| Abelmoschus manihot (L.) Medicus from the family of Malvaceae is an annual herbal plant widely distributed in the whole country and firstly recorded in "Jiayou Materia Medica". A survey of the ethnopharmacology literature shows that the roots and aerial parts of Abelmoschus manihot are extensively used in traditional medicine with the efficacy of clearing heat and promoting diuresis, detumescence and detoxification. Modern pharmacological researches have showed that it possessed better therapeutic effect in the treatment of nephritis, cardiovascular disease and cancer. Phytochemical studies have revealed that Abelmoschus manihot contained various resource constituents including flavonoids, polysaccharides, organic acids, cellulose, long chain hydrocarbon, and so on. However, the most common application mode of Abelmoschus manihot in the present is picking its flowers for medicine, which no doubt causes enormous waste of resources and environmental pollution as numbers of Abelmoschus manihot roots, stems, leaves are discarded or burned. The reason for this situation is that most studies focus on the flowers and the flavonoids, while the ingredients in other parts of Abelmoschus manihot such as roots, stems and leaves have been little investigated, not to mention the contents at different stages of growth. Thus, this dissertation was confined the discussion to the chemistry, biosynthesis and biotransformation of non-medicinal parts to find out the scientific basis for the development and utilization of Abelmoschus manihot.1 Study on the resource chemistry of non-medicinal parts of Abelmoschus manihot1.1 The distribution and dynamic accumulation of total flavonoids33 samples of Abelmoschus manihot roots, stems, leaves and flowers in 10 growing periods was determined and analtied by the NaNO2-Al(NO3)3-NaOH color colorimetric method. The results showed that the contents of total flavonoids in leaves with the range from 20.87 mg/g to 50.54 mg/g were significantly higher than those of roots and stems, but lower than the medicinal parts of flowers with the from 80.69 mg/g to 92.00 mg/g. As for the dynamic accumulation of total flavonoids in different growth stages, it was found that the content of total flavonoids in stems, leaves and flowers showed an increasing trend with the extension of growth period, while the samples of roots reached their peak in the bud stage, and then gradually reduced. The contents of total flavonoids in stems and leaves of Abelmoschus manihot could accumulate to 50-60 mg/g in yellow withered stages, which indicated that stems and leaves of Abelmoschus manihot can be collected after picking the flowers for the using of the flavonoids resources.1.2 The distribution and dynamic accumulation of total polysaccharidesThe contents of neutral polysaccharide (NP) and acidic polysaccharides (AP) in the 33 samples were tested by the phenol-sulfuric acid method and the carbazole-sulfuric acid method, and the contents of total polysaccharides were the sum of both. The result revealed that the contents of NP and AP in the different parts of Abelmoschus manihot showed a fluctuation during different growing periods and there was no obvious regularity on the whole trend. The contents of total polysaccharides in non-medicinal parts were significantly higher those of the total flavonoids, while the contents of these two compounds in flowers was equivalent. In addition, the contents of NP (28.51-93.44 mg/g) was higher than those of AP (12.63-55.35 mg/g), and the range of total polysaccharides were from 43.64 to 145.29 mg/g. As for the different parts, the samples of flowers had the highest contents of total polysaccharides. For the different growing periods, the contents of polysaccharides in the leaves were higher in the early growth period. Moreover, the sum of polysaccharides in the non-medicinal parts were much higher than the flowers. Therefore, the exploitation of polysaccharides in the non-medicinal parts of Abelmoschus manihot has a huge positive significance for the development and utilization of Abelmoschus manihot.1.3 The distribution and dynamic accumulation of nucleotides, nucleosides and nucleobasesAn UPLC-TQ-MS/MS method was applied for the detection of the twelve nucleotides, nucleosides and nucleobases. The validated method was successfully applied to identify the 12 analytes in different parts of Abelmoschus manihot harvested at ten growth periods. It turned out that the contents of 12 investigated compounds was in the range between 49.90 and 1901.17 μg/g. The data demonstrated that the distribution and concentration of the 12 compounds in Abelmoschus manihot four parts were arranged in a decreasing order as leaf> flower> stem> root. Typically, the concentrations of nucleobases were much higher than those of nucleosides and nucleotides in all samples. Moreover, thymine, uracil and adenine were found to be the most abundant compounds in most samples.2’-deoxyinosine was not detected in all of the 33 samples. Thymidine and 2’-deoxyadenosine were just contained in early periods of root and stem samples. And 2’-deoxyadenosine-5’-monophosphate and cytidine-5’-monophosphate were absent or at a very low content in different A. manihot samples. Based on the results, the leaves and flowers of A. manihot could be developed as health products possessed nutraceutical and bioactive properties in the future.1.4 The distribution and dynamic accumulation of amino acidsUPLC-TQ-MS/MS has been established for the simultaneous determination of 22 amino acids in different parts harvested at ten growth periods. The results revealed that the ranges of the amino acid contents in Abelmoschus manihot roots, stems, leaves and flowers were from 232.7 to 3829.4 μg/g,334.9 to 925.9μg/g,1214.0 to 4222.6μg/g and 1865.7 to 4461.9 μg/g, respectively. The accumulation of amino acids in Abelmoschus manihot parts above ground occurred mainly from G5 to G8, while their accumulation in the roots happened from G8 to G10, the deciduous periods of Abelmoschus manihot. Moreover, it turned out to be that gaminobutyric acid, glutamine, lysine, proline, asparagine and aspartic acid were the predominant types of total amino acids in all of the non-medicinal parts, and the glutamine, lysine and serine were those of flowers samples. The characterization of amino acids in Abelmoschus manihot roots, stems and leaves would be very helpful for their potential value improvements as food and medicine to provide amino acids.2 Study on the biosynthesis process of flavonoids contained in Abelmoschus manihot2.1 The distribution and dynamic accumulation of precursorsThe determination of phenylalanine and tyrosine was carried out by the method of UPLC-TQ-MS/MS. The results showed that tyrosine had higher levels in all samples than that of phenylalanine. The data also demonstrated that the distribution and concentration of these 2 compounds in Abelmoschus manihot four parts were mostly arranged in a decreasing order as leaf> flower> stem> root, moreover, the contents of leaves and flowers were significantly higher than those of stems and roots. In terms of the different growing periods, the whole change of the contents in roots was not obvious, and both of the two compounds showed the same changing tendency, which was similar to the situation of stems. The contents of two kinds of precursors in leaves were greatly changed. The contents of phenylalanine reached the peak at G7, and then the content decreased, while tyrosine got the peak at G8. Both of the two precursor compounds had the highest levels in leaves, which indicated that the site of flavonoids biosynthesis might happen in the leaf part of Abelmoschus manihot. In addition, it is presumed that after being generated by shikimic acid, the prephenate is mostly converted to tyrosine.2.2 The distribution and dynamic accumulation of intermediates and active componentsA method of UPLC-TQ-MS/MS was established for the simulation of two key intermediate compounds (cinnamic acid and p-hydroxycinnamic acid) and five main flavonoid compositions of Abelmoschus manihot (hyperoside, isoquercitrin, rutin, quercetin and myricetin). The results of the method validation showed that it was feasible to detect the contents of the 7 components in the samples by using the method established in this section. Moreover, cinnamic acid were not detected in all of the 33 samples, and the contents of p-hydroxycinnamic acid in roots and stems were higher than those of leaves and flowers. The contents of flavonoid glycosides in the samples were significantly higher than those of aglycones, and specifically with the order as hyperoside> isoquercitrin> rutin> quercetin> myricetin. The data also demonstrated that the distribution and concentration of these 5 compounds in Abelmoschus manihot four parts were mostly arranged in a decreasing order as flower> leaf> stem> root. The peak of total flavonoids contained in the flowers appeared at the period of G8, and that of the leaves occured at G9-G10. As a result, it is proved that the leaves of Abelmoschus manihot can be used to extract monomer flavonoid extract components in further.2.3 The activities of key enzymes in 33 amples of Abelmoschus manihotThree key enzymes (PAL, C4H and 4CL) of the biosynthesis process of flavonoids were extracted and their enzyme activities were determined according to the related literature. It was found that in addition to the peak 396 U/g at the G7 of the leaf sample, the PAL activities didn’t have significant differences between four parts and ten growing periods with the values of 11 to 118 U/g. Combined with the determination of precursors phenylalanine and intermediates cinnamic acid, it is conjectured that PAL was not the rate limiting enzyme of the biosynthesis process of flavonoids in Abelmoschus manihot. The activities of C4H and 4CL in the samples of leaves were both highest, with the range from 130 to 3490 U/g and 337 to 2268 U/g, respectively. Only three roots samples were detected for 4CL activity. The activities of 4CL in stems and leaves were positively correlated with the contents of p-hydroxycinnamic acid during the early stage of growing periods, and they were negatively correlated after the G5 period. After flowering, the 4CL activity in the leaves significantly increased, while in the medicinal parts flowers,4CL activities were decreased. Therefore, it is suggesting that the Abelmoschus manihot flavonoids synthesis mainly happen in the leaf part and then the active ingredients were transported to the flowers.3 Study on the biotransformation of the extract of non-medicinal parts of Abelmoschus manihot by the plant endophytes3.1 Population structure and ecological distribution of Abelmoschus manihot endophytes and rhizospheric microorganisms in different parts during ten growing periodsThe plant endophyte and rhizospheric microorganisms are the important parts of the plant body, including bacteria, fungi and actinomycetes. From four parts of Abelmoschus manihot during nine growth periods,142 strains of endophytic bacteria,176 strains of endophytic fungi and 118 strains of endophytic actinomycetes were isolated. And from the rhizosphere soil,30 strains of endophytic bacteria,34 strains of endophytic fungi and 45 strains of endophytic actinomycetes were obtained. After integrating the strains with similar morphology,47 strains of fungi,39 strains of bacteria and 24 strains of actinomycetes were acquired from the Abelmoschus manihot samples. 10 strains of fungi,24 strains of bacteria and 13 strains of actinomycetes were gained from the rhizosphere soil samples. And it was found that the leaf samples contained the largest number of plant endophytes. In addition, the number of endophytic bacteria was bigger than that of endophytic fungi at the early growth periods, while was simaller at the flowering and defoliating periods. The total number of plant endophytes and rhizospheric microorganisms showed an increasing trend during the periods of the accumulation of Abelmoschus manihot flavonoids.3.2 Biological transformation of the Abelmoschus manihot non-medicinal parts extract by the isolated endophytes and rhizosphere microorganismsUPLC-Q-TOF/MS and MetabolynxTM software were used to detect and analyse the metabolites produced by the co-culture of the Abelmoschus manihot non-medicinal parts extract and plant microorganisms. The results showed that 15 compounds were detected, and the main metabolic pathways including deglycosylation, methylation, hydroxylation, methoxy methylation, acetylation, oxidation and et al. In addition,12 strains of fungi showed the excellent transformation activity of the extract.3.3 Isolation, purification and identification of the endophytes and rhizosphere microorganisms with the transformation activityThe culture of insert and plate were used to obtain the colony morphology and mycelial microstructure of the 12 strains which showed the excellent transformation activity of the Abelmoschus manihot non-medicinal parts extract. The result indicated that the fungi including 4 strains of Penicillium,3 strain of Aspergillus,2 strain of Oospora,1 strain of Rhizopus,1 strain of Helicostylum, and 1 strain of Mycelia sterlia. |
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