Withania somnifera (L.) Dunal: Enhanced production of withanolides and phenolic acids from hairy root culture after application of elicitors.

Withania somnifera (L.) Dunal is an important indigenous medicinal plant with extensive pharmaceutical potential. The root is the main source of major bioactive compounds of this plant species including withanolides, withanine, phenolic acids, etc. Hairy root culture (HRC) is a crucial method for low-cost production of active compounds on a large scale. Four different Agrobacterium rhizogenes strains have been used for the hairy root induction. Maximum transformation efficiency (87.34 ± 2.13%) was achieved with A4 bacterial strain-mediated transformed culture. The genetic transformation was confirmed by using specific primers of seven different genes. Seven HR (Hairy root) lines were selected after screening 29 HR lines based on their fast growth rate and high accumulation of withanolides and phenolic acids content. Two biotic and three abiotic elicitors were applied to the elite root line to trigger more accumulation of withanolides and phenolic acids. While all the elicitors effectively increased withanolides and phenolic acids production, among the five different elicitors, salicylic acid (4.14 mg l-1) induced 11.49 -fold increase in withanolides (89.07 ± 2.75 mg g-1 DW) and 5.34- fold increase in phenolic acids (83.69 ± 3.11 mg g- 1 DW) after 5 days of elicitation compared to the non-elicited culture (7.75 ± 0.63 mg g-1 DW of withanolides and 15.66 ± 0.92 mg g-1 DW of phenolic acids). These results suggest that elicitors can tremendously increase the biosynthesis of active compounds in this system; thus, the HRC of W. somnifera is cost-effective and can be efficiently used for the industrial production of withanolides and phenolic acids.

Identification and characterization of a novel tyrosine aminotransferase gene (SmTAT3-2) promotes the biosynthesis of phenolic acids in Salvia miltiorrhiza Bunge.

Rosmarinic acid (RA) and salvianolic acid B (SAB) are main phenolic acids in Salvia miltiorrhiza Bunge have been widely used in the treatment of cardiovascular and cerebrovascular diseases due to their excellent pharmacological activity. RA is a precursor of SAB, and tyrosine transaminase (TAT, EC 2.6.1.5) is a crucial rate-limiting enzyme in their metabolism pathway. This study identified a novel TAT gene, SmTAT3-2, and found that it is a new transcript derived from unconventional splicing of SmTAT3. We used different substrates for enzymatic reaction with SmTAT1, SmTAT3 and SmTAT3-2. Subcellular localization of SmTAT1 and SmTAT3-2 was completed based on submicroscopic techniques. In addition, they were overexpressed and CRISPR/Cas9 gene edited in hairy roots of S. miltiorrhiza. Revealed SmTAT3-2 and SmTAT1 showed a stronger affinity for L-tyrosine than SmTAT3, localized in the cytoplasm, and promoted the synthesis of phenolic acid. In overexpressed SmTAT3-2 hairy roots, the content of RA and SAB was significantly increased by 2.53 and 3.38 fold, respectively, which was significantly higher than that of overexpressed SmTAT1 strain compared with EV strain. These findings provide a valuable key enzyme gene for the phenolic acids metabolism pathway and offer a theoretical basis for the clinical application.

SmJAZ4 interacts with SmMYB111 or SmMYC2 to inhibit the synthesis of phenolic acids in Salvia miltiorrhiza.

Jasmonic acid (JA), as an important plant hormone, can induce the synthesis of phenolic acids in Salvia miltiorrhiza Bunge, a model medicinal plant, but the specific mechanism remains to be further elucidated. JA-responsive SmMYB111 positively regulates the biosynthesis of salvianolic acid B (SalB), but the molecular mechanism is unclear. Here, we found that SmMYB111 directly binds to the promoters of SmTAT1 and SmCYP98A14 and activates their transcription. Yeast two hybrid and bimolecular fluorescent complementation assay indicated that SmMYB111 interacts with SmJAZ4. Furthermore, we systematically characterized the function of SmJAZ4, which was highly expressed in flowers and roots and located in the nucleus and cell membrane. The contents of phenolic acids in the SmJAZ4-overexpressed transgenic plantlets and SmJAZ4-overexpressed transgenic hairy roots decreased significantly. SmJAZ4 interacts with SmMYC2 or SmMYB111 to repress their transcriptional activation activity on target enzyme genes of the biosynthesis pathway of phenolic acids. Overall, the molecular mechanism of SmJAZ4-SmMYC2/SmMYB111 module participating in JA signaling regulation of SalB biosynthesis was elucidated, which give a clue for the molecular regulation of phenolic acids biosynthesis in S. miltiorrhiza.

MAPKK2/4/5/7-MAPK3-JAZs modulate phenolic acid biosynthesis in Salvia miltiorrhiza.

Phenolic acids are the major bioactive metabolites produced in Salvia miltiorrhiza, a traditional Chinese medicine called Danshen. Many phytohormone elicitor treatments induce phenolic acid biosynthesis, even though the underlying mechanism remains obscure. Expression pattern analysis showed that SmMAPK3 was highly expressed in leaves, and SmMAPK3 was significantly induced by salicylic acid (SA) and methyl jasmonate (JA). Bioinformatics analysis revealed that SmMAPK3 belongs to group A and contains a TEY motif in the activation loop together with three conserved regions (P-loop, C-loop and CD-domain). A previous study speculated that SmMAPK3 is likely a positive regulator in the biosynthesis of phenolic acids in S. miltiorrhiza. In this study, overexpression of SmMAPK3 increased phenolic acid biosynthetic gene expression and enhanced the accumulation of phenolic acids in S. miltiorrhiza plantlets. Yeast two-hybrid (Y2H) analysis and firefly luciferase complementation imaging (LCI) assays revealed that SmMAPKK2/4/5/7-SmMAPK3-SmJAZs form a cascade that regulates the accumulation of phenolic acids. In summary, this work deepens our understanding of the posttranscriptional regulatory mechanisms of phenolic acid biosynthesis and sheds new light on metabolic engineering in S. miltiorrhiza.

Research progress on intervention effect and mechanism of protocatechuic acid on nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) has become a surge burden worldwide due to its high prevalence, with complicated deterioration symptoms such as liver fibrosis and cancer. No effective drugs are available for NALFD so far. The rapid growth of clinical demand has prompted the treatment of NAFLD to become a research hotspot. Protocatechuic acid (PCA) is a natural secondary metabolite commonly found in fruits, vegetables, grains, and herbal medicine. It is also the major internal metabolites of anthocyanins and other polyphenols. In the present manuscript, food sources, metabolic absorption, and efficacy of PCA were summarized while analyzing its role in improving NAFLD, as well as the mechanism involved. The results indicated that PCA could ameliorate NAFLD by regulating glucose and lipid metabolism, oxidative stress and inflammation, gut microbiota and metabolites. It was proposed for the first time that PCA might reduce NAFLD by enhancing the energy consumption of brown adipose tissue (BAT). However, the PCA administration mode and dose for NAFLD remain inconclusive. Fresh insights into the specific molecular mechanisms are required, while clinical trials are essential in the future. This review provides new targets and reasoning for the clinical application of PCA in the prevention and treatment of NAFLD.

Effects of various artificial agarwood-induction techniques on the metabolome of Aquilaria sinensis.

BACKGROUND: Agarwood is a highly sought-after resinous wood for uses in medicine, incense, and perfume production. To overcome challenges associated with agarwood production in Aquilaria sinensis, several artificial agarwood-induction treatments have been developed. However, the effects of these techniques on the metabolome of the treated wood samples are unknown. Therefore, the present study was conducted to evaluate the effects of four treatments: fire drill treatment (F), fire drill + brine treatment (FS), cold drill treatment (D) and cold drill + brine treatment (DS)) on ethanol-extracted oil content and metabolome profiles of treated wood samples from A. sinensis. RESULTS: The ethanol-extracted oil content obtained from the four treatments differed significantly (F < D < DS < FS). A total of 712 metabolites composed mostly of alkaloids, amino acids and derivatives, flavonoids, lipids, phenolic acids, organic acids, nucleotides and derivatives, and terpenoids were detected. In pairwise comparisons, 302, 155, 271 and 363 differentially accumulated metabolites (DAM) were detected in F_vs_FS, D_vs_DS, F_vs_D and FS_vs_DS, respectively. The DAMs were enriched in flavonoid/flavone and flavonol biosynthesis, sesquiterpenoid and triterpenoid biosynthesis. Generally, addition of brine to either fire or cold drill treatments reduced the abundance of most of the metabolites. CONCLUSION: The results from this study offer valuable insights into synthetically-induced agarwood production in A. sinensis.

Borate and phosphite treatments of potato plants (Solanum tuberosum L.) as a proof of concept to reinforce the cell wall structure and reduce starch digestibility.

Potatoes are one of the main sources of carbohydrates in human diet, however they have a high glycaemic index (GI). Hence, developing new agricultural and industrial strategies to produce low GI potatoes represents a health priority to prevent obesity and related diseases. In this work, we investigated whether treatments of potato plants with elicitors of plant defence responses can lead to a reduction of tuber starch availability and digestibility, through the induction of cell wall remodelling and stiffening. Treatments with phosphites (KPhi) and borate were performed, as they are known to activate plant defence responses that cause modifications in the architecture and composition of the plant cell wall. Data of suberin autofluorescence demonstrated that potato plants grown in a nutrition medium supplemented with KPhi and borate produced tubers with a thicker periderm, while pectin staining demonstrated that KPhi treatment induced a reinforcement of the wall of storage parenchyma cells. Both compounds elicited the production of H2O2, which is usually involved in cell-wall remodelling and stiffening reactions while only KPhi caused an increase of the total content of phenolic compounds. A two-phase digestion in vitro assay showed that treatment with KPhi determined a significant decrease of the starch hydrolysis rate in potato tubers. This work highlights the ability of cell wall architecture in modulating starch accessibility to digestive enzymes, paving the way for new agronomic practices to produce low GI index potatoes.

Phenylalanine Increases the Production of Antioxidant Phenolic Acids in Ginkgo biloba Cell Cultures.

The aims of this study were to evaluate the antioxidant properties, to investigate the content of major secondary metabolites in Ginkgo biloba cell cultures, and to determine the change in the production of phenolic acids by adding phenylalanine to the culture medium. Three in vitro methods, which depend on different mechanisms, were used for assessing the antioxidant activity of the extract: 1,1-diphenyl-2-picrylhydrazil (DPPH), reducing power and Fe2+ chelating activity assays. The extract showed moderate activity both in the DPPH and in the reducing power assays (IC50 = 1.966 ± 0.058 mg/mL; ASE/mL = 16.31 ± 1.20); instead, it was found to possess good chelating properties reaching approximately 70% activity at the highest tested dose. The total phenolic, total flavonoid, and condensed tannin content of G. biloba cell culture extract was spectrophotometrically determined. The phenolic acid content was investigated by RP-HPLC, and the major metabolites-protocatechuic and p-hydroxybenzoic acids-were isolated and investigated by 1H NMR. The results showed that phenylalanine added to G. biloba cell cultures at concentrations of 100, 150, and 200 mg/150 mL increased the production of phenolic acids. Cultures that were grown for 3 weeks and collected after 4 days of phenylalanine supplementation at high concentration showed maximal content of phenolic acids (73.76 mg/100 g DW).

Metabolic and process engineering for microbial production of protocatechuate with Corynebacterium glutamicum.

3,4-Dihydroxybenzoate (protocatechuate, PCA) is a phenolic compound naturally found in edible vegetables and medicinal herbs. PCA is of high interest in the chemical industry and has wide potential for pharmaceutical applications. We designed and constructed a novel Corynebacterium glutamicum strain to enable the efficient utilization of d-xylose for microbial production of PCA. Shake flask cultivation of the engineered strain showed a maximum PCA titer of 62.1 ± 12.1 mM (9.6 ± 1.9 g L-1 ) from d-xylose as the primary carbon and energy source. The corresponding yield was 0.33 C-mol PCA per C-mol d-xylose, which corresponds to 38% of the maximum theoretical yield. Under growth-decoupled bioreactor conditions, a comparable PCA titer and a total amount of 16.5 ± 1.1 g PCA could be achieved when d-glucose and d-xylose were combined as orthogonal carbon substrates for biocatalyst provision and product synthesis, respectively. Downstream processing of PCA was realized via electrochemically induced crystallization by taking advantage of the pH-dependent properties of PCA. This resulted in a maximum final purity of 95.4%. The established PCA production process represents a highly sustainable approach, which will serve as a blueprint for the bio-based production of other hydroxybenzoic acids from alternative sugar feedstocks.

Physiological and transcriptomic analyses to reveal underlying phenolic acid action in consecutive monoculture problem of Polygonatum odoratum.

BACKGROUND: The root rot of fragrant solomonseal (Polygonatum odoratum) has occurred frequently in the traditional P. odoratum cultivating areas in recent years, causing a heavy loss in yield and quality. The phenolic acids in soil, which are the exudates from the P. odoratum root, act as allelochemicals that contribute to the consecutive monoculture problem (CMP) of the medicinal plant. The aim of this study was to get a better understanding of P. odoratum CMP. RESULTS: The phenolic acid contents, the nutrient chemical contents, and the enzyme activities related to the soil nutrient metabolism in the first cropping (FC) soil and continuous cropping (CC) soil were determined, and the differentially expressed genes (DEGs) related to the regulation of the phenolic acids in roots were analyzed. The results showed that five low-molecule-weight phenolic acids were detected both in the CC soil and FC soil, but the phenolic acid contents in the CC soil were significantly higher than those in the FC soil except vanillic acid. The contents of the available nitrogen, available phosphorus, and available potassium in the CC soil were significantly decreased, and the activities of urease and sucrase in the CC soil were significantly decreased. The genomic analysis showed that the phenolic acid anabolism in P. odoratum in the CC soil was promoted. These results indicated that the phenolic acids were accumulated in the CC soil, the nutrient condition in the CC soil deteriorated, and the nitrogen metabolism and sugar catabolism of the CC soil were lowered. Meantime, the anabolism of phenolic acids was increased in the CC plant. CONCLUSIONS: The CC system promoted the phenolic acid anabolism in P. odoratum and made phenolic acids accumulate in the soil.

SmSPL6 Induces Phenolic Acid Biosynthesis and Affects Root Development in Salvia miltiorrhiza.

Salvia miltiorrhiza is a renowned model medicinal plant species for which 15 SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) family genes have been identified; however, the specific functions of SmSPLs have not been well characterized as of yet. For this study, the expression patterns of SmSPL6 were determined through its responses to treatments of exogenous hormones, including indole acetic acid (IAA), gibberellic acid (GA3), methyl jasmonic acid (MeJA), and abscisic acid (ABA). To characterize its functionality, we obtained SmSPL6-ovexpressed transgenic S. miltiorrhiza plants and found that overexpressed SmSPL6 promoted the accumulation of phenolic acids and repressed the biosynthesis of anthocyanin. Meanwhile, the root lengths of the SmSPL6-overexpressed lines were significantly longer than the control; however, both the fresh weights and lateral root numbers decreased. Further investigations indicated that SmSPL6 regulated the biosynthesis of phenolic acid by directly binding to the promoter regions of the enzyme genes Sm4CL9 and SmCYP98A14 and activated their expression. We concluded that SmSPL6 regulates not only the biosynthesis of phenolic acids, but also the development of roots in S. miltiorrhiza.

Transcription factor SmSPL7 promotes anthocyanin accumulation and negatively regulates phenolic acid biosynthesis in Salvia miltiorrhiza.

Plant-specific SQUAMOSA promoter-binding protein-like (SPL) transcription factors play critical regulatory roles during plant growth and development. However, the functions of SPLs in Salvia miltiorrhiza (SmSPLs; a model medicinal plant) have not been reported. Here, the expression patterns and functions of SmSPL7 were characterized in S. miltiorrhiza. SmSPL7 was expressed in all parts of S. miltiorrhiza, with the highest expression level in the leaves, and could be inhibited by multiple hormones, including methyl jasmonate, auxin, abscisic acid, and gibberellin. SmSPL7 is localized within the nucleus and exhibits robust transcriptional activation activity. Transgenic lines overexpressing SmSPL7 demonstrated pronounced growth inhibition, accompanied by increased anthocyanin accumulation via the genetic activation of the anthocyanin biosynthesis pathway. However, SmSPL7 overexpression significantly decreased salvianolic acid B (SalB) production by inhibiting the transcripts of genes implicated in its biosynthesis pathway. Further analysis indicated that SmSPL7 directly binds to SmTAT1 and Sm4CL9 promoters and blocks their expression to inhibit the biosynthesis of SalB. Taken together, these results indicate that SmSPL7 is a negative regulator of SalB biosynthesis but positively regulates anthocyanin accumulation in S. miltiorrhiza. These findings provide new insights into the functionality of the SPL family while establishing an important foundation for further uncovering the crucial roles of SmSPL7 in the growth of S. miltiorrhiza.

Enzymatic Synthesis of Lipophilic Esters of Phenolic Compounds, Evaluation of Their Antioxidant Activity and Effect on the Oxidative Stability of Selected Oils.

The aim of the study was to compare the effect of the substituent and its position in the aromatic ring on the antioxidant activity of hexanoic acid esters obtained in reactions catalyzed by immobilized lipase B from Candida antarctica. 4-Hydroxybenzyl hexanoate, 2-hydroxybenzyl hexanoate, 4-methoxybenzyl hexanoate, and vanillyl hexanoate were obtained with conversion yields of 50 to 80%. The antioxidant activity of synthesized esters, their alcohol precursors and BHT (Butylated HydroxyToluene) was compared with DPPH (2,2-diphenyl-1-picrylhydrazyl), CUPRAC (cupric ion reducing antioxidant capacity), and CBA (crocin bleaching assay) methods. Furthermore, it was investigated whether the presence of vanillyl hexanoate in a concentration of 0.01 and 0.1% affected the oxidative stability of sunflower and rapeseed oils in the Rancimat test. It was observed that the antioxidant activity of hexanoic acid esters depends on the presence and position of the hydroxyl group in the aromatic ring. The highest activities were found for vanillyl alcohol, vanillyl hexanoate, and BHT. The addition of the ester and BHT significantly extended the induction times of the tested oils, and these compounds exhibited similar activity. Vanillyl hexanoate increased the induction time from 4.49 to 5.28 h and from 2.73 to 3.12 h in the case of rapeseed and sunflower oils, respectively.

Indole primes defence signalling and increases herbivore resistance in tea plants.

Upon herbivore attack, plants emit herbivore-induced plant volatiles (HIPVs). HIPVs can prime defences and resistance of intact plants. However, how HIPVs are decoded and translated into functional defence responses is not well understood, especially in long-lived woody plants. Here, we investigated the impact of the aromatic HIPV indole on defence-related early signalling, phytohormone accumulation, secondary metabolite biosynthesis and herbivore resistance in tea plants. We find that tea plants infested with tea geometrid caterpillars release indole at concentrations >450 ng*hr-1 . Exposure to corresponding doses of synthetic indole primes the expression of early defence genes involved in calcium (Ca2+ ) signalling, MPK signalling and jasmonate biosynthesis. Indole exposure also primes the production of jasmonates and defence-related secondary metabolites. These changes are associated with higher herbivore resistance of indole-exposed tea plants. Chemical inhibition of Ca2+ and jasmonate signalling provides evidence that both are required for indole-mediated defence priming and herbivore resistance. Our systematic assessment of the impact of indole on defence signalling and deployment shows that indole acts by boosting Ca2+ signalling, resulting in enhanced jasmonate-dependent defence and resistance in a woody plant. Our work extends the molecular basis of HIPV-induced defence priming from annual plants to an economically important tree species.

Metabolomics characterizes the metabolic changes of Lonicerae Japonicae Flos under different salt stresses.

Salt stress affects the metabolic homeostasis of medicinal plants. However, medicinal plants are sessile organisms that cannot escape from salt stress. They acclimatize themselves to the stress by reprogramming their metabolic pathways. Lonicerae Japonicae Flos (LJF) with strong antioxidant activity is commonly used in traditional Chinese medicine, tea, and beverage. Nevertheless, the variation of integrated metabolites in LJF under different salt stresses remains unclear. In this study, High Performance Liquid Chromatography tandem triple time-of-flight mass spectrometry (HPLC- triple TOF-MS/MS) coupled with multivariate statistical analysis was applied to comparatively investigate the metabolites changes in LJF under different salt stress (0, 100, 200, 300 mM NaCl). Total 47 differential metabolites were screened from 79 metabolites identified in LJF under different salt stress. Low salt-treated group (100 mM NaCl) appeared to be the best group in terms of relative contents (peak areas) of the wide variety in bioactive components. Additionally, the phenylpropanoid pathway, monoterpenoid biosynthesis, glycolysis, TCA cycle, and alkaloid biosynthesis were disturbed in all salt-stress LJF. The results showed that LJF metabolisms were dramatically induced under salt stress and the quality of LJF was better under low salt stress. The study provides novel insights into the quality assessment of LJF under salt stress and a beneficial framework of knowledge applied to improvement the medicinal value of LJF.

Valorization of Gleditsia triacanthos Invasive Plant Cellulose Microfibers and Phenolic Compounds for Obtaining Multi-Functional Wound Dressings with Antimicrobial and Antioxidant Properties.

Gleditsia triacanthos is an aggressive invasive species in Eastern Europe, producing a significant number of pods that could represent an inexhaustible resource of raw material for various applications. The aim of this study was to extract cellulose from the Gleditsia triacanthos pods, characterize it by spectrophotometric and UHPLC-DAD-ESI/MS analysis, and use it to fabricate a wound dressing that is multi-functionalized with phenolic compounds extracted from the leaves of the same species. The obtained cellulose microfibers (CM) were functionalized, lyophilized, and characterized by ATR-FTIR and SEM. The water absorption and retention capacity as well as the controlled release of phenolic compounds with antioxidant properties evaluated in temporal dynamics were also determined. The antimicrobial activity against reference and clinical multi-drug-resistant Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterobacter cloacae, Candida albicans, and Candida parapsilosis strains occurred immediately after the contact with the tested materials and was maintained for 24 h for all tested microbial strains. In conclusion, the multi-functionalized cellulose microfibers (MFCM) obtained from the reproductive organs of an invasive species can represent a promising alternative for the development of functional wound dressings with antioxidant and antimicrobial activity, as well as being a scalable example for designing cost-effective, circular bio-economy approaches to combat the accelerated spread of invasive species.

The gut microbiome drives inter- and intra-individual differences in metabolism of bioactive small molecules.

The origin of inter-individual variability in the action of bioactive small molecules from the diet is poorly understood and poses a substantial obstacle to harnessing their potential for attenuating disease risk. Epidemiological studies show that coffee lowers the risk of developing type 2 diabetes, independently of caffeine, but since coffee is a complex matrix, consumption gives rise to different classes of metabolites in vivo which in turn can affect multiple related pathways in disease development. We quantified key urinary coffee phenolic acid metabolites repeated three times in 36 volunteers, and observed the highest inter- and intra-individual variation for metabolites produced by the colonic microbiome. Notably, a urinary phenolic metabolite not requiring the action of the microbiota was positively correlated with fasting plasma insulin. These data highlight the role of the gut microbiota as the main driver of both intra- and inter-individual variation in metabolism of dietary bioactive small molecules.

Moderate Wine Consumption Reduces Faecal Water Cytotoxicity in Healthy Volunteers.

There are some studies that suggest that moderate consumption of wine, as part of a healthy and balanced diet, has a favourable effect on intestinal health. This study evaluates the effect of moderate wine consumption on faecal water (FW) cytotoxicity as a parameter of gut health. To that end, faecal samples before and after a red wine intervention study (250 mL of wine/day, 4 weeks) in healthy volunteers (n = 8) and in a parallel control group (n = 3) were collected and assayed for in vitro FW cytotoxicity. Two reference compounds, phenol and p-cresol, were used for assessing the cytotoxicity assays using two colon epithelial cell lines (HT-29 and HCT 116) and different assay conditions (FW dilution and incubation time). For the two cell lines and all assay conditions, the means of percentage cell viability were higher (lower cytotoxicity) for samples collected after the red wine intervention than for those collected before, although significant (p < 0.05) differences were only found in certain assay conditions for both cell lines. Significant positive correlations between the percentage cell viability and the contents of some faecal metabolites (short-chain fatty acids (SCFA) and phenolic acids (PA)) were found for the more resistant cell line (HCT 116), suggesting that the reduction in FW cytotoxicity observed after moderate red wine consumption was related to the production of microbial-derived metabolites such as SCFA and PA, whose faecal contents have been shown to increase after wine consumption. FW cytotoxicity can be deemed as a holistic biomarker that involves diet, gut microbiota and host.

Identification of the Metabolites in Rat Urine after Oral Administration and Elucidation of the Metabolic Process of Naozhenning Granule Using LC-MS.

Naozhenning (NZN) granule, a Chinese herbal formula, is widely used to treat craniocerebral trauma and promote functional recovery. In our previous study, the chemical components, as well as the serum metabolites in the male Sprague-Dawley rats of the NZN granule after oral administration were characterized. In this study, the urine metabolites in the male Sprague-Dawley rats were further investigated by ultrahigh-performance liquid chromatography-Q Exactive hybrid quadrupole-Orbitrap high-resolution accurate mass spectrometry. In order to identify the urine metabolites comprehensively, three sample preparation methods were used, including solid-phase extraction, protein precipitation method and solvent partition. Based on the accurate molecular weight and the fragmentation information from the MS spectra, a total of 76 urine metabolites were identified, which including 17 prototypes and 59 metabolites. The results showed that the detected urine metabolites were different for the different pretreatment methods, as some metabolites could only be detected in the particular pretreatment method. In addition, the metabolic processes of the components from NZN granule to the serum and urine were also elucidated and discussed. The results will provide useful information for further studying the relationship between the chemical components and pharmacological activity of NZN granule.

The Content of Phenolic Acids and Flavonols in the Leaves of Nine Varieties of Sweet Potatoes (Ipomoea batatas L.) Depending on Their Development, Grown in Central Europe.

The aim of the study was the qualitative and quantitative analysis of the bioactive components present in the leaves of 9 sweet potato cultivars grown in the moderate climate in Poland, which were harvested at different growth stages according to the BBCH (Biologische Bundesanstalt, Bundessortenamt und Chemische Industrie) scale (14, 51, 89). It was found that sweet potato leaves contained 7 polyphenolic compounds, including 5 chlorogenic acids-neochlorogenic acid (5-CQA), chlorogenic acid (3-CQA), 4-cryptochlorogenic acid (4-CQA), 34-di-O-caffeoylqunic acid (3,4-CQA), 3,5-di-O-caffeoylqunic acid (3,5-CQA)-and 2 flavonoids, quercetin-3-O-galactoside (Q-3-GA) and quercetin-3-O-glucoside (Q-3-GL). Their content depended on the genotype of the examined cultivars and on the stage of leaf development. The mean content of the identified polyphenolic compounds in the examined cultivars ranged from 148.2 to 14.038.6 mg/100 g-1 DM for the leaves harvested at growth stage 14 according to the BBCH scale. In the case of leaves harvested at BBCH stage 51, the concentration of polyphenolic compounds ranged from 144.76 to 5026.8 mg/100 g-1 DM and at BBCH stage 89 from 4078.1 to 11.183.5 mg/100 g-1 DM. The leaves of the Carmen Rubin cultivar collected at stage 14 contained the highest amount of polyphenolic compounds, while Okinava leaves had the highest amount of these compounds at stage 51. The highest content of polyphenolic compounds in leaves at BBCH growth stage 89 was found in the Radiosa variety. The highest concentration levels were found for 3-CQA at all stages of leaf development. Significant correlations between polyphenol content and antioxidant activity measured by 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and ferric reducing/antioxidant power (FRAP) were found. The results of this experiment revealed that the growth stages and genetic properties of cultivars have a very significant influence on the content of phenolic acids and flavonols in sweet potato leaves. The results are innovative and can have a practical application, as the knowledge of the content of the substances under study makes it possible to determine the optimal management practice of sweet potato leaf harvest in order to obtain more top-quality raw material.