Flavonoids contribute most to discriminating aged Guang Chenpi (Citrus reticulata 'Chachi') by spectrum-effect relationship analysis between LC-Q-Orbitrap/MS fingerprint and ameliorating spleen deficiency activity.

To further explore the mechanism of "the longer storage time, the better bioactivity" of aged Guang Chenpi, the dry pericarp of Citrus reticulata 'Chachi' (CRC), a series of activity assessments were performed on spleen deficiency mice. The constituents in CRC with different storage years were analyzed by LC-Q-Orbitrap/MS. A total of 53 compounds were identified, and CRC stored for more than 5 years showed higher flavonoid content, especially that of polymethoxyflavones. Anti-spleen deficiency bioactivity analysis among various CRC with different storage years showed aged CRC (stored for more than 3 years) could significantly alleviate fatigue and depression behaviors much better, increase D-xylose and gastrin secretion, and upregulate the expression of the linking protein occludin in the colon walls. Results from 16S rDNA sequencing showed that aged CRC could downregulate the abundance of Enterococcus, Gemmata, Citrobacter, Escherichia_Shigella, and Klebsiella, which were significantly overrepresented in the model group. Bacteroides, Muribaculum, Alloprevotella, Paraprevotella, Alistipes, Eisenbergiella, and Colidextribacter were downregulated in the model group but enriched in the CRC groups. At last, the spectrum-effect relationship analysis indicated that flavonoids such as citrusin III, homoeriodictyol, hesperidin, nobiletin, and isosinensetin in aged CRC showed the highest correlation with better activity in ameliorating spleen deficiency by regulating gut microbiota. Flavonoids contribute most to discriminating aged CRC and could disclose the basis of "the longer storage time, the better bioactivity" of aged Guang Chenpi.

Combined analysis of plasma metabolome and intestinal microbiome sequencing to explore jiashen prescription and its potential role in changing intestine-heart axis and effect on chronic heart failure.

Background: Heart failure (HF) is a syndrome with global clinical and socioeconomic burden worldwide owing to its poor prognosis. Jiashen Prescription (JSP), a traditional Chinese medicine (TCM) formula, exhibits unambiguous effects on treating HF. Previously, we have reported that underlying mechanisms of JSP by an untargeted metabolomics approach, but the contribution of gut microbiota and metabolic interaction to the cardioprotective efficacy of JSP remains to be elucidated. Materials and methods: Firstly, the rat model of heart failure was established by the permanent ligation of the left anterior descending coronary artery. The efficacy evaluation of JSP in treating HF rats was per-formed by left ventricular ejection fraction (LVEF). Then, 16S rRNA gene sequencing and LC/MS-based metabolomic analysis were utilized to explore the characteristics of cecal-contents microecology and plasma metabolic profile, respectively. After that, the correlation between intestinal micro-ecological characteristics and plasma metabolic characteristics was analyzed to explore the potential mechanism of the JSP treatment in HF. Results: JSP could improve the cardiac function of heart failure rats and thus ameliorate heart failure via enhancing rat LVEF. Results of intestinal flora analysis revealed that JSP not only adjusted gut microbiota disturbances by enriching species diversity, reducing the abundance of pathogenic bacteria (such as Allobaculum, Brevinema), as well as increasing the abundance of beneficial bacteria (such as Lactobacillus, Lachnospiraceae_NK4A136_group), but also improved metabolic disorders by reversing metabolite plasma levels to normality. Through the conjoint analysis of 8 metabolites and the OTUs relative abundance data in the 16srRNA sequencing results by WGCNA method, 215 floras significantly related to the eight compounds were identified. The results of the correlation analysis demonstrated a significant association between intestinal microbiota and plasma metabolic profile, especially the significant correlation of Ruminococcaceae_UCG-014 and Protoporphyrin IX, Ruminococcaceae_UCG-005, Christensenellaceae_R-7_group and nicotinamide, dihydrofolic acid. Conclusion: The present study illustrated the underlying mechanism of JSP to treat heart failure by affecting intestinal flora and plasma metabolites, provide a potential therapeutic strategy against heart failure.

[Effects of drought stress on physiological and biochemical and chemical components of Cinnamomum cassia seedlings].

Six month old Cinnamomum cassia seedlings were used to simulate drought stress with polyethylene glycol(PEG 6000). The physiological indicators(osmotic substances, antioxidant enzymes, etc.) and chemical components of seedlings under different drought levels and the correlation between the two were studied. The results showed that the chlorophyll content and relative water content decreased gradually with the increase of PGE 6000(0, 5%, 10%, 15%) concentration and time(3, 5, 7 d), while the soluble protein content, soluble sugar content and catalase(CAT) activity increased, but the rising rate slowed down with the time. The activities of peroxidase(POD), superoxide dismutase(SOD), malondialdehyde(MDA) and proline content increased at first and then decreased. The content of coumarin, cinnamaldehyde, cinnamic acid and dimethoxycinnamaldehyde decreased, while the content of cinnamyl alcohol continued to increase.Under drought stress, the fluorescence signals of reactive oxygen species and no contents in roots of C. cassia seedlings were significantly stronger than those of the control.Further correlation analysis showed that coumarin content, di-methoxycinnamaldehyde content and osmoregulation substance content were significantly negatively correlated(P<0.05), cinnamic acid content was significantly negatively correlated with POD and SOD activities(P<0.01).It was found that C. cassia seedlings showed a certain degree of drought tolerance under short-term or mild drought stress, but if the drought exceeded a certain degree, the physiological metabolism of the seedlings would be unbalanced.

Characterizing microRNAs and their targets in different organs of Camellia sinensis var. assamica.

To comprehensively annotate miRNAs and their targets in tea plant, Camellia sinensis, we sequenced small and messenger RNAs of 9 samples of Camellia sinensis var. assamica (YK-10), a diploid elite cultivar widely grown in southwest China. In order to identify targets of miRNAs, we sequenced two degradome sequencing profiles from leaves and roots of YK-10, respectively. By analyzing the small RNA-Seq profiles, we newly identified 137 conserved miRNAs and 23 species specific miRNAs in the genome of YK-10, which significantly improved the annotation of miRNAs in tea plant. Approximately 2000 differently expressed genes were identified when comparing RNA-Seq profiles of any two of the three organs selected in the study. Totally, more than 5000 targets of conserved miRNAs were identified in the two degradome profiles. Furthermore, our results suggest that a few miRNAs play roles in the biosynthesis pathways of theanine, caffeine and flavonoid. These results enhance our understanding of small RNA guided gene regulations in different organs of tea plant.

Optimization of Flavonoids Extraction Process in Panax notoginseng Stem Leaf and a Study of Antioxidant Activity and Its Effects on Mouse Melanoma B16 Cells.

The Panax notoginseng (P. notoginseng) stem leaf is rich in flavonoids. However, because of a lack of research on the flavonoid extraction process and functional development of P. notoginseng stem leaf, these parts are discarded as agricultural wastes. Therefore, in this study, we intend to optimize the extraction process and develop the skin-whitening functions of P. notoginseng stem leaf extracts. The extraction process of the stem and leaf of P. notoginseng flavonoid (SLPF) is optimized based on the Box⁻Behnken design (BBD) and the response surface methodology (RSM). The optimum extraction conditions of the SLPF are as follows: the extraction time, the ethanol concentration, the sodium dodecyl sulfate (SDS) content and the liquid material ratio (v/w, which are 52 min, 48.7%, 1.9%, and 20:1, respectively. Under the optimal extraction conditions, the average total SLPF content is 2.10%. The antioxidant activity and anti-deposition of melanin of mouse B16 cells of P. notoginseng stem leaf extracts are studied. The results indicate that the EC50 values of reducing activity, 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activities, the superoxide anion removal ability, and the 2,2-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) free radical removal ability are 7.212, 2.893, 2.949, and 0.855 mg/mL, respectively. The extracts IC50 values of the tyrosinase and melanin synthesis are 0.045 and 0.046 mg/mL, respectively. Therefore, the optimal processing technology for the SLPF obtained in this study not only increases its utilization rate, but also decreases material costs. The extracts from the P. notoginseng stem leaf may be developed as food or beauty products.

Phased secondary small interfering RNAs in Panaxnotoginseng.

BACKGROUND: Recent results demonstrated that either non-coding or coding genes generate phased secondary small interfering RNAs (phasiRNAs) guided by specific miRNAs. Till now, there is no studies for phasiRNAs in Panax notoginseng (Burk.) F.H. Chen (P. notoginseng), an important traditional Chinese herbal medicinal plant species. METHODS: Here we performed a genome-wide discovery of phasiRNAs and its host PHAS loci in P. notoginseng by analyzing small RNA sequencing profiles. Degradome sequencing profile was used to identify the trigger miRNAs of these phasiRNAs and potential targets of phasiRNAs. We also used RLM 5'-RACE to validate some of the identified phasiRNA targets. RESULTS: After analyzing 24 small RNA sequencing profiles of P. notoginseng, 204 and 90 PHAS loci that encoded 21 and 24 nucleotide (nt) phasiRNAs, respectively, were identified. Furthermore, we found that phasiRNAs produced from some pentatricopeptide repeat-contain (PPR) genes target another layer of PPR genes as validated by both the degradome sequencing profile and RLM 5'-RACE analysis. We also found that miR171 with 21 nt triggers the generations of 21 nt phasiRNAs from its conserved targets. CONCLUSIONS: We validated that some phasiRNAs generated from PPRs and TASL genes are functional by targeting other PPRs in trans. These results provide the first set of PHAS loci and phasiRNAs in P. notoginseng, and enhance our understanding of PHAS in plants.

Small RNA profiles from Panax notoginseng roots differing in sizes reveal correlation between miR156 abundances and root biomass levels.

Plant genomes encode several classes of small regulatory RNAs (sRNAs) that play critical roles in both development and stress responses. Panax notoginseng (Burk.) F.H. Chen (P. notoginseng) is an important traditional Chinese herbal medicinal plant species for its haemostatic effects. Therefore, the root yield of P. notoginseng is a major economically important trait since the roots of P. notoginseng are the parts used to produce medicine. To identify sRNAs that are critical for the root biomass of P. notoginseng, we performed a comprehensive study of miRNA transcriptomes from P. notoginseng roots of different biomasses. We identified 675 conserved miRNAs, of which 180 pre-miRNAs are also identified, and three TAS3 loci in P. notoginseng. By using degradome sequencing, we identified 79 conserved miRNA:target or tasiRNA:target interactions, of which eight were further confirmed with the RLM 5'-RACE experiments. More importantly, our results revealed that a member of miR156 family and one of its SPL target genes have inverse expression levels, which is tightly correlated with greater root biomass contents. These results not only contributes to overall understanding of post-transcriptional gene regulation in roots of P. notoginseng but also could serve as markers for breeding P. notoginseng with greater root yield.

[Effect of different water conditions on Panax notoginseng seeds after-ripening and germination physiology].

Effect of different water conditions on the physiological indexes (e.g.seed water content, vigor, antioxidase activities)of Panax notoginseng seeds were studied under process of after-ripening and germination.The results showed show that compared with 2.5% treatment, under the treatment of 5%, P.notoginseng seeds possessed stable seed water content, the seed vigor was exceed by 51%,variation of antioxidant enzyme (SOD, POD, CAT) activity and malondialdehyde (MDA) content were small, crude fat and total sugar content decreased significantly.With the increase of PEG 6000 concentration, the germination characteristic indexes obviously decreased, antioxidase activities increased firstly and decreased afterwards, content of MDA, soluble protein and total sugar increased obviously.There were significant positive correlation between germination characteristic indexes and osmotic substance content(r>0.900, P<0.01), and significant negative correlation with MDA (r>0.900, P<0.01).In conclusion, because the characteristic of dehydration intolerance of P.notoginseng seeds, 5% water content of sand burying stratification treatment was the best for after-ripening, 15% concentration of PEG 6000 treatment was the highest tolerance limit of germination process.

[Physiological response and bioaccumulation of Panax notoginseng to cadmium under hydroponic].

The physiological response and bioaccumulation of 2-year-old Panax notoginseng to cadmium stress was investigated under a hydroponic experiment with different cadmium concentrations (0, 2.5, 5, 10 µmol · L(-1)). Result showed that low concentration (2.5 µmol · L(-1)) of cadmium could stimulate the activities of SOD, POD, APX in P. notoginseng, while high concentration (10 µmol · L(-1)) treatment made activities of antioxidant enzyme descended obviously. But, no matter how high the concentration of cadmium was, the activities of CAT were inhibited. The Pn, Tr, Gs in P. notoginseng decreased gradually with the increase of cadmium concentration, however Ci showed a trend from rise to decline. The enrichment coefficients of different parts in P. notoginseng ranked in the order of hair root > root > rhizome > leaf > stem, and all enrichment coefficients decreased with the increase of concentration of cadmium treatments; while the cadmium content in different parts of P. notoginseng and the transport coefficients rose. To sum up, cadmium could affect antioxidant enzyme system and photosynthetic system of P. notoginseng; P. notoginseng had the ability of cadmium enrichment, so we should plant it in suitable place reduce for reducing the absorption of cadmium; and choose medicinal parts properly to lessen cadmium intake.

[Effect of Panax notoginseng seedlings physiological response under simulated drought stress by polyethylene glycol (PEG 6000)].

The physiological effects of Panax notoginseng seedlings under simulated drought stress by PEG 6000 on antioxidant enzymes, osmotic substances and root activities were studied. The results showed that the activity of POD and APX in roots and leaves kept rising with increasing processing concentration and time. However, on the one hand, at the same processing time, SOD in roots and leaves firstly increased and then decreased with the increase of processing concentration. On the other hand, at the same processing concentration, SOD kept rising with the extension of processing time. In addition, the activity of CAT in roots and leaves tended to increase with the increasing concentration at the same processing time, while it increased at first and then decreased with the extension of time at the same concentration. The activity of SOD and APX in stem did not change obviously, whereas CAT activity in stem increased with the increasing processing time and concentration. With the increase of processing concentration and the extension of processing time, the MDA, soluble protein, proline content and root activity in leaves and roots apparently rose. Moreover, fluorescence signal of H2O2 and NO in root tip enhanced as the processing concentration increased after treated for 1 d. In summary, P. notoginseng seedlings could deal with drought stress by means of adjusting the system of antioxidant enzyme, permeating stress substances and impeded stress signal substances. Thus, when the concentration of PEG 6000 was more than 5%, it would have harm on P. notoginseng seedlings.

[Effects of stereoscopic cultivation on soil microorganism, enzyme activity and the agronomic characters of Panax notoginseng].

Compartments of soil microorganism and enzymes between stereoscopic cultivation (three storeys) and field cultivation (CK) of Panax notoginseng were carried out, and the effects on P. notoginseng agronomic characters were also studied. Results show that concentration of soil microorganism of stereoscopic cultivation was lower than field cultivation; the activity of soil urea enzyme, saccharase and neutral phosphatase increased from lower storey to upper storey; the activity of soil urea enzyme and saccharase of lower and upper storeys were significantly lower than CK; agronomic characters of stereoscopic cultivated P. notoginsengin were inferior to field cultivation, the middle storey with the best agronomic characters among the three storeys. The correlation analysis showed that fungi, actinomycetes and neutral phosphatase were significantly correlated with P. notoginseng agronomic characters; concentration of soil fungi and bacteria were significantly correlated with the soil relative water content; actinomycete and neutral phosphatase were significantly correlated with soil pH and relative water content, respectively; the activities of soil urea enzyme and saccharase were significantly correlated with the soil daily maximum temperature difference. Inconclusion, The current research shows that the imbalance of soil microorganism and the acutely changing of soil enzyme activity were the main reasons that caused the agronomic characters of stereoscopic cultivated P. notoginseng were worse than field cultivation. Thus improves the concentration of soil microorganism and enzyme activity near to field soil by improving the structure of stereoscopic cultivation is very important. And it was the direction which we are endeavoring that built better soil ecological environment for P. notoginseng of stereoscopic cultivation.