in cells. Again, the direct effect and the protective effect after the induction of oxidative stress with H20 2 were studied.
    This study was carried out only with cold extracts, and following the
    25 indications of the Cayman glutathione assay (Chemical Company) (Baker al., 1990), based on the enzymatic recycling of GSSG to GSH in the presence of the enzyme glutathione reductase for the quantification of GSH.
    The incubation of the cells with the extracts was carried out in 6-well plates in which
    30,000,000 cells / well and 3 mL of EMEM medium supplemented with 2 mM glutamine, 1% fetal bovine serum, 1% non-essential amino acids and 1% antibiotics (10,000 U of penicillin and 10 mg / mL of streptomycin) were placed ). After incubating for 24 h in an incubator at 37 oC and 5% CO2, the medium was aspirated, medium was added with 6 different concentrations of the extract between 1 mg / mL and 40 mg / mL in medium
    35 culture and incubated in the above conditions for another 20 h. After this time, the medium was aspirated, washed with PBS and the cells were collected by scraping in 1 mL of PBS.
    To study the protective effect, after 20 hours of pretreatment with the extract, the medium was aspirated and replaced with 3 mL of culture medium with H202 (200 IJM) and incubated for 3 h. The medium was then aspirated, washed with P8S and the cells were collected by scraping in 1 mL of P8S.
    To perform the assay it is necessary to lyse the cells and make the intracellular GSH accessible. For this, the cells were sonicated for 10 seconds in 1 mL of 50 mM phosphate buffer pH 6-7, with 1 mM EDTA. Then, the cell debris sediments when centrifuged at 10,000 g for 15 minutes at 4 oC and discarded, keeping the
    10 supernatant on ice.
    Deproteinization is continued, which is necessary to avoid interference of thiol groups present in cellular proteins. For this purpose, equal volumes of 10% metaphosphorus were mixed in water and the supernatant, stirred in vortex and allowed to stand at
    15 room temperature for 5 minutes. It was centrifuged at 2,000 g for two minutes and the supernatant was collected. 1 mL of supernatant was added 50 IJL of 4M triethanolamine to raise the pH of the solution.
    A calibration line was prepared with commercial GSSG at different concentrations. 50 IJL
    20 of each of the treatments (different concentrations of saffron for the study of the direct effect and the protective effect) and of the dilutions of the standard were placed in a 96-well plate and 150 IJL of the reaction mixture (MES buffer) was added. (0.4 M (Nmorpholino) -etanesulfonic acid, 2 mM 0.1 MY EDTA phosphate, pH 6), NADP +, 9-Icosa-6 phosphate, glutathione reductase, glucose-6-phosphate dehydrogenase, water and DTN8). The plate is
    25 incubated in the dark for 30 minutes and then absorbance at 405 nm was measured in Versa Max plate reader (Molecular devices).
    All trials were performed in triplicate. The results were expressed as a concentration of GSH / GSSG after interpolation in a calibration line. The analysis
    The statistical data was carried out with a repeated measures ANOVA, using the IBM SPSS Statistics 20 program. The pairwise comparison based on the estimated marginal averages was carried out by means of adjustment for multiple Bonferroni comparisons (p <0, Q5). Results:
    When studying the direct effect of the extract, no significant differences were observed between the control and the treatments with the different concentrations of saffron extracts. The
    results suggest that there is no direct antioxidant effect in terms of aincrease in GSH levels in treatments with saffron extracts at any ofthe concentrations tested. As for the protective effect, a marking was foundstatistically significant increase in the levels of total GSH with an effect5 dose dependent that increases with the extract concentration. One of the mainEndogenous antioxidants of the organism is the GSH, due to the presence of the thiol group of theCysteine that makes it an excellent proton donor. In this way the tripeptideGSH interacts with ROS as hydrogen peroxide, either directly or mediatedby glutathione peroxidase activity, reducing the oxidative stress caused by these 10 radicals
    The graph in Figure 11 shows the commented effect of the extracts on reduced glutathione (GSH) levels. Values are expressed as% versus unstressed control (100%). Different letters indicate significant differences (ANOVA-Benferroni test, p <0.05).
    BIBLOGRAPHY.-
    Valeria, R., Parenti, F., Tugnol, V., Schenetti, L., Mucci, A. 2015. Crocus sativus Petals:
    Waste or Valuable Resource The Answer of High-Resolution and High-Resolution Magic
    20 Angle Spinning Nuclear Magnetic Resonance. J. Agric. Food Chem., 63 (38), pp 8439-S444. DOI: 10.1021 /acs.jalc.5b03284
    Zeka, K, Ruparelia, K.C., Continenza, M.A., Stagos, D., Veglió, F., Arroo, R.R. 2015. Petals of Crocus sativus L. as a potential source of the antioxidants crocin and kaempferol.
    25 Phytotherapy, 107: 128-34. DOI: 10.1 016 / j.fitote. 2015.05.014.
    Omidi, A., Riahinia, N., Montazer Torbati, M.B., Behdani, M.A. 2014. Hepatoprotective effect of Crocus sativus (saffron) petals extract against acetaminophen toxicity in male Wistar rats.
    Avicenna J Phytomed, 4 (5): 330-336.
    Montoro, P., Maldini, MT, Luciani, L., Tuberoso, CIG, Congiu, F., Pizza, C. 2012. Radical Scavenging Activity and LC-MS Metabolic Profiling of Petals, Stamens, and Flowers of Crocus sativus L. Journal of Food Science, 10.1111 /j.1750-3841.2012.02803.x
    Termentzi, A., Kokkalou, E. 2008. LC-DAD-MS (ESI +) analysis and antioxidant capacity 01 35 Crocus sativus petal extracts. Med plant ,. 74 (5): 573-81. doi: 10.1055 / 8-2008-1074498.
    Hosseinzadeh, H., Ghenaati, J. 2006. Evaluation of the antitussive effect of stigma and petals of saffron (Crocus sativus) and its components, safranal and crocin in guinea pigs. Phytotherapy. 77 (6): 446-8.
    5 Li, C.Y., Lee, E.J., Wu, T.S. 2004. Antityrosinase principies and constituents of the petals of Croeus sativus. J Nat Prod., 67 (3): 437-40.
    Fatehi, M., Rashidabady, T., Fatehi-Hassanabad, Z. 2003. Effects of Crocus sativus petals' extract on rat blood pressure and on responses induced by electrical field stimulation in the 10 rat isolated vas deferens and guinea-pig ileum. J Ethnopharmacol., 84 (2-3): 199-203.
    Hosseinzadeh, H., Younesi, H.M. 2002. Antinociceptive and anti-inflammatory effects of Crocus sativus L. stigma and petal extracts in mice. BMC Pharmacol., 15; 2: 7.
    15 Kubo, l., Kinst-Hori, 1. 1999. Flavonols from saffron flower: tyrosinase inhibitory activity and inhibition meehanism. J Agrie Food Chem., 47 (10): 4121-5.
    Serrano-Diaz, J., Estevan, C., Sogorb, M.A., Carmona, M., Alonso, G.L., Vilanova, E. 2014. Cytotoxic effect against 3T3 fibroblasts cells of saffron floral bio-residues extracts. Food 20 Chem. 15; 147: 55-9. doi: 10.1016 / j.foodehem. 2013.09.130.
    Serrano-Díaz, J., Sánchez, A.M., Maggi, L., Martinez-Tomé, M., García-Diz, L., Murcia, M.A., Alonso, G.L. 2012. Increasing the applications of Crocus sativus flowers as natural antioxidants. J Food SeL, 77 (11): C1162-8. doi: 10.1111 / j.1750-3841 .2012.02926.x.
    Rubio Moraga, A., Ahrazem, O., Rambla, J.L., Granell, A., Gómez Gómez, L. 2013. Crocins with high levels of sugar conjugation contribute to the yellow colors of early-spring flowering eroeus tepals. PLoS One, 13; 8 (9): e71946. doi: 10.1371 / journal.pone.0071946.
    权利要求:
    Claims (5)
    [1]
    1.-Saffron bio-waste extracts, obtained from frozen and fresh samples ofthe floral debris of Crocus sativus once separated the red stigmas of the flower in the5 spice collection process, both by continuous hot extraction, and bycold alcohol extraction, used methanol as solvent, CHARACTERIZED bypresent a phenol content determined by the Folin-Ciocalteau method of117.10 mg eq gallic acid Ig extract obtained in cold and 51, 39-42.5 mg eq gallic acid Igextract obtained in hot, and a content in flavonoids and anthocyanides determined
    10 per colorimetric assay, of 20.12 ± 4.45 mg eq epicatequinalg extract obtained in cold, and 9.3013 ± 0.764 mg cyanidin 3-glycoside Ig extract obtained in cold, respectively.
    [2]
    2. Saffron bio-waste extracts, according to claim 1, CHARACTERIZED for their antioxidant capacity against cellular oxidative stress, when presented in the in vitro assay
    15 of the DPPH, a capacity to capture the DPPH radical. compared with gallic acid used as a reference substance, with ICs of 5,664 ± 0.232 IJg / mL, 27% for hot extracts and 37% for cold extracts, at an extract concentration of 500 IJg / mL in methanol
    3. Saffron bio-waste extracts, according to claim 1, CHARACTERIZED for their antioxidant capacity against cellular oxidative stress, by presenting in the in vitro test of Xanthine / Xanthine oxidase, the ability to capture the super6xide radical 02-in comparison with gallic acid with ICso of 7.01 ± 0.4731 j.Jg / mL, and the ability to inhibit Xanthine Oxidase compared to gallic acid at a concentration of 8.33 IJg / mL,
    25 in percentages for different concentrations of hot extract, frozen and fresh samples.
    [4]
    4. Saffron bio-waste extracts, according to claim 1, CHARACTERIZED for their antioxidant capacity against cellular oxidative stress, when presenting in the test on
    30 MTT cell culture, the effect on the viability of Hep G2 cells under normal conditions and under oxidative stress induced by H20 2 (200 j.JM) in percentages of cell growth inhibition versus control with PBS (100%) for different hot extract concentrations, frozen and fresh samples.
    35 5. Saffron bio-waste extracts, according to claim 1, CHARACTERIZED for their antioxidant capacity against cellular oxidative stress, when presenting in the cell culture assay of diacetate 2-7-dichlorodihydrofluorescin (DCFH-DA), the ability to reduce Intracellular ROS concentration levels in Hep G2 culture in fluorescence percentages at 90 minutes with respect to the PBS control (100%), in the presence and absence of H20 2 (stressed and unstressed control), for different concentrations of frozen and fresh extract obtained hot, in fluorescence indices throughout the
    5 time for different concentrations of extract obtained cold.
    [6]
    6. Saffron bio-waste extracts, according to claim 1, CHARACTERIZED for their antioxidant capacity against cellular oxidative stress, by presenting in the Cayman glutathione cell culture assay the ability to increase the levels of
    10 concentration of GSH in Hep G2 culture in percentages of GSH versus control in the absence of H20 2 (control without stress).
    [7]
    7.-Use of saffron bio-waste extracts, according to previous claims, as active ingredients of antioxidant cosmetic products, for human body application 15 in order to reduce or prevent oxidative stress of the cells against external contaminating factors.
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