• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
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    • 专利摘要:
    The present invention describes the process of obtaining and the characteristics of the corresponding extract from the by-products of the industry of obtaining cloudy turkey juice. Also, the extract obtained has a series of beneficial physiological effects on the harmful effects of the so-called metabolic syndrome. In the trials with a diet enriched in fats with respect to the control study, it reduces the percentage of cells transformed into adipocytes, the increase in weight, the increase in insulin that occurs in these diets; reduces the content of cholesterol, lipids and triglycerides in them, with an increase in the level of interferon gamma and a reduction in the levels of interleukin 6; and allows a better control of blood glucose. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2694437A1
    申请号:ES201700646
    申请日:2017-06-20
    公开日:2018-12-20
    发明作者:Domingo Saura López；Nuria Martí Bruñá；Manuel Valero Roche；Sara GEA BOTELLA；David MULA MUÑOZ；Jesús Tómas OLIVARES ROLDÁN；Salud VEGARA GÓMEZ；Nieves Muñoz Mateo；Concha MARTÍNEZ MADRID；Ignacio GONZALEZ OCHOA；Daniel Ramón VIDAL；María Dolores HERRANZ LÓPEZ；Bernat SORIA ESCOM；Francisco Martin Bermudo
    申请人:Mitra Sol Technologies SL；
    IPC主号:
    专利说明:

    DESCRIPTION

    Obtaining and purification of carotinoids from by-products of the industrialization of persimmon and application in foods and beverages of a functional nature.
     5
    Sector of the technique

    The present invention describes the obtaining and purification of an extract rich in carotenoids, especially P-cryptoxanthin, from the byproducts obtained from the industrial processing thereof, as well as its use as a functional ingredient. 10

    State of the art

    Persimmon (Diospyros kaki Thunb), a fruit species belonging to the Ebanaceae family, is native to China and is widely spread throughout Asia. In Spain 15 began to be cultivated as an ornamental tree until later its cultivation led to the obtaining of a fruit for fresh consumption of pleasant flavor. The cultivation of persimmon was centralized in the Mediterranean region, going from a 2000 ha crop in 2010 to 9650 ha in 2012.
     twenty
    Persimmon (Diospyros kaki Thunb) has a great nutritional importance due to its high content of vitamins, carotenoids (P-cryptoxanthin, carotene, druptoxanthin, zeaxanthin, lutein and lycopene) (Daood et al, 1992), organic acids and fiber and is also a good source of phenolic compounds such as p-coumaric acid, catechin, epicatechin, epigallocatechin and proanthocyanidins (Giordani et al, 2011; Dembitsky et al, 2011; Mallavadhani et al., 1998), which have been implicated in the reduction of degenerative human diseases (Van Poppel, 1993, Giovannucci et al, 1995, Steinmetz and Potter, 1996) due to their antioxidant and free radical scavenging properties (Boileau et al, 1999, Matsumoto et al, 2010), antidiabetic activity (Dewanjee , 2009) and also has a high hypolipidemic effect (Zou et al, 2012). Also, persimmon contains abundant condensed tannins (Macheix et al., 1990) 30 that appear to have physiological functions including the inhibitory effects on human lymphoid leukemia cells (Achiwa et al., 1997) and inhibitory effects on mutagenicity (Achiwa et al. al., 1996).

    The metabolic syndrome per se is not a disease, but rather a set of well-defined alterations that increase the risk of suffering from cardiovascular diseases, type 2 diabetes and certain types of tumors. Obesity, alterations in the lipid profile, insulin resistance, low-grade inflammation and oxidative stress seem to be behind the increase in the risk of suffering the aforementioned diseases. Currently, a fundamental aspect in the treatment of these clinical alterations is found in the modification of the lifestyle, mainly exercise and dietary modifications (Samson and Garber, 2014).

    Among the changes related to diet, several systematic reviews and meta-analysis studies have indicated that the consumption of fruits, vegetables, legumes and monounsaturated fats significantly reduce the clinical manifestations of the metabolic syndrome (Ko et al., 2013 ). In this sense, there are compounds present in foods of plant origin, such as carotenoids that have a beneficial role against the metabolic syndrome. It has been seen that a Mediterranean type diet increases the plasma levels of carotenoids and that this increase improves the oxidative stress present in people with metabolic syndrome (Barona et al., 2012). In addition, a study conducted in Australia has shown that people with metabolic syndrome had circulating serum levels of five carotenoids, significantly lower than people who did not have a syndrome.
    metabolic (Coyne et al., 2009). Results similar to the previous study were found in the Japanese population (Suzuki et al., 2011).

    Our invention focuses on obtaining an extract rich in carotenoids, compounds related to the color and maturation state of the persimmon (Zhou, 2011, Gorinstein, 2001), 5 being the -cructoxanthin the most abundant and in the assay of the activity biological effect of -criptoxanthin in a murine model with metabolic syndrome and in a nutritional intervention in males with metabolic syndrome. This carotenoid is found in both the skin and pulp of persimmon as well as in other fruits and vegetables (Ebert, 1985, Gorinstein, 1999) and seems to have beneficial effects on obesity (Shirakura, 2012; Takayanagi, 2011; Iwamoto, 10; 2012) and in different types of cancers (Miyazawa, 2007, Vrouenraets, 2000, Yuan, 2003). 

    The extraction of carotenoids from natural sources has been studied extensively, but mainly for lycopene and -carotene. Carotenoids oxidize very easily due to their strong antioxidant capacity as previously described by other authors 15 (Kitakawa, 2004) and they are also sensitive to light and heat. These characteristics reduce the possible range of extraction methodologies. Various methods of extraction and purification can be applied to obtain natural carotenoids, such as extraction with solvents, supercritical fluid extraction (SFE), distillation, membrane separation, chromatography, or crystallization (Patents CA2395319, US6344573, 20 US2003044499, AU2003287701, ES2241503, US20097572468, CA2305091, AU2369002, US2005266132). Of all of them, the extraction with solvents has been the most used in the industry due to its simplicity and its low cost. However, techniques that minimize the use of organic solvents to produce food products are beginning to be used. SPEAKING ABOUT FLASH 25 

    On the other hand, as regards the biological activity of extracts rich in p-cryptoxanthin, there are studies that refer to their role in the prevention of obesity (Patent US20100273727 A1), but in this case the extract is obtained from the tangerine.
     30
    The process object of the present invention is based on the use of a process that does not require the use of organic solvents for the extraction and purification of these compounds from the by-products of the persimmon industry. The process subjects the product to destruction and crushing by bursting the hot material by exposing it suddenly to high vacuum conditions. Subsequently the product is subjected to tangential filtration and extraction by adsorption resins, which manage to isolate and purify the carotenoids contained in the product. The equipment used in this extraction process has been commonly used in the wine-making industry.

    Bibliography. 40

    Achiwa, T., Hibasami, H., Katsuzaki, H., Imai, K., & Komiya, T. (1997). Inhibitory effects of persimmon (Diospyros kaki) extract and related polyphenol compounds on growth of human lymphoid leukemia cells. Bioscience Biotechnology and Biochemistry, 61, 1099-1101.
     Four. Five
    Achiwa, T., Hibasami, H., Katsuzaki, H., Kada, H., & Komiya, T. (1996). Inhibitory effects of persimmon (Diospyros kaki) and constituents on the mutagenicity

    of C-nitro and C-nitroso compounds formed by reaction of sorbic acid with sodium nitrite. Journal of the Japanese Society for Food Science and Technology, 43, 493-501. fifty

    Barona, J., Jones, J.J., Comperatore, M., Andersen, C., Schwartz, S.J., Lerman,
    R. H., Fernandez, M.L. (2012) A mediterranean-style low-glycemic-load diet increases plasma carotenoids and decreases LDL oxidation in women with metabolic syndrome. Journal Nutritional Biochemistry, 23, 609-615.

    Boileau, T. W. M., Moore, A. C., & Erdman, J. W. (1999). Carotenoids and Vitamin A. In 5 Antioxidant Status, Diet, Nutrition and Health; Papas, M., Ed .; CRC Press; New York

    Coyne, T., Ibiebele, T.I., Baade, P.d., McClintock, C., Shaw, J.E. (2009). Metabolyc syndrome and serum carotenoids: findings of a cross-sectional study in Queensland, Australia. British Journal of Nutrition, 102, 1688-1677. 10

    Daood, H.G., Biacs, P., Czinkotai, B., & Hoschke, A. (1992). Chromatographic investigation of carotenoids, sugars and organic acids of Diospyros kaki fruits. Food Chemistry, 45, 151-155.

    Dembitsky, V.M., Poovarodom, S., Leontowicz, H., Leontowicz, M., Vearasilp, 15
    S., Trakhtenberg, S., & Gorinstein, S. (2011).

    The multiple nutrition properties of some exotic fruits: Biological activity and active metabolites. Food Research International, 44, 1671-1701.
     twenty
    Dewanjee, S., Das, A.K., Sahu, R, & Gangopadhyay, M. (2009). Antidiabetic activity of Diospyros peregrina fruit: Effect on hyperglycemia, hyperlipidemia and augmented oxidative stress in experimental type 2 diabetes. Food and Chemical Toxicology, 47, 2679-2685.

    Ebert, G. and Gross, J. (1985). Carotenoid changes in the peel of ripening persimmon 25 Diospyros kaki) cv.Triumph. Phytochemistry, 24, 29-32.

    Giordani, E., Doumett, S., Nin, S., & Del Bubba, M. (2011). Selected primary and secondary metabolites in fresh persimmon {Diospyros kaki Thunb): A review of analytical methods and current knowledge of fruit composition and health benefits. Food Research International, 44, 30 1752-1767.

    Giovannucci, E., Ascherio, A., Rimm, E. B., Stampfer, M. J., Colditz, G. A., & Willet, W. C. (1995). Intake of carotenoid and retinol in relation to prostate cancer risk. Journal of the National Cancer Institute, 87, 1767-1776. 35

    Gorinstein, S. (1999). Comparative content of total polyphenols and dietary fiber in tropical fruits and persimmon. Journal of Nutritional Biochemistry, 10, 367-371.

    Gorinstein, S., Zachwieja, Z., Folta, M., Barton, H., Piotrowicz, J., Zemser, M., Weisz, M., 40 Traktenberg, S. and Martin-Belloso, O., J. (2001). Comparative content of dietary fiber, total phenolics, and minerals in persimmons and apples. Journal of Agriculture Food Chemistry, 49, 952-957.

    Iwamoto, M., Imai, K., Ohta, H., Shirouchi, B., Sato, M. (2012). Supplementation of highly concentrated 45 P-cryptoxanthin in a satsuma mandarin beverage improves adipocytokine profiles in obese Japanese women. Lipids in Health and Disease, 11, 52.

    Kitakawa, N. S., Kato, H., Takahashi, A. and Yonemoto, T. J. (2004). Missing title American Oil Chemistry Society, 81, 389. 50

    Ko. B.J., Park, K.E., Mantzoros, C.S. (2013). Diet patterns, adipokines and metabolism: where are we and what is next . Metabolism, 63, 168-177.
    Mallavadhani, U.V., Panda, A.K. and Rao, Y.R. (1998). Pharmacology and chemotaxonomy of Diospyros. Phytochemistry, 49, 901-951.
    Macheix, J., Fleuriet, A., & Billot, J. (1990). Fruit Phenolics CRC Press. Florida, 378

    Matsumoto, K., Kadowaki, A., Ozaki, N., Takenaka, M., Ono, H., Yokoyama, S. -I., & Gato, N. 5 (2011). Bile Acid-binding Ability of Kaki-tannin from Young Fruits of Persimmon (Diospyros kaki) In Vitro and In Vivo. Phytotherapy Research, 25, 624-628.

    Miyazawa, K., Miyamoto, S., Suzuki, R., Yasui, Y., Ikeda, R., Kohno, H., Yano, M., Suzuki, K. (2007). Dietary P-cryptoxanthin inhibits N-butyl-N- (4-hydroxybutyl) nitrosamine-induced urinary 10 bladder carcinogenesis in male ICR mice Oncology Reports, 17, 297-304.

    Samson, S.L., Garber, A.J. (2014). Metabolic syndrome. Endocrinology Metabolic Clinical North America, 43, 1-23.
     fifteen
    Shirakura, Y., Takayanagi, K., Mukai, K., Tanabe, H., Inoue, M. (2012). p- Cryptoxanthin suppresses the adipogenesis of 3T3-L1 cells via RAR activation. Journal of Nutritional Science and Vitaminology, 57, 426-431.

    Steinmetz, K. A., Potter, J. D. (1996). Vegetables, fruit, and cancer prevention: A review. 20 Journal of the American Dietetic Association, 53, 536-543.

    Suzuki, k., Ito, Y., Inoue, T., Hamajima, N. (2011). Inverse association of serum carotenoids with prevalence of metabolic syndrome among Japanese. Clinical Nutrition, 30, 369-375.
     25
    Takayanagi, K., Morimoto, S.-I., Shirakura, Y., Mukai, K., Sugiyama, T., Tokuji, Y., Ohnishi, M. (2011). Mechanism of visceral fat reduction in Tsumura Suzuki obese, diabetes (TSOD) mice orally administered P-cryptoxanthin from Satsuma mandarin oranges (Citrus unshiu Marc). Journal of Agricultural and Food Chemistry, 59, 12342-12351.
     30
     Van Poppel, G. (1993). Carotenoids and cancer: An update with emphasis on human intervention studies. European Journal of Cancer, 29A, 1335-1344.

    Vrouenraets, M.B., Visser, G.W.M., Loup, C., Meunier, B., Stigter, M., Oppelaar, H., Stewart, F.A., Van Dongen, G.A. (2000). Suppression of azoxymethane-induced colon carcinogenesis in 35 male F344 rats by mandarin juices rich in P-cryptoxanthin and hesperidin. International Journal of Cancer, 88, 146-150.

    Yuan, J.-M., Stram, D O, Arakawa, K., Lee, H.-P., Yu, M.C. (2003). Dietary cryptoxanthin and reduced risk of lung cancer: The Singapore Chínese health study. Cancer Epidemiology 40 Biomarkers and Prevention, 12, 890-898.

    Zhou, C., Zhao, D., Sheng, Y., Tao, J., Yang, Y. Carotenoids in fruits of different persimmon cultivars. Molecules, 2011, 16, 624-636.
     Four. Five
    Zou, B, Li, C.M., Chen, J.Y., Dong, X.Q., Zhang, Y., & Du, J. (2012). High molecular weight persimmon tannin is a potent hypolipidemic in high-cholesterol diet fed rats. Food Research International, 48, 970-977.

    fifty
    Detailed description of the invention.

    The carotenoids obtained from the persimmon, preferably -criptoxanthin, were subjected to studies with laboratory animals (mice) and were used for a nutritional intervention trial in humans. 5 
    The carotenoids of the present invention are not particularly limited and for example, -carotene, -carotene, -carotene, cryptoxanthin, astaxanthin, canthaxanthin, lutein, zeaxanthin, lycopene, tunaxanthin and Fucoxanthin 
     10
    The cryptoxanthin of the present invention is not particularly limited and for example a-cryptoxanthin and P-cryptoxanthin should be included. In addition, citrus fruits should be considered as sources of these cryptoxanthin and especially persimmon (Diospyros kaki Thunb), which has a high production is particularly desirable.
     fifteen
    The extracts were produced in the following way:

    The by-product of persimmon used for the production of the carotenoid extract comes from the industrialization residue of persimmon for the production of turbid juice. Next, Figure 1 shows a summary of the production process of persimmon juice and the stage of the process from which the by-products used in this work come from.

    In the tests carried out, the same type of by-product is used, the only difference being that these are divided into two types, according to the point of the process obtained during the industrial processing of the persimmon. From the industrialization of persimmon, the skin, peduncle, pulp and seeds are obtained as by-products, distributed in two groups of by-products, 1 and 2. The first is obtained after the sudden expansion, 10 crushing and sifting, and the second after of the enzymatic treatment and decantation, as can be seen in figure 1.
     30
    Table 1. Percentage (%) of total carotenoids present in the extracts of persimmon obtained from the different subproducts:

     By-product  Total Carotenoid Solvent Treatment% SD
     one  Wash Ethanol 96% 6.09 0.73
     Acetone  13.67 0.51
     Ethanol / Acetone (1: 1)  2.85 0.58
     Hexane / Acetone / Ethanol (50:25:25)  0.83 0.07
     Crushed + Washing  Ethanol 96% 8.19 0.57
     Acetone  6.7 0.47
     Crushed + Wash + Sudden Expansion  Ethanol 96% 19.53 0.34
     Acetone  21.97 0.35
     Ethanol / Acetone (1: 1)  6.28 0.28
     two  Wash Ethanol 96% 1.45 0.52
     Acetone  0.34 0.13
     Ethanol / Acetone (1: 1)  2.11 0.59
     Crushed + Washing  Ethanol 96% 1.95 0.57
     Acetone  1,66 0,47
     Crushed + Wash + Sudden Expansion  Ethanol 96% 6.61 0.35
     Acetone  1.79 0.39
     Ethanol / Acetone (1: 1)  4.65 0.34

    Table 2. Content (g / kg of extract) of carotenoids present in the 10 extracts of persimmon obtained from the different subproducts:

     Subp.  Trat. Solvent β-cryptoxant β-carotene-carotene 
     Wash Ethanol 96% 24.6 5.3 0.38
     Acetone 54.8 12.92 2.16
     Ethanol / Acetone (1: 1) 5.54 4.13 0.23
     Crushed + Ethanol Wash 96% 24.62 9.81 0.86
     one  Acetone 20.72 4.11 0.23
     Crushed + Wash + Sudden expansion Ethanol 96% 58.39 1.33 0.1
     Acetone 26.64 4.57 0.33
     Ethanol / Acetone (1: 1) 35.32 10.53 0.73
     Wash Ethanol 96% 2,41 4 1,15
     Acetone 0.34 1.9 0.4
     Ethanol / Acetone (1: 1) 4.01 4.1 1.15
     Crushed + Wash Ethanol 96% 7.31 7.4 2.6
     two  Acetone 2.03 2.03 0.7
     Crushed + Wash + Sudden expansion Ethanol 96% 5,72 5,72 0,31
     Acetone 2.61 2.61 1.01
     Ethanol / Acetone (1: 1) 3.46 3.46 2.2
     5
    The biological studies carried out were:

    I) In laboratory animals: weight with a standard scale; glycemia using a portable Accu-check glucometer (Roche); insulin through an enzyme-linked immunosorbent assay (ELISA) with a specific kit to detect mouse insulin (Mercodia Mouse Insulin ELISA kit); 10
    insulin resistance using the HOMA index equation (HOMA index = fasting insulin (IU / ml) x fasting glucose (mM) / 22.5); endocrine function through an intraperitoneal glucose overload test; total cholesterol and triglycerides using the reflectance photometry technique with a Reflotron Plus (Roche); LDL cholesterol by the method of precipitation with a commercial kit for the detection of LDL cholesterol (Biosystems); 5 interferon gamma (IFN-) and interleukin 6 (IL-6) by the ELISA technique using the specific V-Plex panel and the Meso QuickPlex SQ120 (MSD) plate reader; total hepatic lipids by extracting the total lipids of the liver with Folch's method and liver damage by measuring the amount of collagen present in the livers by means of a histological study using the "Sirius red" stain. In all cases blood was obtained from the tail of animals and the animals were fasting overnight. 

    In the present invention, unless specified, all quantities, ratios or percentages will always refer to the weight.
     fifteen
    In the oral administration form of the composition of the present invention, in which the carotenoids and the cryptoxanthins are found, their amounts are not limited. For example, carotenoids and cryptoxanthines can be at levels of between 0.00001 to 70%, preferably from 0.0001 to 50% and more preferably from 0.01 to 30%. twenty

    The oral administration form of the present invention may contain substances other than carotenoids and cryptoxanthins and which may influence the beneficial effects on the metabolic syndrome described in the present invention. In this sense it is desirable that these substances are at concentrations ranging between 0.01 and 90%. 25

    When administered orally, the composition of the present invention induces a reduction in body weight, an improvement in the lipid profile, a decrease in insulin resistance and circulating levels of glucose and insulin, a better control of postprandial glycemia, a decrease in low-grade inflammation, less oxidative stress and less liver damage in animal models and in people with metabolic syndrome.

    As regards the method of oral administration of the composition of the present invention it can be ingested directly as such alone or it can be ingested processed in the form of powders, tablets, granules, hard and soft capsules, gels, pastes, syrups, 35 suspensions, emulsions, drinks and the like. A standard of the amounts to be ingested can be selected based on the age, sex, weight and other considerations of the individuals. In the case of an adult individual, daily doses may range from 0.01 mg to 10 g of carotenoids and from 0.01 mg to 10 g of cryptoxanthins.
     40
    The period in which the effects of the present invention can be obtained after intake is not particularly limited, but it is desirable that the number of days ingested per year be one day or more or 365 days or less, preferably between 14 days or more and 365 days or less and more preferably between 60 days or more and 365 days or less.
     Four. Five
    The objects to which the present invention can be administered are not particularly limited, but preferably are warm-blooded animals, especially mammals and more preferably humans.

    The extract of carotenoids and / or cryptoxanthins of the present invention can be used as ingredients in the preparation of various types of foods, beverages, healthy foods and beverages, foods and beverages for specific uses in the field of health, functional foods, food supplements. and parapharmacy food products.
    In the present invention, functional food is understood as that which is consumed as part of a normal diet and that contains biologically active ingredients, which offer health benefits and reduce the risk of suffering from chronic diseases.

    In the present invention, food supplements are understood as those foods whose purpose is to complement the normal diet and which consist of concentrated sources of nutrients (vitamins and minerals) or of other substances that have a nutritional or physiological effect, in simple or combined form. .

    In the present invention it is understood as parapharmacy food products those 10 food products that are not medicines, are consumed and made available to users, in accordance and according to what is established, in specific technical-sanitary regulations of the different categories of products that exist in the market, as well as in the general regulations in force in the matter.
     fifteen
    Throughout the description and the claims the word "comprises" and its variants do not intend to exclude other technical characteristics, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention.
     twenty
    Exemplary embodiments

    The following specific examples that are provided in this patent document serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and are not to be construed as limitations on the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting the scope of application thereof.

    For biological tests the following extraction system was used, whose description is not intended to be limiting, but exhaustive. 30

    Process of obtaining the extract of carotenoids of persimmon.

    The persimmon is a fruit that has a high amount of sugars that can influence the subsequent drying process of the obtained extract. Therefore, an initial step was the washing of the raw material to obtain a product with a low amount of sugars.

    The water of the raw material is a very important factor for the subsequent extraction process. The industrial extraction is generally carried out with dry material because it reduces the complications that may occur during the extraction stage. The removal of the water can be carried out by using a vacuum oven at a maximum temperature of 50 ° C for 12 h or a zeodratation treatment that allows drying at room temperature.

    For the extraction of carotenoids from the sample, a hydroalcoholic medium 45 (Ethanol 95%) is used and kept under agitation to increase the efficiency of the process at a temperature between 25 and 45 ° C, and a solid-liquid ratio 1 :3. Once homogenized, the dough is introduced into a sudden expansion equipment allowing a greater rupture of the material and increasing the extraction performance of the same. The extraction proceeded in a nitrogen-rich environment to inhibit the potential oxidation processes that may occur during the extraction of some of the active ingredients.

    The obtained suspension is filtered and the solvent is recovered by a vacuum filtration system. The obtained liquid was concentrated 100 times in a vacuum evaporator until a concentrate rich in carotenoids and other compounds was obtained.

    The saponification of the obtained concentrate was carried out by the addition of 40% KOH in methanol for 45 min at 50 ° C to favor the process and decrease the time.

    The partition was made with 1 volume of ethyl ether with a 10% saline solution, which could be NaCl or Na 2 SO 4.
     10
    Subsequently, the washing with water is carried out and the organic phase is dried with MgSO 4 or Na 2 SO 4, to be subsequently dried under vacuum.

    Composition of the extracts obtained by solid-liquid extraction.
     fifteen
    For the HPLC analysis of the extracts, solutions of X g / L (in hexane or ethanol) were prepared. The extract was homogenized in a vortex until completely dissolved and filtered through a 0.22 m filter. The composition of the extracts was analyzed by liquid chromatography RRLC 1200 series (Agillent Technologies, 40 CA) coupled to time-of-flight mass spectrometry (TOF) using electrospray as interface 20 (Bruker Daltonics, GmbH, Germany). The compounds were separated by a YMC30 column of 2.1x150 mm and 3 m pore size, at 35 ° C and a flow of 0.5 ml / min, with an injection volume of 10 l. The diode array detector coupled to the chromatography system was set in a spectrum range between 120 and 950 nm. The mobile phase consisted of a gradient with A: methanol / acetone (60:40) and B: acetone / water (60:40); (0-3 min 60-30% B, 25 3-22 min 30% B, 22-26 min30-10% B, 26-41.5 min 10-60% B). The conditions of the mass spectrometer were a positive mode ionization, a capillary voltage of 4500 V, drying gas temperature of 290 ° C, drying gas flow of 9 L / min and nebulizer gas pressure at 40 p.s.i. The mass range was 150-640. 
     30
    Examples

    In all the examples carried out in experimental animals, the procedure was the same. Male mice were taken from the 5 week old C57BL / 6J strain. These mice were fed two types of diet: i) control diet, consisting of the diet "Teklad Global 35 14% Protein Rodent Maintenance Diet 2014" (Harland). This diet supposes a contribution of fats equivalent to 13% (2.7% of them are saturated fats) of the total caloric intake. This diet was administered to the control groups, throughout the study (6 months) and ii) high-fat diet (HFD), consisting of the diet 'Teklad Global 14% Protein Rodent Maintenance Diet 2014' (Harland) supplemented with fats saturated. This diet supposes a contribution of 40 fat equivalent to 49% (46.3% of them are saturated fats) of the total caloric intake. This diet was administered to the HFD groups throughout the study (6 months). A group of mice fed a control diet were administered 0.8 mg / kg body weight / day of P-cryptoxanthin (β-CRX), throughout the study (6 months). This was the control group + P-CRX. In addition, a group of mice fed with the HFD diet were administered 0.8 mg / kg of 45 body weight / day of β-CRX, from the 3rd month of the study and during the remaining 3 months of the study. This was the group HFD + β-CRX. The β-CRX was prepared in an emulsion of olive oil and injected each day into 4 g pieces of food. The mice ate a piece of 4 g of food each day. Each group of mice was made up of 8 mice.
     fifty
    Example 1. Effect of β-cryptoxanthin on body weight in animals subjected to a standard diet and a diet rich in saturated fats. The mice were weighed on an empty stomach overnight, between 9:00 and 11:00 am. During the first 3 months the weighing was carried out once
    a month. In the following 3 months, the animals were weighed every 15 days. The data show that animals fed a diet rich in saturated fats, supplemented with β-CRX (HFD-β-CRX) have a significant decrease in weight (** p <0.01) with respect to animals fed the diet rich in saturated fats (HFD). On the other hand, the animals fed with the diet rich in saturated fats (HFD) show a significant increase in weight (* p <0.001) with respect to the control and control groups + β-CRX. The administration of β-CRX (control + β-CRX) did not affect the weight of the control animals. Figure 1.

    Example 2. Effect of p-cryptoxanthin on glycemia in animals subjected to a standard diet and a diet rich in saturated fats. Blood glucose was measured fasting for 10 nights, between 9:00 and 11:00 am. During the first 3 months the measurement was performed once a month. In the following 3 months, blood glucose was determined every 15 days. Blood glucose was determined from a drop of blood from the tail using a portable glucometer Accu-Check (Roche). The data show that animals fed a diet rich in saturated fats, supplemented with β-CRX (HFD-β-CRX) have a significant decrease in glycaemia (** / p <0.001) with respect to animals fed with the diet rich in saturated fats (HFD). On the other hand, animals fed the diet rich in saturated fats (HFD) show a significant increase in blood glucose (* p <0.01) with respect to the control and control + β-CRX groups. The administration of β-CRX (control + β-CRX) did not affect the glycemia of the control animals. Figure 2. 20

    Example 3. Effect of P-cryptoxanthin on plasma insulin in animals subjected to a standard diet and a diet rich in saturated fats. Plasma insulin was measured fasting overnight, between 9:00 and 11:00 am. During the first 3 months the measurement was performed once a month. In the following 3 months, plasma insulin was determined every 15-25 days. Insulin was determined from drops of blood from the tail. 4 drops of blood were obtained. To obtain the plasma, the blood samples were centrifuged at 1500 g for 15 minutes. Plasma samples were frozen at -80 ° C until use. Insulin determination was done by ELISA using a specific kit to detect mouse insulin (Mercodia Mouse Insulin ELISA kit). The data show that animals fed a diet rich in saturated fats, supplemented with β-CRX (HFD-β-CRX) have a significant decrease in plasma insulin values (** / p <0.01) with respect to to animals fed the diet rich in saturated fats (HFD) On the other hand, animals fed the diet rich in saturated fats (HFD) show an increase in plasma insulin (* p <0.001) with respect to the control groups and control + β-CRX. The administration of β-CRX (control + β-CRX) did not affect the plasma insulin values of the control animals. Figure 3

    Example 4. Effect of p-cryptoxanthin on insulin resistance (HOMA index) in animals subjected to a standard diet and a diet rich in saturated fats. The HOMA index 40 was calculated from the blood glucose and plasma insulin values taken fasting overnight, between 9:00 and 11:00 am. During the first 3 months the measurement was performed once a month. In the following 3 months, plasma insulin was determined every 15 days. To calculate the HOMA index, the following equation was used:
     Four. Five
    HOMA index = fasting insulin (IU / ml) x fasting glucose (mM) / 22.5. 

    The data show that animals fed a diet rich in saturated fats, supplemented with β-CRX (HFD-β-CRX) have a significant decrease in the values of the HOMA index (** p <0.01) with respect to the animals fed the diet rich in saturated fats (HFD). On the other hand, animals fed the diet rich in saturated fats (HFD) show an increase in the value of the HOMA index (* p <0.001) with respect to the control and control + β-CRX groups. The administration of β-CRX (control + β-CRX) did not affect the HOMA index values in the control animals. Figure 4
    Example 5. Effect of p-cryptoxanthin on endocrine function in animals subjected to a standard diet and a diet rich in saturated fats. Endocrine function was calculated by an intraperitoneal glucose overload. This overload was done with the animals fasting overnight, between 9:00 and 11:00 am. During the first 3 months the measurement was performed once a month. In the following 3 months, the blood glucose was determined every 5 15 days. To do this, the blood glucose was measured at time 0, subsequently, each animal was injected into the peritoneum 2 g of glucose / kg of weight, dissolved in 200 l of saline. Subsequently, blood glucose was measured at 15, 30, 60 and 120 minutes. Blood glucose was determined from a drop of blood from the tail using a portable glucometer (Accu-Check, Roche). The data show that animals fed a diet rich in saturated fats, 10 supplemented with β-CRX (HFD-β-CRX) have a better glycemic control after glucose overload compared to animals fed the rich diet in saturated fats (HFD). On the other hand, animals fed the diet rich in saturated fats (HFD) show a worse glycemic control after glucose overload (* p <0.05) with respect to the control and control + β-CRX groups. The administration of β-CRX (control + β-CRX) 15 did not affect the regulation of glycemia after glucose overload in the control animals. Figure 5. 

    Example 6. Effect of -criptoxanthin on the levels of total cholesterol in animals subjected to a standard diet and a diet rich in saturated fats. The total cholesterol was determined by means of the reflex photometry technique with a Reflotron Plus (Roche) device, in a drop of blood obtained from the tail of the animal. The drop of blood was obtained between 9: 00-11: 00 in the morning and after subjecting the animals to a night of fasting. The data show that animals fed a diet rich in saturated fats, supplemented with β-CRX (HFD-β-CRX) have a significant decrease in total cholesterol values 25 (** / p <0.05) with respect to to animals fed a diet rich in saturated fats (HFD). On the other hand, animals fed the diet rich in saturated fats (HFD) show a significant increase in total cholesterol values (* p <0.01) with respect to the control and control + β-CRX. The administration of β-CRX (control + β-CRX) did not affect the total cholesterol values of the control animals. The measurements were made at 3 and 30 6 months after starting the study. Figure 6. 

    Example 7. Effect of p-cryptoxanthin on LDL-cholesterol levels in animals subjected to a standard diet and a diet rich in saturated fats. LDL cholesterol was measured in serum samples, obtained from 200 l of blood from the tail of animals. 35 The blood was obtained between 9:00 and 11:00 am in the morning and after subjecting the animals to a night of fasting. To obtain the serum, the blood samples were centrifuged at 3500 rpm for 15 minutes. LDL cholesterol was determined by the precipitation method, using a commercial kit (LDL cholesterol detection kit, Biosystems). The data show that animals fed a diet rich in saturated fats, supplemented with P-40 CRX (HFD-β-CRX) have a significant decrease in LDL cholesterol values (** p <0.05) with respect to Animals fed the diet rich in saturated fats (HFD). On the other hand, animals fed the diet rich in saturated fats (HFD) show a significant increase in LDL cholesterol values (* / p <0.001) with respect to the control and control + β-CRX. The administration of β-CRX (control + β-CRX) did not affect the 45 total cholesterol values of the control animals. The measurements were made 3 and 6 months after starting the study. Figure 7. 

    Example 8. Effect of p-cryptoxanthin on triglyceride levels in animals subjected to a standard diet and a diet rich in saturated fats. The triglycerides were determined by means of the reflex photometry technique with a Reflotron Plus (Roche) equipment, in a drop of blood obtained from the tail of the animal. The drop of blood was obtained between 9: 00-11: 00 in the morning and after subjecting the animals to a night of fasting. The data show
    that animals fed a diet rich in saturated fats, supplemented with β-CRX (HFD-β-CRX) have a significant decrease in triglyceride values (** p <0.05) with respect to animals fed the diet rich in saturated fats (HFD). On the other hand, animals fed the diet rich in saturated fats (HFD) show a significant increase in triglyceride values (* p <0.001) with respect to control and control groups + β-CRX. The administration of β-CRX (control + β-CRX) did not affect the triglyceride values of the control animals. The measurements were made 3 and 6 months after starting the study. Figure 8

    Example 9. Effect of -criptoxanthin on the plasma levels of interferon gamma in 10 animals subjected to a standard diet and a diet rich in saturated fats. The IFN-y was measured in serum samples, obtained from 50 l of blood from the tail of the animals. The blood was obtained between 9:00 and 11:00 am in the morning and after subjecting the animals to a night of fasting. To obtain the plasma, the blood samples were centrifuged at 1500 g for 15 minutes. The plasma samples were frozen at -80 ° C until the time of their use. IFN-y was determined by ELISA, using the specific V-Plex panel for mice and the Meso QuickPlex SQ120 (MSD) plate reader. The data show that animals fed a diet rich in saturated fats, supplemented with β-CRX (HFD-β-CRX) have a significant decrease in plasma IFN-y values (** p <0.05) with respect to to animals fed a diet rich in saturated fats (HFD). On the other hand, 20 animals fed the diet rich in saturated fats (HFD) show a significant increase in the plasma levels of IFN-y (* / p <0.01) with respect to the control and control + β- CRX The administration of β-CRX (control + β-CRX) did not affect the IFN-y values in the control animals. The measurements were made 3 and 6 months after starting the study. Figure 9. 25 

    Example 10. Effect of -criptoxanthin on the plasma levels of interleukin 6 in animals subjected to a standard diet and a diet rich in saturated fats. IL-6 was measured in serum samples, obtained from 50 l of blood from the tail of the animals. The blood was obtained between 9:00 and 11:00 am in the morning and after subjecting the animals to a 30-night fast. To obtain the plasma, the blood samples were centrifuged at 1500 g for 15 minutes. Plasma samples were frozen at -80 ° C until use. IL-6 was determined by ELISA, using the specific V-Plex panel for mice and the Meso QuickPlex SQ120 (MSD) plate reader. The data show that animals fed a diet rich in saturated fats, supplemented with β-CRX (HFD-β-CRX) 35 have a significant decrease in plasma IL-6 values (** / p <0.05) with respect to animals fed the diet rich in saturated fats (HFD). On the other hand, animals fed the diet rich in saturated fats (HFD) show a significant increase in the plasma levels of IL-6 (* p <0.01) with respect to the control and control + β-CRX. The administration of β-CRX (control + β-CRX) did not affect IL-6 40 values in the control animals. The measurements were made 3 and 6 months after starting the study. Figure 10. 

    Example 11. Effect of -criptoxanthin on the amount of total lipids in the livers of animals subjected to a standard diet and a diet rich in saturated fats. The amount of total hepatic lipids was carried out by extracting the total lipids from the liver by the Folch method. The data show that animals fed a diet rich in saturated fats, supplemented with β-CRX (HFD-β-CRX) have a significant decrease in the amount of total hepatic lipids (** p <0.05) with respect to Animals fed the diet rich in saturated fats (HFD). On the other hand, the animals fed the diet rich in saturated fats (HFD) show a significant increase in the amount of total hepatic lipids (* p <0.01) with respect to the control and control + β-CRX groups. The administration of β-CRX (control + β-CRX) did not affect the amount of total lipids
    hepatic in control animals. The measurements were made 3 and 6 months after starting the study. Figure 11

    Example 12. Effect of p-cryptoxanthin on the level of liver damage in the livers of animals subjected to a standard diet and a diet rich in saturated fats. Liver damage was determined by measuring the amount of collagen present in the livers. To do this, a histological study was done using the "Sirius red" stain. Liver sections included in paraffin were used. The nuclei were stained with hematoxylin (Weigert) for 8 minutes. Staining with "picro-sirius red" was performed for 60 minutes. Subsequently, the stained sections were dehydrated with 100% ethanol. Finally, the samples were cleaned with xylene and mounted on resin. The counting was done in a bright field microscope, with polarized light. The data were expressed in terms of the percentage of positive cells with respect to the total number of cores counted. The data show that animals fed a diet rich in saturated fats, supplemented with β-CRX (HFD-β-CRX) have a significant decrease in the number of cells positive for "sirius red" (** p <0.01) with 15 with respect to animals fed the diet rich in saturated fats (HFD). On the other hand, animals fed the diet rich in saturated fats (HFD) show a significant increase in the number of cells positive for "sirius red" (* p <0.001) with respect to the control and control groups + β-CRX Administration of β-CRX (control + β-CRX) did not affect the number of cells positive for "sirius red" in the control animals. The measurements were made at 3 and 6 months after starting the study. Figure 12

    Brief description of the figures

    Figure 1. Shows the variation in the weight of the different groups of animals throughout the study. Four groups of animals were established: i) control: animals that followed a standard diet; ii) HFD: animals that followed a diet high in saturated fats; iii) control + β-CRX: animals that followed a standard diet supplemented with 0.8 mg / kg of weight / day of β-cryptoxanthin and iv) HFD + β-CRX: animals that followed a diet high in saturated fats supplemented with 0.8 mg / kg of weight / day of β-cryptoxanthin. The data are the mean ± SEM. n = 30 8 animals per condition. * p <0.001 vs rest of the groups. ** / p <0.01 HFD- β-CRX vs FIFD.

    Figure 2. Shows the variation of fasting glycemia of the different groups of animals throughout the study. Four groups of animals were established: i) control: animals that followed a standard diet; ii) HFD: animals that followed a diet high in saturated fats; iii) control + 35 β-CRX: animals that followed a standard diet supplemented with 0.8 mg / kg weight / day of P-cryptoxanthin and iv) HFD + β-CRX: animals that followed a diet high in saturated fat supplemented with 0.8 mg / kg of weight / day of β-cryptoxanthin. The data are the mean ± SEM. n = 8 animals per condition. * p <0.001 vs rest of the groups. ** p <0.01 HFD- β-CRX vs. HFD.
     40
    Figure 3. Shows the variation of the fasting plasma insulin of the different groups of animals throughout the study. Four groups of animals were established: i) control: animals that followed a standard diet; ii) FIFD: animals that followed a diet high in saturated fats; iii) control + β-CRX: animals that followed a standard diet supplemented with 0.8 mg / kg weight / day of β -criptoxanthin and iv) HFD + β-CRX: animals that followed a diet high in saturated fat supplemented with 0.8 mg / kg weight / day of p-cryptoxanthin. The data are the mean ± SEM. n = 8 animals per condition. * p <0.001 vs rest of the groups. ** / p 0.01 HFD- β -CRX vs. HFD.

    Figure 4. Shows the variation in the value of insulin resistance, measured as HOMA index 50 of the different groups of animals throughout the study. Four groups of animals were established: i) control: animals that followed a standard diet; ii) HFD: animals that followed a diet high in saturated fats; iii) control + β-CRX: animals that followed a standard diet supplemented with 0.8 mg / kg weight / day of P-cryptoxanthin and iv) HFD + β-CRX:
    animals that followed a diet high in saturated fats supplemented with 0.8 mg / kg weight / day of β-cryptoxanthin. The data are the mean ± SEM. n = 8 animals per condition. * / p <0.001 vs rest of the groups. ** / K 0.01 HFD- β-CRX vs. HFD.

    Figure 5. Shows the modification of the glycemia values after an intraperitoneal glucose tolerance test, performed in fasting, of the different groups of animals, carried out at the end of the study. Four groups of animals were established: i) control: animals that followed a standard diet; ii) HFD: animals that followed a diet high in saturated fats; iii) control + β-CRX: animals that followed a standard diet supplemented with 0.8 mg / kg weight / day of β-cryptoxanthin and iv) HFD + β-CRX: animals that followed a diet high in saturated fat supplemented with 0.8 mg / kg of weight / day of β-cryptoxanthin. The data are the mean ± SEM. n = 8 animals per condition. * p <0.05 vs rest of the groups.

    Figure 6. Shows the values of total cholesterol in blood at 3 and 6 months of the study, of the different groups of animals. Four groups of animals were established: i) control: animals that followed a standard diet; ii) HFD: animals that followed a diet high in saturated fats; iii) control + β-CRX: animals that followed a standard diet supplemented with 0.8 mg / kg weight / day of p-cryptoxanthin and iv) FIFD + β-CRX: animals that followed a diet high in saturated fat supplemented with 0.8 mg / kg of weight / day of β-cryptoxanthin. The data are the mean ± SEM. n = 8 animals per condition. * / p <0.01 vs rest of the groups. ** p <20 0.05 HFD- β-CRX vs. HFD.

    Figure 7. Shows the values of LDL cholesterol in blood at 3 and 6 months of the study, of the different groups of animals. Four groups of animals were established: i) control: animals that followed a standard diet; ii) HFD: animals that followed a diet high in saturated fats; iii) control + β-CRX: animals that followed a standard diet supplemented with 0.8 mg / kg of weight / day of β-cryptoxanthin and iv) HFD + β-CRX: animals that followed a diet high in saturated fats supplemented with 0.8 mg / kg weight / day of P-cryptoxanthin. The data are the mean ± SEM. n = 8 animals per condition. * p <0.001 vs rest of the groups. ** / p <0.05 HFD- β-CRX vs. HFD. 30

    Figure 8. Shows the values of triglycerides in blood at 3 and 6 months of the study, of the different groups of animals. Four groups of animals were established: i) control: animals that followed a standard diet; ii) HFD: animals that followed a diet high in saturated fats; iii) control + β-CRX: animals that followed a standard diet supplemented with 35 0.8 mg / kg weight / day of β-cryptoxanthin and iv) HFD + β-CRX: animals that followed a diet high in saturated fat supplemented with 0.8 mg / kg of weight / day of β-cryptoxanthin. The data are the mean ± SEM. n = 8 animals per condition. * p <0.001 vs rest of the groups. ** p <0.05 HFD- β-CRX vs. HFD.
     40
    Figure 9. Shows the IFN-y values in plasma at 3 and 6 months of the study, of the different groups of animals. Four groups of animals were established: i) control: animals that followed a standard diet; ii) HFD: animals that followed a diet high in saturated fats; iii) control + β-CRX: animals that followed a standard diet supplemented with 0.8 mg / kg weight / day of β-cryptoxanthin and iv) HFD + β-CRX: animals that followed a diet high in saturated fat supplemented with 0.8 mg / kg of weight / day of -criptoxanthin. The data are the mean ± SEM. n = 8 animals per condition. * p <0.01 vs rest of the groups. ** / p <0.05 HFD- β-CRX vs. HFD. 

    Figure 10. It shows the values of 11-6 in plasma at 3 and 6 months of the study, of the different 50 groups of animals. Four groups of animals were established: i) control: animals that followed a standard diet; ii) HFD: animals that followed a diet high in saturated fats; iii) control + β-CRX: animals that followed a standard diet supplemented with 0.8 mg / kg of
    weight / day of p-cryptoxanthin and iv) HFD + β-CRX: animals that followed a diet high in saturated fats supplemented with 0.8 mg / kg of weight / day of β-cryptoxanthin. The data are the mean ± SEM. n = 8 animals per condition. * p <0.001 vs rest of the groups. ** / p <0.05 HFD- β-CRX vs. HFD.
     5
    Figure 11. Shows the amount of total lipids, in percentage terms, at 3 and 6 months of the study, in the livers of the different groups of animals. Four groups of animals were established: i) control: animals that followed a standard diet; ii) HFD: animals that followed a diet high in saturated fats; iii) control + β-CRX: animals that followed a standard diet supplemented with 0.8 mg / kg weight / day of β-cryptoxanthin and iv) HFD β-CRX: 10 animals that followed a diet high in saturated fat supplemented with 0.8 mg / kg weight / day of p-cryptoxanthin. The data are the mean ± SEM. n = 8 animals per condition. * p <0.01 vs rest of the groups. ** / p <0.05 HFD- β-CRX vs. HFD.

    Figure 12. Shows the percentage of positive cells against "sirius red" staining, in percentage terms, at 3 and 6 months of the study, in the livers of the different groups of animals. Four groups of animals were established: i) control: animals that followed a standard diet; ii) HFD: animals that followed a diet high in saturated fats; iii) control + β-CRX: animals that followed a standard diet supplemented with 0.8 mg / kg weight / day of β-cryptoxanthin and iv) HFD + β-CRX: animals that followed a diet high in saturated fat 20 supplemented with 0.8 mg / kg of weight / day of β-cryptoxanthin. The data are the mean ± SEM. n = 8 animals per condition. * p <0.001 vs rest of the groups. ** p <0.01 HFD- β-CRX vs. HFD.
    权利要求:
    Claims (16)
    [1]

    1. Procedure for obtaining extracts enriched in persimmon carotenoids (Diospyros kaki) characterized by:
     5
    • Use by-products derived from the production of persimmon juice such as skins, peduncle, pulp and seeds.
    • Treatment with a team of sudden expansion of the previous subproducts previously crushed and sieved. 10
    • Addition of one of the following solvents:
    - Water-ethanol in relation 4:96 to 0: 100, including 40:60
     fifteen
    - Acetone
    - Acetone-ethanol in relation
    • Extraction treatment with these solvents. twenty
    • Isolation of the liquid part of the solid by filtration or centrifugation.
    • Evaporation of the liquid in vacuum until complete elimination of the solvent.
     25
    [2]
    2. Process according to claim 1, characterized in that the derivative by-product can originate from the destry of the fresh commercialization of the persimmon (Diospyros kaki).

    [3]
    3. Process according to claim 1, characterized in that the monohydroxy alcohol used is a low molecular weight alcohol. 30

    [4]
    4. Preparation based on kaki extract obtained by the method described in claims 1 to 3, characterized by having a composition of carotenoids in the following range: a minimum percentage of carotenoids between 25 and 0.5%
     35
    [5]
    5. An oral administration composition that produces the following biological effects: reduction of body weight, improvement of the lipid profile, decrease in insulin resistance and circulating levels of glucose and insulin, better control of postprandial glycemia, decrease in low grade inflammation, less oxidative stress and less liver damage in animal models and in people with metabolic syndrome. 40

    [6]
    6. An oral administration composition that produces the effects described in the first claim and that contains carotenoids.

    [7]
    7. A composition for oral administration that produces the effects described in the first claim and which contains especially cryptoxanthins within the carotenoids.

    [8]
    8. A composition for oral administration that produces the effects described in the first claim and which especially contains cryptoxanthin-cryptoxanthin. 
     fifty
    [9]
    9. The composition described in claims 1-4 is obtained from persimmon (Diospyros kaki Thunb).

    [10]
    10. Use of the composition described in claims 1-4 for use in the manufacture of meals.

    [11]
    11. Use of the composition described in claims 1-4 for use in the manufacture of beverages. 5

    [12]
    12. Use of the composition described in claims 1-4 for use in the manufacture of healthy foods and beverages.

    [13]
    13. Use of the composition described in claims 1-4 for use in the manufacture of foods and beverages that are used in a specific way in the field of health.

    [14]
    14. Use of the composition described in claims 1-4 for use in the manufacture of functional foods.
     fifteen
    [15]
    15. Use of the composition described in claims 1-4 for use in the manufacture of food supplements.

    [16]
    16. Use of the composition described in claims 1-4 for use in the manufacture of parapharmacy food products. twenty
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    同族专利:
    公开号 | 公开日
    ES2694437B2|2020-10-22|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US20070116818A1|2005-11-22|2007-05-24|Toyo Seikan Kaisha, Ltd.|Extract containing beta-cryptoxanthin component from persimmon fruit|
    ES2593258A1|2015-06-05|2016-12-07|Universidad Miguel Hernández De Elche|Extraction of biologically active compounds from waste from the wine industry |
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