英文版花色素结构假说.doc

anthocyanidin configuration and biosynthetic pathways hypothesisTIAN Xiao-Yan(Shenyang Agriculture University Food Institute, Liaoning Province Shenyang,110161, in China)MAbstract This study was carried out on the mass spectrometry analysis of Nanguo pear fruit peel anthocyanidin, after detailed calculation of MS and +MS2, hypothesized that the anthocyanidin configuration in nature exist is in the form of lantern body or globule body, and its compositions either only anthocyanidin unit, or associating with glucoses. The lantern body or globule body is connected between the cell membrane or cytoplasm membrane or garna membrane and photosynthesis system. These anthocyanidins have specific molecular weight, shape and specific spatial configuration. Six kinds of anthocyanidins, as usually proposed, called pelargonidin, cyanidin, delphinidin, peonidin, petunidin, malvidin, perhaps only very little exist in nature at monomers, almost all anthocyanidins exist in nature at lantern body or globule body. Anthocyanidin is a reticular macromolecules consisting of phenyl benzopyrane connected through oxygen bridge similar as a lantern body or globule body. A layer 4B rings link a layer 4phenyl benzopyranes through 4 C of B ring and 5 C of A ring of phenyl benzopyrane by oxygen bridge tied together longitudinally, while 2C, 7C of A ring of phenyl benzopyrane by oxygen bridge hand in hand tied together horizontally, repeating this combination units until its lenghth meeting the needs. Some 4phenyl benzopyranes of lantern body anthocyanidin link with Mg2+, its function resembles some chlorophyll a, can absorb and convert light energy. If 3C or 5C of B ring has no oxygen bridge linking each other, after acidulated, get pelargonidin; if 3 and 5 of B ring only link one or two oxygen bridges, get cyanidin and pelargonidin; if 3 and 5 of 4B rings has three or four oxygen bridges, get delphinidin and cyanidin; if position 3 of B ring only links one methoxy, get peonidin; if 3 of B ring links one methoxy, while position 5 of B ring links one oxygen bridge, get petunidin; if 3 and 5 of B ring link two methoxy, get malvidin. In general, pelargonidin, cyanidin, delphinidin exist in inner of anthocyanidin; peonidin, petunidin, malvidin exist in top of anthocyanidin for connecting with the cell membrane or the photosynthesis system, so the content of peonidin, petunidin, malvidin is relatively rare. Color differences of flowers and fruits, lie on the contents of anthocyanidin and abundance of oxygen bridges. While the contents of anthocyanidin and abundance of oxygen bridges are higher, spectral blue shift. The major function of anthocyanidin is absorbing ultraviolet rays, transforming high light energy to low energy for the photosynthesis, so prevent the ultraviolet rays injure on flowers and fruits. The study considers that flowers and fruits possess the features of photosynthesis. Anthocyanidin macromolecules just like Sky-net covering on the breakage of chloroplast membranes or granum membranes damaged by Uv, Uv high-energy photons activate high-energy electrons, when high-energy electrons pass through p- conjugation rings of anthocyanidin macromolecules, rotating with high speed in p- conjugation rings, producing heat and released out, at the same time, high-energy electrons turned into low-energy electrons transporting to the carbon reaction system, so as to ensuring the carbon reaction system can effectively carry out photosynthesis. This hypothesis considers anthocyan biosynthesis main substrates is glucose and acetyl coA. introduction:A large number of experiments have been carried out in orchard and laboratory aiming at raising the contents of anthocyanidin of Nanguo pear peel. Nanguo pear is special local product of Anahan city and Haicheng city in Liaoning Province of China, Nanguo pear is a variant of Pyrus ussuriensis Maxim, its aroma is wonderful and rich tasting, can be the king of pear. But, it has a fatal defect, that is fruit depigmentation when meeting autumn rains in maturation period, accompanying with fruit quality sharp declining, and again colouring is hard. For solveing the difficult problem, carried out on the mass spectrometry analysis of Nanguo pear fruit peel anthocyanidin, accidently, discovered the reasonable framework of anthocyanidin in nature. It is a complete net macromolecule or globule, not fragment. 1 Materials and Methods1.1 materialsTen Nanguo pear fruits were picked up from 10-12 years Nanguo pear, in the south direction, middle part, and equal height, 3 repeats, On august 25, 2010, in Song village of Jiewen town of Haicheng city in Liaoning province.Chemical reagents: Hydrochloric acid, 95% ethanol, muriate, anhydrous sodium acetate is analytical reagent of the shenyang chemical reagent factory production; D101 macroporous resin coming from the Chinese medicine group chemical reagent co., LTD; ethyl alcohol, methane acid is chromatographicall pure coming from the Chinese medicine group chemical reagent co., LTD; Instrument: N-1001 type rotary evaporation apparatus; SHB- circulating water type vacuum pump; DHL-A type computer constant flow pump; Christ vacuum freeze drier; 1100 liquid chromatograph/mass spectrometer (LC/MS).1.2 methods1.2.1 extraction and purification of Nanguo pear peel anthocyanidin Peel 10g, fully grinding, adding in 250ml 1% hydrochloric acid ethanol solution, 50 constant temperature water bath 4 h, 4000r.min-1centrifugal 10min, residue by the same extraction method extracting again to filtrate colorless, mergeing the filtrate and constant volume to the same volume, pH differential method was used for determination of the contents of anthocyanins 9. Crude extract decompression concentration, purification passing through D101 macroporous resin, With Secondary distilled water washing to remove impurity substance, such as sugar, Organic acid and protein，and so on. Then 60% ethanol elution, Vacuum concentration eluent, freeze drying to dark red powder.1.2.2 HPLC-DAD-ESI-MS identification of Nanguo pear anthocyanidinChromatograph conditions: chromatographic column: Agilent SB-C18(2.1mm150mm, 3.0 m); mobile phase: A liquid is methyl alcohol, B liquid is 0.5% methane acid; Linear gradient elution order: 015min, A liquid 15%40%; 1530min, A liquid 40%90%; 3035min, A liquid 90%90%; Sample quantity: 5L; Column temperature: 30; velocity: 0.2mL.min-1; DAD detector detection wavelength: 530nm.Mass spectrum conditions: cation scanning: ESI+, M/Z 501000; Break voltage: 1.0V; Gas curtain gas: Nebalizer 30psi; Drygas: 6.0L.min-1; Drytemp: 320.2 result and analysis2.1 anthocyanidin MS+ and MS2+ analysis hypothesis premiseThe study considers that one peak or relative one line of MS+ or MS2+ is certain anthocyanidin macromolecule, just because different contents lead to either one peak or relative one line. In our MS+ or MS2+ map, one relative line corresponds contents between 103-105, while one peak corresponds contents between 105-108. Anthocyanidin macromolecule is a specific macromolecule, anthocyanidin basic unit contains O+, after primary mass spectrum analysis, the chemical bond on O+ both sides, possiblly random fractures and makes the intramolecular rearrangement, or hydrogenation results O+ to recover for O2-, so the charge numbers of anthocyanidin change on a large scale. The crack type of chemical bond and hydrogenation lie on the size of anthocyanidin and the amount of oxygen bridges. primary mass spectrum fracture mode determine the size of MS2+ fragments. The hypothesis core idea considers: the sum of all fragments of one peak or one relative line is the whole molecular of one anthocyanidin. But some fragmentss intens is 103 while others is 104 in one peak, how to add? The hypothesis considers that all of these fragments should on the highest intens, is the different detection ratio of different fragments deciding different intens.Combining primary mass spectrum with secondary mass spectrum, the study considers that the sample of Nanguo pear peel contains 61 kinds of anthocyanidin macromolecule. It is easy to differenttiate between peaks, while relative line, how to differentiate each other? Book of Changes (the Yi Jing): digital comes into being appearance, digital changes while appearance changes. Combining digital changes of primary mass spectrum with secondary mass spectrum, ascertain the origination of every one anthocyanidin.Any anthocyanidin macromolecule has top, Either phenyl benzopyran or glucose as the top Connected to the cell membrane or chloroplast membrane, to initial anthocyanidin macromolecule biosynthesis. Firstly, synthetize the first layer 4 B ring, B ring links with top together by oxygen bridge at 4C position, while 4 B rings link together by four oxygen bridge hand in hand at 3, 5 C, Or not link or two B ring connected each other by one oxygen berdge. The amount of oxygen bridges depends on the content of glucose and the activity of the oxygen bridge enzyme. This hypothesis considers anthocyan biosynthesis main substrates is glucose and acetyl coA; Secondly, synthetize the second layer 4 benzopyrans; thirdly, synthetize 4 B rings again; fourthly, synthetize 4 benzopyrans again，Continuous cycle down. layers depends on the distance between the cell membrane and the chloroplast membrane, or chloroplast double membrane spacing or chloroplast membrane and granum membrane. anthocyanidin macromolecule like the NvWaBuTian seven colour stone of Chinese mythology, it is flowers or fruit colors sky-net. The premise of this hypothesis is that the truth is the most simple. The sunlight shines on the whole world, so plants accept all spectrums. Plants wont waste any light sources, all of light source will be used. Leaves use flavonoids in cytoplasm to absorb ultraviolet light, and release heat energy to reduce the energy, while transmit light energy to chloroplast for photosynthesis. Leaves use flavonoids to absorb ultraviolet light, while flowers and fruits use anthocyanidin to absorb ultraviolet light. Flowers and fruits are organs of generation, take on organ specificity, possess photosynthesis ability, and possess enzymes of synthetising anthocyan, for example, oxygenase, B ring synthetases, and so on. In the hypothesis, flowers and fruits possess highly efficient photosynthesis.When some flavonoids are directly used for synthesis of anthocyanin, two OH of flavonoids link together by dehydration; when flavonoids are used up, glucose and CoA can be used for the anthocyanidins biosynthesis, oxygen bridges connects anthocyanidin units together directly by oxygenase. The hypothesis use pelargonidin, cyanidin, delphinidins concept and molecular weight to calculate, only for calculating conveniently. Pelargonidin can provide 4-OH, cyanidin can provide 5-OH, delphinidin can provide 6-OH for dehydration, we here say the dehydration reaction is just the convenient say.A layer B rings is equivalent to 4 benzene rings, while a layer benzopyrans is equivalent to 8 benzene rings. Electron flow transmission in layers, just likes water flow, converges through 4 benzene rings, while diffuses through 8 benzene rings. In the converging process, the electron flow accelerates, while in the diffusion process, the electron flow decelerates, release thermal energy resulting to electric energy cutting down. Continuous converging and diffusion, guarantee the electron flow with low energy state quickly getting to chlorophyll for photosynthesis. Such space configuration guarantees for chlorophyll photosynthesis efficiency.Benzene ring height is 0.58 nm, a anthocyanidin unit height is 0.58 * 2 = 1.16 nm. Plant cell size is 10-100 m, chloroplast diameter is 4-10 m, chloroplast contains double membranes, each layer membrane thickness is 6-8nm, space between inside and outside layer membranes is 10-20nm.Based on the above hypothesis, comes to a decision that the sample contains 61 kinds anthocyanidins macromolecule. Now discusses its structure, in cell position and membrane connection mode.2.2 hypothesis explanation of primary mass spectrum analysis of Nanguo pear peel anthocyanidins in detail Figure 1 primary mass spectrum Figure 2 21.0-21.8min peak +MS of random sampleUsing 21.0-21.8min peak as an example, the primary mass spectrum highest nuclear mass ratio is 382.9, 395, 464.9. Illustrate that charge numbers change causing nuclear mass ratio change. As long as controlling conditions of primary mass spectrum, we can get complete anthocyanidins with fragments into the secondary mass spectrum, even fragments like flying banner, but still connectting within the macromolecules. Controlling conditions of the secondary mass spectrum, can get the sum of fragments just is complete anthocyan macromolecules weight. 2.3 hypothesis explanation of secondary mass spectrum analysis of Nanguo pear peel anthocyanidins in detail2.3.1 explanation of spatial structure and plan sketch of Nanguo pear peel anthocyanidins in detailFigure 3 the secondary mass spectrumThe secondary mass spectrum compareing with primary mass spectrometry, has lost a lot of anthocyanidin molecules. Such as 33.5-33.6min peak, primary mass spectrometry total ion spectrum is about 1.2108 level; but secondary mass spectrum total ion spectrum is about 106 level; Lost 102 level molecules, where they go? They were smashed, were caught by the detector, and not into the secondary mass spectrum. Figure 4 33.5-33.6min peak +MS2Figure 4 is the complete +MS2 map of 33.5-33.6min peak. Nuclear mass ratio is 398.8, 399.3, 367.3, 351.5. 398.8 and 399.3 fragments intens is 104, 367.3 fragment intens is103, 351.5 fragment intens is 106. The hypothesis considers that they are 4 fragments for a whole, their intens all should be 106, why reduce? because different fragments have different again cyclization process, its formation probability and monitored ratio are different; If fragments have no time to cyclization, which lead to negative existing forms and cannot be detected. 398.8+399.3+367.3+351.5= 1516.9, through the calculation, 4* delphinidin +2* pelargonidin +（-O-CH2-）+（-O-）+4H+-（4*6+2*4）/2*H2O=4*303+2*271+30+16+4-288=1516, it is a globule composing with 6 anthocyanidin units.Length calculation: 1*anthocyanidin unit+2*top+2*O-C-+1*-C-H+1*O-H，which 2*top length is similar with a benzene length. 1*1.16nm+0.58nm+2*0.143nm+1*0.1nm+1*0.096nm 2.222nm, according to the length, the globule exists between granum membranes. 1 methoxy- link with membraneCH3-O-2.222nm1 pelargonidin: as top4 delphinidin: that is 4 B ring link by 4 -O-1 pelargonidin: as bottom-OH1-OH link with membraneFigure 5 33.5-33.6min peak anthocyanidin spatial structure sketch mapSpread the globule for plane, its splitting way as arrow shown in figure 6, which 1 link with 2, and 1 link with 2 in nature. respectively get a367, b351, c399, d399 fragments, H+ reset during splitting. O3 position breaks. During mass spectrometric analysis, solvent can provide H+, 2H+ for C4，1H+ for O3；1H+ for C6, and directly causing globule to reduce 2 positive charges. Figure 6 33.5-33.6min peak anthocyanidin globule spreading planThe up top a is peonidin that people concepted. The down top bis cyaniding. The hypothesis consider that anthocyanidin synthetic initial enzyme links -CH2-OH to phosphatide-OH of cell membrane or chloroplast membrane, comes into being HO-CH2-O-phosphatide, -CH2-OH comes from glucose. With HO-CH2-O-phospholipid as the origin, synthetises one B ring, and then synthetises one benzopyran ring, as the top. Again synthetises a layer 4 B rings, again synthetises a layer 4 benzopyran rings, Alternate synthesis, in turn, has been repeated to chloroplast membrane or granum membrane. If 4 B ring not connected, after hydrolysis, can get pelargonidin; If 4 B ring is linked together by 4 oxygen bridge, after hydrolysis, can get delphinidin; If 4 B ring is linked together by 1 or 2 or 3 oxygen bridge, after hydrolysis, can get cyaniding or delphinium. The oxygen bridge numbers depend on the activity of oxygen bridge synthesis enzyme, 1-3 oxygen bridges reflect the anthocyanidin association is underway. During the secondary mass spectrum analysis process, anthocyanidin breaks into four fragments, and occurring molecular rearrangement, namely 367, 351, 399, 399. The arrow in figure 6 show fracture mode. Possible fragments structure as below: a367 fragment(contains 15H+) b351 fragment(contains 11H+) c399fragment(contains 7H+) d399fragment(contains 7H+)Figure 7 367, 351, 399 fragments possible framework2.3.2 explanation of secondary mass spectrum molecule ion of Nanguo pear peel anthocyanidins in detailAll molecule ion nuclear mass ratio of Nanguo pear peel anthocyanidins conclude follow:Table 1 All molecule ion nuclear mass ratio of Nanguo pear peel anthocyanidins0-100100-200200-300300-359360-399400-500500-600600-70060.885.0102.1：85.0117.0126.7：85.0147.0155.0157.0163.0183.0185.0190.0200203204205207209210212213217221227：155231：185243：102245250：84253255257：117258259266271272273274275277279282283284：250285293299：271301303307311：250314315316317：301318319327：183329：301331333335336：14