Research On Chemistry Constituents Of Wheat (Triticum Aestivum L.) Seed Coat And Mechanism Of Its Pigment Constituents Biosynthesis

There are various types of the pigments present in wheat seed coat, which may beassociated with seed quality traits. Some pigment constituents play a specific role inpharmacological effects. A study was conducted to evaluate the correlation betweenwheat seed coat pigment contents and seed quality traits, characterize the chemistryconstituents and pharmacological effects of wheat seed coat. Three groups of wheatcultivars with different grain color, red kernel wheat (Yangmai 158, Ningmai 9 andS105), white kernel wheat (Yumai 18, Wenmai 6 and Lainong 2066), and the purplekernel wheat heimai 76 were used to study their seed coat pigments dynamics, theenzyme kinetics corresponded to the pigments biosynthesis during the development ofseed, and the relationship between the pigments and seed quality traits. The correlationsbetween pigment contents and quality traits were analyzed in different wheat cultivars.Based on these studies, the physiology mechanism and regulatory pathways were furthercharacterized during seed coat pigment biosynthesis. The wheat seed coat's chemicalconstituents analyzed systematically and screened for their pharmacological effects.The anti-tumor activities of total flavonoids and alkylphenols in wheat bran weredemonstrated both in vitro and in vivo.The main results were summarized as follows:1 Biosynthesis of pigment constituent in seed coats and its relationship with grainquality in different wheat cultivars1.1 The biosynthesis of seed coat pigments in wheat cuitivars with different graincolorThe main pigments in wheat seed coat are chlorophyll, lutein, carotenoids, flavonoids andanthocyanin. The result showed that the content of chlorophyll, lutein and carotenoids inall three groups changed as a single peak curve, increased to the high peak value quicklyat the beginning and mid of grain filling, then descended quickly, kept to the lower lever during the ripening process until maturity. In mature grain, the content of chlorophyll,lutein and carotenoids in purple kernel wheat Heimai 76 was the highest among the threegroups. There were no significant differences observed in pigment contents betweenred kernel wheat and white kernel wheat.In contrast, the content of total flavonoids increased at the beginning of grain filling,decreased gradually in mid, increased sharply until maturity. In mature grain, thecontent of total flavonoids in the purple kernel Heimai 76 was 1.6-3.9 folders more thanother cultivars. The total flavonoids contents followed the order of purple kernelwheat＞red kernel wheat＞white kernel wheat. The results of variance analysis showedthat there were significant differences in the contents of total flavonoids among threegroups.The content of anthocyanin in purple kernel Heimai 76 increased slowly at the beginningof grain filling, quickly to tiptop at late stage until maturity. Anthocyanin production atthe beginning of grain filling in Yumai 18, Ningmai 9 and S105 reached to a pinnacle,and then decreased quickly, started to increase at later stage of grain filling till maturity.The content of anthocyanin of Yangmai 158, Wenmai 6,and Lainong 2066 increasedslowly at the beginning of grain filling, decreased in mid, increased again at later. It wasfound that the anthocyanin contents followed the order of purple kernel wheat＞red kernelwheat＞white kernel wheat. The results of variance analysis demonstrated that there weresignificant differences in the contents of anthocyanin among each group.The data showed that the main pigments constituted for seed coat color were chlorophyll,lutein, carotenoids, flavonoids and anthocyanin. The grain color is determined by theamount and types of the pigments that are present in seed coat, and the contents of totalflavonoids and anthocyanin are the main factors responsible for seed coat color.1.2 The activity of pigment synthesis enzymes in wheat seed coats with differentcolorsThe activity of Polyphcnol Oxidase (PPO) in seed coat trended as "M" during the wheatgrowth in each wheat types with different color. In mature seeds, the activity of PPO in Heimai 76 was higher than red kernel wheat, which was higher than white kernel wheatin total. However, there were no significant differences observed in the seed coat PPOactivity among three groups. The activity of Tyrosinase was high at the beginning ofgrain filling, and then decreased gradually, finally reached to the minimum in matureseeds. The activity of PPO in Heimai 76 was much higher than red kernel wheat, whichwas higher than white kernel wheat as a whole.Phenylalanine ammonia lyase (PAL) activity during the seed development showed an "N"variational curve with change of low-high-low-high basically. The activity of PAL inHeimai 76 was much higher than red kernel wheat, which was higher than white kernelwheat in total. There were significant differences observed in the seed coat PAl. activityamong three groups. The activity of PPO and tyrosinase decreased in nearly matureseeds, while PAL activity increased. The result suggested that the different color ofseed coat was formed mainly due to the difference in PAL activity.1.3 The relationship of pigments, enzymes, seed quality traits with different graincolorsThree groups of wheat varieties with different grain color, red kernel wheat (Yangmai158, Ningrnai 9 and S105), white kernel wheat (Yumai 18, Wenmai 6 and Lainong 2066),and the purple kernel wheat Heimai 76 were used to analyze the relationship betweenseed coat pigment (as chlorophyll A and B, lutein, carotenoids, anthocyanin and flavonoids)and correspondent enzymes, the pigments and quality traits of mature seeds. The resultsshowed that the contents of chlorophyll A, chlorophyll B, lutein, type carotene,flavonoids were positively correlated, which was much or most with each other. Thecontent of anthocyanin was significantly correlated with the contents of chlorophyll A,chlorophyll B, and flavonoids, while the correlations with lutein and type carotene wereweak. The content of every pigment correlated significantly with the activity ofPhenylalanine ammonla-lyase, but weakly with Polyphenol Oxidase and tyrosinase.The pigments did not show significant correlation with seed quality traits except thecontents of chlorophyll A and anthocyanin present significant positive correlation withfalling number. The results showed that the wheat bran colors could not reflect seedquality traits in general. 2 The chemistry constituents and pharmacological effects of wheat seed coat2.1 Chemistry constituents of wheat seed coatThe bran of Ningmai 9 was used to study chemisty constituents systemically by tools ofspectrum (such as ¹H-NMR, ¹³C-NMR, DEFT, HMQC, HMBC, ESI-MS, GC/MS, IR,UV etc.),physics and chemistry constant, standard compound antitheses.,. Altogether,31 compounds were separated and the structures of 29 among those compounds wereidentificated. Among them six compounds were isolated for the first time fromTriticum L., while nine compounds were isolated from wheat firstly. Two of them werenew compounds and one was a new natural metabolism compound. There were fiveflavonoid compounds as Corymboside (WB-8), Isocorymboside (WB-9), TriticusideA (WB-10A), Triticuside B (WB-10B), Apigenin (WB-13); two phenolic acidcompounds as trans-3,4-dimethoxycinnamic acid (WB-11), Ferulic acid (WB-12);five alkylphenols compounds as 5-heptadecylresorcinol (WB-3),5-nonadecylresorcinol (WB-4), 5-heneicosylresorcinol (WB-5), 5-tricosylresorcinol(WB-6), 5-pentacosylresorcinol (WB-7); seventeen steroids compounds as4-Methylstigmast-4-en-3-one (WS-1), Stigmasta-4,22E-dien-3-one (WS-2),5a-Stigmast-24(28)-en-3-one (WS-3), 5a-Stigmast-22-en-3-one (WS-4),Stigmast-4-en-3-one (WS-5), Stigmasta-4,24(28)-dien-3-one (WS-6),24-Ethyl-5a-cholestan-3-one (WS-7), 24-Methyl-5a-cholestan-3-one (WS-8),Spinasterone (WS-9), 4-Methylstigmasta-4,24(28)-dien-3-one (WS-10),4-Methylstigmasta-4,22-dien-3-one (WS-11), 4-Methyl-4a,5a-cholestan-3-one(WS-12),4-Methylstigmast-6-en-3-ol (WS-13), Sitosterol (WS-14,WB-2),Stigmasterol (WS-15,WB-1), Sitostanol (WS-16), 4,4-Dimethylcholestan-3-ol(WS-17). Among them, WB-1, WB-2, WB-9, WB-10A, WB-10B, WB-11 wereisolated from Triticum L. for the first time. WS-1, WS-3, WS-4, WS-9, WS-10,WS-11, WS-12, WS-13, WS-17 were identified from wheat for the first time.WB-10A and WB-10B were new compounds. WS-17 was a new natural metabolismcompound. 2.2 Preparation and pharmacological effects of flavonoids and alkylphenols in wheatbranThe bran of Ningmai 9 was used to study extraction, enrichment, purification technologyof effective ingredients flavnoids and alkylphenols. The yield of wheat bran totalflavonoids was 2.1‰, the content reached to 76%. The productivity of wheat bran totalalkylphenols was 4.5‰, the content reached to 72%. The total flavonoids (the contentis 76%) and alkylphenols (the content is 72%) were applied to further pharmacologicalexperiments for anti-tumor functional analysis.It is found that total flavonoids (TF) and total Alkylphenol (TW) inhibited tumor growthof S₁₈₀ mice, correspondently reduced tumor weight of S₁₈₀ mice, prolonged lifetime ofS₁₈₀ mice, relieved symptom of ascites, and showed potential therapical effect on S₁₈₀cancer. In vivo, five Alkylphenol compounds showed inhibitory effect on the HepG2and MAD-MB-231 cell growth. The efficiency of inhibition is up as increasingconcentration of the samples. The activity of 5-heptadecylresorcinol is the mostnotable among five Alkylphenol compounds, its IC₅₀ to HepG2 and MAD-MB-231 cellare 156.11μg/ml and 136.45μg/ml respectively. In summary, we demonstrated thatTF and TW of Triticum aestivum L. showed significantly inhibitory effect on tumorgrowth both in vitro and in vivo...