Patent Application: US-201414769652-A

Abstract:
disclosed is the use of pyrimidines , especially uridine and cytidine chemicals of pyrimidines in stimulation of plant growth and development , as well as use of pyrimidines especially uridine in enhancement of stress tolerance , reduction of stress , repair of stress - related injury and inhibition of stress , and the methods thereof .

Description:
in this detailed description , the preferred embodiments of the use of pyrimidine compounds of the invention in promoting growth and development of plants and increasing their stress tolerances are described for better understanding of the invention without forming any limiting effect . the invention relates to use of uridine and cytidine chemicals and other pyrimidine compounds in agriculture sector ; especially for promoting growth and development of plants and increasing their stress tolerances . pyrimidine is the general name of nitrous aromatic bases generally found in nucleic acids and also in some coenzymes and vitamins . the most basic pyrimidine structure is c 4 h 4 n 2 and the pyrimidines are the derivatives of this main structure . three pyrimidine bases ( cytozine , thymine , and uracil ) are found in biologic systems . uracil is only found in ribonucleic acid ( rna ), thymine in deoxyribonucleic acid ( dna ), and cytozine in both dna and rna . the shapes and sizes of pyrimidines - and also the ability of forming hydrogen bonds with the purines provide the three - dimensional structures and the biological functions of nucleic acids . besides their uracil , cytozine , and thymine base forms , pyrimidine compounds can have the structure of nucleoside such as uridine , cytidine , and thymidine , respectively , which are formed by addition of a ribose ring to these bases through beta - n - glycosidic bond ; deoxy nucleoside structure such as deoxyuridine , deoxycytidine , and deoxythymidine , respectively , which are formed by addition of a deoxyribose ring to these bases through beta - n - glycosidic bond ; nucleotide structure such as uridine - 5 ′- monophosphate , uridine - 5 ′- diphosphate , uridine - 5 ′- triphosphate , cytidine - 5 ′- monophosphate , cytidine - 5 ′- diphosphate , cytidine - 5 ′- triphosphate , thymidine - 5 ′- monophosphate , thymidine - 5 ′- diphosphate , thymidine - 5 ′- triphosphate , which are the one -, two -, or three - phosphate added forms of these nucleosides ; deoxy forms of these nucleotides ; and structures such as cytidine - 5 ′- diphosphate choline , cytidine - 5 ′- diphosphate ethanolamine , uridine - adenosine tetraphosphate , which are the structures wherein choline , ethanolamine , adenosine etc . are added to these nucleotides . cytidine is a pyrimidine nucleoside . it is found in plant ( ross , 1965 ) and animal ( traut , 1994 ) tissues . in plants , it takes part in synthesis of cytidine - 5 ′- diphosphate ( cdp ) and cytidine - 5 ′- triphosphate ( ctp ) ( ross and cole , 1968 ). it is included in the structure of rna in the same ratio with uridine ( ross and cole , 1968 ). in addition , following deamination reaction in plants , some part of the cytidine is transformed into uridine ( ross and cole , 1968 ). while cytidine is the major pyrimidine in blood circulation of rats ( traut , 1994 ), in human blood circulation the major pyrimidine is uridine ( wurtman et . al ., 2000 ). moreover , as in the plants , also in humans , cytidine provided externally to the body is quickly transformed into uridine as a result of deamination ( wurtman et . al ., 2000 ). cytidine is transformed into ctp and cytidine - 5 ′- diphosphate choline ( cdp - choline ) through kennedy pathway and thus takes part in membrane phospholipid synthesis ( kennedy and weiss , 1956 ). cdp - choline , which is derived from cytidine , is studied extensively in terms of its neuroprotective effects in animal experiments and some clinical studies . cdp - choline reduces damage in hypoxic and ischemic brain injuries and improves the learning and memory functions which are impaired with aging ( secades , 2011 ). with these features , it is suggested to be useful as neuroprotective in cases of stroke , traumatic brain injury , and alzheimer disease ( secades , 2011 ). uridine is a pyrimidine nucleoside and a constituent of plant ( ross , 1965 ) and animal ( pelling , 1959 ) tissues . uridine is also the constituent of nucleotides comprising mono -( uridine - 5 ′- monophosphate [ ump ]), di -( uridine - 5 ′- diphosphate [ udp ]) and tri - phosphate ( uridine - 5 ′- triphosphate [ utp ]), nucleotide sugars ( udp - glucose and udp - galactose ) ( ross and cole , 1968 ) and phospholipid intermediate metabolites ( kennedy and weiss , 1956 ) cytidine - 5 ′- triphosphate ( ctp ) ( genchev and mandel , 1974 ) and cytidine - 5 ′- diphosphate choline ( cdp - choline ) ( cansev et . al ., 2005 ) compounds . uridine plays role in various physiological functions such as glycogen and phospholipid biosynthesis and protein and lipid glycosylation ( lecca and ceruti , 2008 ). rna synthesis has a vital role in plant growth and development ( oota , 1964 ) and experimental disintegration of rna affects growth and development ( brachet , 1954 ). membrane phospholipids are also the most important components of cell membranes and cell growth and reproduction are associated with the increase of membrane phospholipid synthesis in both plants ( xue et . al ., 2009 ) and animal cells ( bashir et . al ., 1992 ) and tissues ( wurtman et . al ., 2009 ). it is also shown that ump , which is a source of uridine , is transformed into uridine after entering into body and reaches the brain ( cansev et . al ., 2005 ) and improves phospholipid production ( wurtman et . al ., 2006 ) or neuron branching and thus neural communication in infant ( cansev et . al ., 2009 ) or adult ( sakamoto et . al ., 2007 ) experimental animals . the uridine added to the neurons in the culture also increases the growth and branching of these cells ( pooler et . al ., 2005 ). with above said characteristics , uridine treatment is found to increase learning and memory functions in experimental animals ( teather and wurtman , 2006 ; holguin et . al ., 2008a ) and also in environmentally impoverished animals ( holguin et . al ., 2008b ). in addition , uridine reduces brain damage of laboratory animals in experimental models . for instance , in experimental parkinson model , uridine administered in the form of ump ameliorated brain lesion and reduced rotational behaviour , which is the typical indication of damage ( cansev et . al ., 2008 ). moreover , uridine treatment significantly reduced the level of damage in infant rats , which are exposed to hypoxic ischemic brain damage ( cansev et . al ., 2013 ). prevention of programmed cell death ( apoptosis ) mechanism of brain cells by uridine mediated to this effect ( cansev et . al ., 2013 ). in the prior art , when uridine is used on humans ; it is known that it causes diarrhea when it is taken in high oral dosage such as 10 g per day ( van groeningen et . al ., 1991 ) and the dose of 10 g / m 2 administered intravenously is known to cause shaking ( leyva et . al ., 1984 ). promotion of plant growth and development through administration of uridine or cytidine in the present invention , said uridine or cytidine chemical is used on plants . said uridine is administered on cucumber ( cucumis sativus ) plants in the preferred embodiment of the invention . said cytidine is administered on cucumber ( cucumis sativus ) plants in the preferred embodiment of the invention . their sowing is preferably made into vials of 72 such that 1 seed would be present per vial . said seeds are germinated at 25 ° c . in plant growth cabin . following the stage of germination , 10 ml of uridine or cytidine solution prepared at 10 or 100 μm concentration is administered to the plants twice a week . water is used as dissolver in order to dissolve uridine or cytidine ( it is preferably dissolved in pure water and at room temperature ). in order to prepare 10 − 9 m ( 1 nano molar ) solution ; 0 . 000244 mg uridine is dissolved in 1 l of water . in order to prepare 1 m ( 1 molar ) solution ; 244000 mg uridine is dissolved in 1 l of water . in order to prepare 10 μm solution ; 2 . 44 mg uridine is dissolved in 1 l of water . in order to prepare 100 μm solution ; 24 . 4 mg uridine is dissolved in 1 l of water . in order to prepare 10 − 9 m ( 1 nano molar ) solution ; 0 . 000243 mg cytidine is dissolved in 1 l of water . in order to prepare 1 m ( 1 molar ) solution ; 243000 mg cytidine is dissolved in 1 l of water . in order to prepare 10 μm solution ; 2 . 43 mg cytidine is dissolved in 1 l of water . in order to prepare 100 μm solution ; 24 . 3 mg cytidine is dissolved in 1 l of water . plants are grown under light for 16 hours at 24 ° c . and 22 ° c . and under dark conditions for 8 hours at 20 ° c . daily in growth cabinet for 3 weeks until they have 2 actual leaves . afterwards , measurement of plant parts are made as shown in fig9 with below given details : in the present invention , said uridine chemical is used on plants . said uridine is administered on cucumber ( cucumis sativus ) plants in the preferred embodiment of the invention . their sowing is preferably made into vials of 72 such that 1 seed would be present per vial . said seeds are germinated at 25 ° c . in plant growth cabin . following the stage of germination , 10 ml of uridine solution prepared at 10 or 100 μm concentration is administered to the plants twice a week . water is used as dissolver in order to dissolve uridine ( it is preferably dissolved in pure water and at room temperature ). in order to prepare 10 − 9 m ( 1 nano molar ) solution ; 0 . 000244 mg uridine is dissolved in 1 l of water . in order to prepare 1 m ( 1 molar ) solution ; 244000 mg uridine is dissolved in 1 l of water . in order to prepare 10 μm solution ; 2 . 44 mg uridine is dissolved in 1 l of water . in order to prepare 100 μm solution ; 24 . 4 mg uridine is dissolved in 1 l of water . plants are grown under light for 16 hours at 24 ° c . and 22 ° c . and under dark conditions for 8 hours at 20 ° c . daily in growth cabinet for 3 weeks until they have 2 actual leaves . for high temperature stress applications , the temperature of the growth cabin is increased gradually to 35 , 40 , and 45 ° c . and kept for 24 hours at each temperature level . following application of 45 ° c ., total amount of soluble protein is measured in the leaf samples taken from the plants . total soluble protein extraction is made by using the method of arora et . al . ( 1992 , 1997 ) with some modifications suggested by gulen and eris ( 2003 ). solution components used in total soluble protein extraction are : 50 mm borax ( sodium tetraborate ) 50 mm ascorbic acid 1 mm pmsf ( phenylmethylsulphonyl ) % 1 β - mecaptoethanol 5 ml of the extraction solution prepared as given above is taken and homogenized together with 1 g of leaf sample in mortar . homogenized samples are taken into 15 ml centrifuge tubes and centrifuged for 1 . 5 hours at 26 000 g and 4 ° c . following centrifuge , the above liquid phase is taken and passed through 0 . 22 μm diameter filters . total soluble protein amount is determined according to bradford ( 1976 ) method as proposed by arora and wisniewski ( 1994 ). the amount of protein in the supernatant obtained from the protein extraction is determined according to spectrophotometric measurements . measurements are made by using single use polycarbonate basins at 595 nm wavelength and 0 , 10 , 20 , 30 , 40 , 50 μg / μl bsa ( bovine serum albumin ) standards are used for calculating total soluble protein amount . bsa stock solution is prepared as 5 mg bsa / ml extraction solution . in a preferred embodiment of the invention ; uridine solutions can be used in the form of application to the soil together with liquid fertilizers at certain ratios ( liquid fertilizer components ). in a preferred embodiment of the invention ; uridine solutions can be used by being added to the plant nutrient components in soilless agriculture applications ( plant nutrient components ). in another preferred embodiment of the invention ; uridine solutions can be used by being buried into soil after tabletting with suitable filling materials ( tablet composition ). in another preferred embodiment of the invention ; uridine solutions can be used together with irrigation water in drip irrigation system ( drip irrigation ). in another preferred embodiment of the invention ; uridine solution can be used in the form of coating by being sprayed onto leaves of plants and to fruits ( spraying ). the effects of uridine on plants depends on concentration of application ( application dose and frequency ) and also application time , the physiological stage of the plant , age of the plant , species or type of the plant , member of the plant and the ecological conditions at the time of application ( temperature , light , moisture , wind , soil etc .). as disclosed in the purpose and the method of application of the invention , the applications can be made when the plants are at the seed phase , plantlet , sapling , seedling , trees at the period of yield etc . different physiological and morphological phases . moreover , it can also be applied on perennial , annual , herbaceous , ligneous , deciduous , evergreen etc . all plant types . as a pyrimidine nucleoside , cytidine can also cause similar impacts with uridine on stress tolerances of plants , since both some part of it is transformed to uridine in plants and also it uses common pathways ( e . g . kennedy pathway ) with uridine during its metabolism . the impacts of cytidine on the plants depends on concentration of application ( application dose and frequency ) and also application time , the physiological stage of the plant , age of the plant , species or type of the plant , member of the plant and the ecological conditions at the time of application ( temperature , light , moisture , wind , soil etc .). as disclosed in the purpose and the method of application of the invention , the applications can be made when the plants are at the seed phase , plantlet , sapling , seedling , trees at the period of yield etc . different physiological and morphological phases . moreover , it can also be applied on perennial , annual , herbaceous , ligneous , deciduous , evergreen etc . all plant types . allard f ., houde m ., krol m ., ivanov a ., huner n . p . a ., sarhan f . 1998 . betaine improves freezing tolerance in wheat . plant cell physiol . 39 , 1194 - 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