During this period, we used OVA-sensitized rats to build the asthma models. We orally or intratracheally administered Lactobacillus paracasei 33 (LP33) to the rats, which reduced the number of total inflammatory cells, lymphocytes, and eosinophils in the bronchoalveolar-lavage fluid, the IgE concentration, and the cytokine levels of TH2 cells, but we found no significant difference in the cytokine levels of TH1 cells. LP33 can be used to prevent asthmatic allergic reactions induced by aerosol particles. Nevertheless, the dosage form or use of LP33 needs to be adjusted to reduce the irritation of lung tissues, which may produce lesions of the trachea. We observed that DEP dosage can alleviate emphysema, and that LP33 has a substantial effect on improving or slowing allergic asthma.Global air pollution and diesel exhaust particles (DEPs) generated by intratracheal instillation aggravate asthma. In this study, we evaluated the effect of probiotics via tracheal- or oral-route administration on allergies or asthma. We continuously perfused rats daily, using the oral and tracheal routes, with approximately 10 Asthma is a chronic pulmonary-inflammatory disease characterized by a large amount of eosinophilic bulb-infiltration and inflammatory reactions in the bronchus, excessive mucus secretion, and obstruction of airflow in the respiratory tract . VariousDEPs are composed of elemental carbon, polycyclic aromatic hydrocarbons, acidic aerosols, volatile organic compounds, etc. After being inhaled by the respiratory system, DEPs penetrate deep into the alveoli and even into the microvessels of the alveoli, and freely penetrate the cells and tissues of the human body, causing systemic effects, especially a substantial increase in the number of lung and bronchial diseases .3 DEPs, high dose: 3 mg/m3 DEPs) for 1 h per day, 5 days per week, for 3 consecutive months had a varied effect on the numbers of neutrophils and lymphocytes. The high-dose DEP group was more affected than the low-dose DEP and control groups. Additionally, the levels of cytokines IL-5, IL-13, and IFN-γ in the low-dose DEP group were higher than those in the control group, which indicated that long-term DEP exposure may increase AHR, the inflammatory response, and pulmonary fibrosis in mice [Numerous researchers have assessed the effect of particulate pollutants, and particularly of DEPs, on the respiratory system in animal models. In a study of the effect of house-dust extract (HDE) on BALB/c mice, the results showed that DEPs increased HDE-induced airway inflammation, airway mucus-production, oxidative response, and inflammatory-cell infiltration, as well as CXC chemokines at bronchoalveolar-lavage concentrations and airway hyperresponsiveness (AHR) . The varLactobacillus Paracasei 33 (LP33) is one of the probiotics that reportedly benefits allergic-disease treatment with immunomodulatory effects in asthmatic animal-models [Probiotics are nonpathogenic microorganisms that have been widely used in premedical research for asthma treatments. Various probiotic strains can alter cytokine production in the gut and related lymphoid tissues. Lactic acid bacteria reduce allergic symptoms in mice caused by ovalbumin (OVA), mainly by increasing the T helper 1 (Th1) immune response, inhibiting the T helper 2 (Th2) immune response, and reducing the allergic response. An animal model was used to explore the prevention or improvement effect of allergic asthma with probiotics, showing that feeding probiotics did not affect the food intake or body weight of mice, and improved the bacterial phase in mouse feces and the cecum. l-models .6 to 109 CFU [9 CFU per mouse [In animal studies, probiotics have generally been orally administered at doses ranging from 10 109 CFU ,8, or 2 er mouse . TherefoWe purchased six-week-old male SD rats from BioLASCO Co., Ltd., Taipei, Taiwan which were allowed to acclimate for a minimum of 1 week before exposure. All the animal experiments and care were approved by the Institutional Animal Care and Use Committee (IACUC) of the Agricultural Technology Research Institute. Rats (weighing 150–250 g) had ad libitum access to rodent chow and water. We set the environment to a 12 h dark/light cycle, with a temperature of 24 ± 2 °C, and 50% ± 20% relative humidity.n = 8 each group) as follows:Group A (IT control group): IT-PBS + IP-challenged PBS + IT PBS,Group B (PO control group): PO-PBS + IP-challenged PBS + IT PBS,Group C (OVA-DEP group): IT-PBS + IP-challenged OVA + IT DEP,7 CFU/mL, 0.1 mL/rat) + IP-challenged OVA + IT DEP,Group D (IT Probiotics group): IT-Probiotics  + IP-challenged OVA + IT DEP,Group E (PO Probiotics group): PO-Probiotics (2.0 × 10Group F: PO-dexamethasone (0.2 mg/kg/rat) + IP-challenged OVA + IT DEP.We randomly assigned 48 male SD rats into six groups , per os (P.O).Note 2: DEP is administrated to rat by intrathecal delivery. Note 3: Intrathecal injection is using a microsprayer.We exposed the rats to OVA for sensitization and challenge; we sensitized and challenged the rats in the control group (Group A and B) using PBS (pH 7.2). On days 1, 14, 21, and 28, we intraperitoneally injected the rats with 1 mL suspension containing 2 mg of freshly prepared OVA and 10% Al (OH)3, in PBS. On day 1, PBS , probiotics (Groups D and E), and dexamethasone (Group F) were continuously given, either orally or via intratracheal instillation, to the rats for 28 days. On day 29, we administered the rats 1.7% OVA via intratracheal instillation for sensitization, then ultrasonic atomizing intratracheal for 30 min with 1% OVA, for 5 consecutive days. On days 30, 32, and 34, we continuously exposed the rats to PBS  or DEP  intratracheally. After the last exposure to DEPs, within 24 h, we sacrificed the rat, and then we collected the abdominal aortic blood, BALF, and lung tissues . 9 colony-forming units of Lactobacillus paracasei subsp. paracasei LP-33) from Taipei, Taiwan, and other ingredients are microcrystalline cellulose, dicalcium phosphate and magnesium stearate. The diesel exhaust particles (DEPs) range in diameter from 0.02 to 0.2 µm. The density is 1.500–1.900 g/cm3 at 20 °C. These particles can adsorb over 450 different organic compounds, including mutagenic and carcinogenic polycyclic-aromatic-hydrocarbons. We purchased DEPs , ovalbumin (OVA), and dexamethasone from Sigma-Aldrich, Saint Louis, MI, USA. The probiotic product  and stored at −70 °C. We used ELISA kits to determine serum-IgE concentration.6 cells/mL, and we smeared and dried the cells after staining with Liu’s stain. We counted and identified at least 200 cells as eosinophils, neutrophils, or lymphocytes, under a light microscope at 200× magnification. We centrifuged the supernatant of BALF 3000 rpm at 4 °C for 10 min, which we then stored at −70 °C. We used ELISA kits to determine the IL-4 and TNF-α concentrations in the BALF.We washed the left lungs three times with 2 mL PBS (6 mL). We centrifuged the BALF at 2000 rpm at 4 °C, for 10 min. We adjusted the cell precipitation to 1 × 10We removed the right lung of the rats, which we then fixed with 10% neutral formalin, embedded in paraffin, sectioned into a thickness of 4−6 μm, and stained with hematoxylin and eosin (H&E) for histopathology. We observed the damage and infiltration of inflammatory cells in the lung tissue, using light microscopy.t-test. We considered p < 0.05 to be statistically significant.Experimental results are reported as mean ± standard deviation (SD). We used SPSS software version 22.0  for data processing. We performed statistical analysis using one-way ANOVA followed by Dunnett’s In this study, we treated rats with probiotics via oral or intratracheal administration every day (106~108 CFU) for four weeks. During this period, the rats were sensitized using ovalbumin (OVA) and the intratracheal administration of diesel exhaust particles , to accelerate asthma . Rats reWe intratracheally administered probiotics (LP33) to the experimental group for 28 days, and induced by OVA  once per week during this period. We monitored body weight before and after the experimental period. During the period, the average weight of each group continued to increase, except for the group of rats treated with dexamethasone via oral administration A,B. Acco4, including eosinophils (0.49 ± 0.41 × 104) and lymphocytes (11.69 ± 7.24 × 104). In Group C, we found notable cell-infiltration (162.65 ± 78.62 × 104), including eosinophils (5.74 ± 1.56 × 104) and lymphocytes (14.47 ± 13.86 × 104). The total cell number (p = 0.002 < 0.01) and eosinophil number (p = 0.000 < 0.001) statistically differed between Groups A and C, suggesting that the OVA/DEP-induced allergic mode increased the expression of inflammatory cells in the BALF. In addition, in Group D  and Group F , the total cell number was 113.67 ± 39.16 × 104, including eosinophils (2.73 ± 1.23 × 104) and lymphocytes (25.06 ± 13.53 × 104). In Group F, the total cell number was 82.93 ± 35.53 × 104, including eosinophils (3.26 ± 2.01 × 104) and lymphocytes (10.55 ± 3.33 × 104). Compared with Group C, Group F showed significantly reduced cell-infiltration (p = 0.021 < 0.05). The total cell and eosinophil numbers in Group D were reduced by 30.1% and 52.4%, respectively; in Group F, they were reduced by 49.0% and 43.2%, respectively  and lymphocytes (3.20 ± 2.75 × 104). In Group C, significant cell-infiltration was found (162.65 ± 78.62 × 104), including eosinophils (5.74 ±1.56 × 104) and lymphocytes (14.47 ± 13.86 × 104). Total cell numbers (p = 0.000 < 0.001), lymphocytes (p = 0.027 < 0.05), and eosinophil (p < 0.01) were statistically different between Group B and Group C, suggesting that the OVA/DEP-induced allergic mode could increase the expression of inflammatory cells in BALF. In addition, in Group E  and Group F , the cell numbers of Group E were the following: total cell number (72.21 ± 24.06 × 104), eosinophil (3.84 ± 2.88 × 104) and lymphocytes (8.23 ± 4.86 × 104), and the cell numbers of Group F were the following: total cell number (82.93 ± 35.53 × 104), eosinophil (3.26 ± 2.01 × 104) and lymphocytes (10.55± 3.33 × 104). Compared with Group C, both Group E and Group F reduced the total cell numbers and showed significant differences . Among them, the total cell number and eosinophil numbers in Group E were reduced by 55.6% and 33.1% respectively, and in Group F they were reduced by 49.0% and 43.2%, respectively . The cytokine expression in Group D rats  was as follows: total protein, 4.5 ± 0.9 mg/mL; IL-4, 23.7 ± 9.1 pg/mL, and TNF-α, 15.9 ± 9.3 pg/mL. The cytokine expression of the Group F rats  was 4.9 ± 2.7 mg/mL total protein, 32.2 ±7.1 pg/mL IL-4, and 16.4 ± 2.4 pg/mL TNF-α. Compared with Group C, only IL-4 in Group D was significantly reduced (p = 0.004 < 0.01). The IL-4- and TNF-α-levels in Group D were reduced by 39.7% and 39.1%, respectively; in Group F, they were reduced by 18.1% and 37.2%, respectively , IL-4 (39.3 ± 7.0 pg/mL), and TNF-α (26.1 ± 9.5 pg/mL). Compared with Group C rats, the expression levels of total protein, IL-4, and TNF-α in the BALF of Group A rats were lower, but only the TNF-α expression level in BALF was statistically different (ectively a–c.p > 0.05). The cytokine expression of Group E was as follows: total protein = 3.8 ± 0.9 mg/mL, IL-4 = 28.8 ± 5.5 pg/mL, and TNF-α = 17.8 ± 4.0 pg/mL. The cytokine expression in the Group F rats was total protein = 4.9 ± 2.7 mg/mL, IL-4 = 32.2 ± 7.1 pg/mL, and TNF-α = 16.4 ± 2.4 pg/mL. Compared with Group C, the IL-4 expression in Group E was lower (p = 0.015 < 0.05), and the TNF-α expression was reduced in Group F (p = 0.037 < 0.05). The IL-4 and TNF-α levels in Group E were reduced by 26.7% and 31.8%. respectively; in Group F, they were reduced by 18.1% and 37.2%, respectively , IL-4 (39.3 ± 7.0 pg/mL), and TNF-α (26.1 ± 9.5 pg/mL). Compared with Group C, the expression levels of total protein, IL-4, and TNF-α in the BALF of Group B were lower than in Group C, but the differences were not statistically significant (ectively a–c.p > 0.05). In Groups D and F, the serum-IgE concentrations were 41.0 ± 19.2 and 30.5± 19.2 ng/mL, respectively; compared with Group C, the IgE concentration was lower in these groups by 51.6% and 64.0%, respectively. The Group F serum-IgE concentration was significantly different from that of Group C (p = 0.035 < 0.05)  A.p = 0.043 < 0.05). In Groups E and F, the serum-IgE concentrations were 55.0 ± 32.8 and 30.5± 19.2 ng/mL, respectively, which were lower than in Group C by 35.1% and 64.0%, respectively. The difference between Groups C and F was significant (p = 0.028 < 0.05)  B.In Group A A, we obsIn Group B A, we obsAsthma is an allergic disease that causes lung inflammation, with common symptoms including intermittent dyspnea, chest tightness, and cough . The patLactobacillus paracasei 33(LP33). Many animal studies have confirmed that Lactobacillus paracasei can inhibit the synthesis of specific immunoglobulin E (IgE) and stimulate interleukin-12 (IL-12) production [In 2016, the World Health Organization indicated that indoor and outdoor air-pollution can damage the respiratory tract, including through acute respiratory-tract infection, chronic obstructive pulmonary disease, and exacerbated allergic-airway response, and is strongly related to cardiovascular disease and cancer . Recentloduction ,19,20, wWe monitored the body weight of the rats before and after the experiment. During this period, the average weight of each group continued to increase, except for that of the rats in the group orally treated with dexamethasone, which agrees with the result reported in the literature . Next, wIn the future, we could monitor TH1/TH2 cytokine levels at different time-points during the experiment or observe the changes in the growth and decline in these levels. In addition, we added air particles to accelerate the induction of asthma in this experiment; this method slightly differs from that used in previous studies. Possibly, we may explore additional TH1/TH2 cytokines and mechanisms. In the histopathological results, we identified mononuclear-inflammatory-cell infiltrate, granulomatous inflammation, and emphysematous lesions. According to the lesion severity score, both OVA and DEP stimulate lesions in the lung tissue, and dexamethasone can effectively reduce these lesions. The typical symptoms of general allergic reactions are bronchial epithelial hyperplasia, pulmonary inflammation, excessive mucus secretion, and bronchial smooth-muscle hyperplasia . TherefoLactobacillus paracasei 33 (LP33) were administered orally or tracheally, we observed its tendency to reduce the total number of inflammatory cells, lymphocytes, and eosinophils in BALF. A reduction in the expression level of TH2 cytokines and IgE was not evident in TH1. We think that LP33 can be used to prevent asthmatic allergic reactions induced by air particles, but the dosage form or use of LP33 needs to be adjusted to reduce pulmonary irritation. In the future, we could try to change the DEP dose to reduce the lesions of pulmonary emphysema so we can better observe the effect of LP33 on improving or alleviating allergic asthma.Based on our results, whether


Dear Editor,1 Very recently, the optogenetic method with a red-shifted Channelrhodopsin was clinically applied for partial recovery of visual function in a blind patient.2 However, major obstacles to achieving optimal optogenetic vision restoration are either the low light sensitivity or the slow kinetics of existing rhodopsin-based optogenetic tools, which can be improved by molecular engineering to enhance the efficacy of fast Channelrhodopsins (ChRs). Here, we present a newly engineered ChR variant PsCatCh2.0, engineered from PsChR,3 which displays inherently high Ca2+ and Na+ conductance and fast kinetics.4 We introduced a novel mutation PsChR L115C (PsCatCh) to enhance its Ca2+ and Na+ permeability further and fused the cleavable N-terminal signal peptide Lucy-Rho  evoked a 14.5 pA  current in PsCatCh2.0-expressing RGCs in rd1 mice , both IEGs c-Fos (red) and Arc (green) were expressed in the light-stimulated retina and V1 cortex of wild-type and PsCatCh2.0-expressing rd1 mice  compared to the wild-type mice , suggesting regained visual function after optogenetic treatment of blind mice.The photosensitivity and kinetics of ice Fig. . It alsoice Fig. , clearlyice Fig. . In contPsCatCh2.0-treated rd1 mice. The fraction of time spent in light boxes, the distance and speed of movement for discovering the hole to the dark box were recorded. PsCatCh2.0-treated rd1 mice in the blue-light chamber could easily find the hole entering to the dark box, with similar performance as the wild-type mice ; wild-type, 40.1% ± 2.1 (n = 11); rd1, 86.1% ± 4.0, (n = 13); one-way ANOVA; Fig. PsCatCh2.0 also rescued the distance and average speed performance of rd1 mice to the wild-type level . Especially, PsCatCh2.0-treated rd1 mice showed visual tracking behavior to the grating flash with an average peak spatial frequency of 0.22 ± 0.02 (c/d), compared to no response of the rd1 littermates, and 0.53 ± 0.02 c/d of the wild-type mice (Fig. PsCatCh2.0-treated rd1 mice improved visual acuity dramatically.Finally, we evaluated visually guided behavior in ice Fig. . Therefo14 photons/cm2 s was all present to induce retinal, cortical and behavioral responses, which is safe for light therapy. In this study, PsCatCh2.0 was expressed in retinal ganglion cells of blind rd1 mice. Visual acuity raised to 0.22 c/d, with a temporal resolution of at least 32 Hz. The faster and larger current PsCatCh2.0 may be an optimal therapeutic option for the treatment of retinal degeneration. Furthermore, the blue-shifted action spectrum of PsCatCh2.0 (Supplementary Fig. 2 to achieve colored vision restoration in the future.A light intensity of 4.7 × 10Supplemental Material


Thyme derived essential oil and its components have numerous applications in pharmaceutical, food, and cosmetic industries, owing to their antibacterial, antifungal, and antiviral properties. To obtain thyme essential oil with different terpene composition, we developed new germplasm resources using the conventional hybridization approach.1 populations derived from Thymus longicaulis (Tl) × T. vulgaris ‘Fragrantissimus’ (Tvf) and T. vulgaris ‘Elsbeth’ (Tve) × T. quinquecostatus (Tq) crosses were selected, with essential oil yield and terpene content as the main breeding goals. Simultaneously, simple sequence repeat (SSR) primers were developed based on the whole-genome sequence of T. quinquecostatus to authenticate the F1 hybrids. A total of 300 primer pairs were selected, and polymerase chain reaction (PCR) was carried out on the parents of the two hybrid populations . Based on the chemotype of the parents and their F1 progenies, we examined the expression of genes encoding two γ-terpinene synthases, one α-terpineol synthase, and maybe one geraniol synthase in all genotypes by quantitative real-time PCR (qRT-PCR).Phenotypic characteristics, including essential oil yield and composition, glandular trichome density, plant type, and fertility, of three wild Chinese and seven European thyme species were evaluated. Male-sterile and male-fertile thyme species were crossed in different combinations, and two FT. quinquecostatus, and screened the expression of monoterpene synthase genes in thyme. The results of this study provide a strong foundation for the creation of new germplasm resources, construction of the genetic linkage maps, and identification of quantitative trait loci (QTLs), and help gain insight into the mechanism of monoterpenoids biosynthesis in thyme.We used hybridization to create new germplasm resources of thyme, developed SSR markers based on the whole-genome sequence of The online version contains supplementary material available at 10.1186/s12870-022-04029-2. Thymus (family Lamiaceae) are commonly used for food, cosmetic, and medicinal purposes [Thymus) species are important aromatic and medicinal plants that have been used as traditional medicine for thousands of years in the Mediterranean basin [p-cymene, 1,8-cineole, linalool, α-terpineol, geraniol, and borneol), sesquiterpenoids , and oxygenated sesquiterpenoids  [Thymus [T. vulgaris.Species belonging to the genus purposes –3. Thymean basin . These san basin –8. The man basin . Generale oxide) , 8, 9. D [Thymus , 9–11, iT. vulgaris) [T. vulgaris clones [Thymus resources is highly diverse, with more than 300 species, which are native to the Mediterranean basin and are widely distributed in the temperate regions of Europe, North Africa, and Asia [Thymus species are mainly distributed in the northwest, north, and northeast regions based on the Flora of China [To date, a few studies have been conducted by scientific research institutions and companies on thyme breeding, especially in European countries, and few of the high-performance industrial and horticultural varieties developed in these studies have been cultivated. In the 4th International Symposium on the Breeding of Medicinal Aromatic Plants, a breeding study was reported, in which male-sterile and male-fertile clones were crossed to optimize the terpenoid content and yield of thyme (ulgaris) . In thisulgaris) . Such hys clones . The gloand Asia . In Chinof China . TherefoT. daenensis and T. fallax are clearly distinct from the other Thymus species, indicating that T. daenensis shares some genetic similarity with T. fallax [Thymus species as well as the phylogenetic relationship among these species [With the rapid development of modern molecular biology approaches and genome sequencing technologies, DNA-based molecular markers have become an important tool for cultivar identification, fingerprinting , 17, and. fallax . Previou species , 17, 22.TPS genes have been identified in several Thymus species, including T. caespititius [T. vulgaris [T. serpyllum [T. albicans [T. citriodorus.Phenolic monoterpenoids in thyme are synthesized via two pathways: the mevalonate (MVA) pathway and 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway . Isopentpititius –30, T. vvulgaris –34, T. serpyllum , T. albialbicans , and T. T. longicaulis (Tl) × T. vulgaris ‘Fragrantissimus’ (Tvf) and T. vulgaris ‘Elsbeth’ (Tve) × T. quinquecostatus (Tq), to generate two F1 populations. Then, simple sequence repeat (SSR) markers were developed based on the whole-genome sequence of T. quinquecostatus [1 individuals. In addition, the expression profiles of two γ-terpinene synthase genes (Tq13G005250.1 and Tq02G002290.1), one geraniol synthase gene (Tq04G005190.1), and maybe one α-terpineol synthase gene (Tq03G001560.1) were analyzed by quantitative real-time PCR (qRT-PCR). Overall, this study provides a valuable collection of new thyme varieties, which could be used for MAS and the verification of TPS gene function in future studies.In this study, male-sterile and male-fertile thyme varieties were crossed in two different combinations, costatus , to authT. rotundifolius (Tr), T. vulgaris ‘Elsbeth’ (Tve), T. thracicus (Tt), and T. vulgaris ‘Fragrantissimus’ (Tvf) are erect-type, and T. serpyllum ‘Aureus’ (Ts), T. guberlinesis (Tg), T. longicaulis (Tl), T. quinquecostatus (Tq), T. quinquecostatus var. przewalskii (Tqp), and T. mongolicus (Tm) are creeping-type. The fertility of different thyme species is shown in Fig. Thyme is a herbaceous perennial or sub-shrub with valuable medicinal and aromatic properties. Thyme plants are of two types, depending on their growth habit: erect-type and creeping-type. These two plant types display remarkable differences in morphology. The plant types of 10 different thyme species are shown in Fig. Glandular trichomes are specialized hairs that originate from the epidermal cells of flowers, leaves, and stems. These organs exist as two types, peltate and capitate, on the surface of approximately 30% of all vascular plants, including lavender, thyme, rosemary, oregano, basil, and other Lamiaceae species . Glandul·100 g− 1), followed by Tt (1.20 mL·100 g− 1), Tr (0.95 mL·100 g− 1), Tve (0.75 mL·100 g− 1), and Tl (0.70 mL·100 g− 1). The essential oil yield of Tq, Tqp, Tm, Tg, and Ts varied between 0.40 mL·100 g− 1 and 0.50 mL·100 g− 1 , respectively. Tqp showed the highest content of carvacrol, which accounted for 48.37% of the total content of volatile compounds. In Tm, thymol was the most abundant volatile compound (38.57%), followed by p-cymene (16.40%). Similarly, in Tve, thymol was the most abundant volatile compound (35.43%), followed by p-cymene (18.42%), and γ-terpinene (13.96%). The thymol contents of Tr, Tt, Tg, and Ts were 36.02%, 41.04%, 26.26%, and 28.96%, respectively. In Tl, geranyl acetate (34.81%) was the most predominant volatile compounds, followed by geraniol (28.54%). The most abundant volatile compounds in Tvf were α-terpineol acetate (45.46%) and α-terpineol (30.84%).The relative contents of essential oil components in 10 different thyme species are shown in Fig. p-cymene, and γ-terpinene contributed greatly to the volatile components of the essential oil of all 10 species. Tq and Tqp grouped together in the first quadrant, and their corresponding characteristic volatile substances included carvacrol, p-cymene, and γ-terpinene; Tvf and Tl were distributed in the third quadrant, which corresponded to volatiles geraniol and α-terpineol; Tm, Tve, Tr, Tt, Tg, and Ts were distributed in the fourth quadrant, and the corresponding predominant volatile was thymol  of which was annotated as highly repetitive [T. quinquecostatus  and 25 bp SSR loci being the least abundant . The number of SSRs gradually decreased with the increase in repeat length  and chromatin conformation capture (Hi-C) technologies revealed 13 chromosomes in petitive . A total1 hybrid populations were obtained from two crosses: T. longicaulis × T. vulgaris ‘Fragrantissimus’  and T. vulgaris ‘Elsbeth’ × T. quinquecostatus   and male-fertile (with pollen) thyme species were crossed as female and male parents, respectively, in different combinations. Finally, two F1 individuals in the two hybrid populations. Progenies with complementary parental bands or only paternal-specific bands were true hybrids, and those with only maternal-specific bands were pseudo-hybrids or inbreds. Based on the genotyping results, 14 lines in the Tl × Tvf progeny , carvacrol , p-cymene , and γ-terpinene . Additionally, lines F1-3, F1-4, F1-5, F1-6, F1-10, F1-11, F1-13, and F1-14 showed the highest contents of geraniol  and geranyl acetate . Thus, the essential oil compositions of these progenies were biased toward the female parent Tl. VOCs with high contents in line F1-7 were geraniol (4.48%), geranyl acetate (27.75%), α-terpineol (3.00%), and α-terpineol acetate (28.03%), thus representing a good aggregation of the dominant compounds found in the two parents. The most abundant compounds in line F1-12 were α-terpineol (9.07%) and α-terpineol acetate (57.44%), indicating a bias toward the male parent Tvf. Cluster analysis of the 17 main chemical compounds found in T1, Tvf, and their 14 F1 lines showed that lines F1-3, F1-4, F1-5, F1-6, F1-10, F1-11, F1-13, and F1-14, which clustered with the female parent Tl, were geraniol-type; F1-12, which grouped with the male parent Tvf, was α-terpineol-type; lines F1-1, F1-2, F1-8, and F1-9 were thymol and carvacrol polymerization-type; and line F1-7 was geraniol and α-terpineol polymerization-type  and geranyl acetate 41.75%) were the most abundant VOCs in the female parent Tl, and α-terpineol (11.76%) and α-terpineol acetate (61.37%) were the most abundant in the male parent Tvf , and γ-terpinene (13.96%) were the most abundant in the female parents Tve, and carvacrol (20.74%), p-cymene (23.23%), and γ-terpinene (11.45%) were the most abundant in the male parent Tq. Among the 11 Tve × Tq F1 lines, F1-3, F1-4, F1-5, F1-8, and F1-11 showed the highest contents of thymol , p-cymene , and γ-terpinene . The compositions of these F1 lines were biased towards the female parent Tve, and the thymol contents of F1-5 and F1-8 were higher than that of the female parent, accounting for 46.19% (F1-5) and 46.38% (F1-8) of the progeny plants. Lines F1-1, F1-2, F1-6, F1-7, F1-9, and F1-10 showed the highest contents of α-terpineol  and α-terpineol acetate , and the sum of the proportions of each of these two compounds (α-terpineol and α-terpineol acetate) in the six above-mentioned F1 lines was more than 90%. Thus, α-terpineol and α-terpineol acetate were the absolute dominant compounds, although these compounds were not dominant in the parents; neither one of the two compounds was detected in the female parent Tve, and only α-terpineol was detected in minor amounts (0.95%) in the male parent Tq. Cluster analysis revealed that, among the 11 progeny lines, F1-3, F1-4, F1-5, F1-8, and F1-11 were thymol-type, while F1-1, F1-2, F1-6, F1-7, F1-9, and F1-10 were α-terpineol-type  and sesquiterpenes C15), respectively. We determined the volatile terpenes in the leaves of Tl × Tvf and Tve × Tq populations. The chemical types found in the Tl × Tvf population were geraniol-type, geraniol/α-terpineol-type, thymol/carvacrol-type, and α-terpineol-type, while those found in the Tve × Tq population were carvacrol-type, thymol-type, and α-terpineol-type Fig. b, d. Conquencing , 69 TPS , respectGES gene  , Tvf (α-terpineol-type), and their progenies F1-6 and F1-12 by qRT-PCR  and female parent (Tve) of Tve × Tq were carvacrol- and thymol-type, respectively. Since γ-terpinene is the catalytic precursor of thymol and carvacrol, we verified the γ-terpinene synthase function of PCR Fig. a. In linsis Fig. a. SimilaThymus genus  with no or completely cut anthers; the latter are completely male-sterile . In diffChinese thyme plants are widely distributed in the northwest, north, and northeast regions in China . TherefoHybrid breeding is an important means of germplasm resource innovation. The advantages of parents can be combined in a single genetic background through interspecific hybridization. In this study, new thyme germplasm resources were generated by hybridizing different chemical types and plant types. SSR markers are simple, time-saving, cost-effective, reproducible, and stable, and therefore have been widely used for the identification of the hybrid progenies in different species , 43. Ear1 progenies derived from the Tl × Tvf cross. Analysis of the same sample using different primer pairs can produce different results, possibly because of the high heterozygosity of thyme, lead to some of the parents can’t dominance or appear some non-parental bands. This may also be caused by the process of meiotic division during gamete formation; exchange of DNA between homologous chromosomes during recombination at marker loci can lead to the disappearance of bands in progenies.The SSR-based genotyping process first involves the screening of co-dominant SSR primers in parents, followed by the identification of progenies . However1 progenies is intermediate between that of the two parents or biased towards the phenotype of the female or male parent, which also provides the possibility of breeding excellent new varieties [1 lines, eight lines showed the same VOC profiles in leaves as their female parent Tl; one line was similar to the male parent Tvf; and five lines were dissimilar to both parents. This conclusion was supported by the 11 F1 lines derived from the Tve × Tq cross; five of these F1 lines showed the same leaf VOC profiles as their female parent Tve, and the remaining six lines were different from both their parents. However, some F1 progenies also showed some volatile compounds that were superior to the both parents or not found in either parent; for example, thymol and carvacrol were found in the progeny of the Tl × Tvf cross, and α-terpineol and α-terpineol acetate were found in the Tve × Tq population. These results suggest that new varieties with related VOC profiles could be developed in thyme through cross breeding. Heterosis may also be present, and the greater the phenotypic differences between parents, the stronger is the heterosis expected to be in their progeny.Generally, the phenotype of Farieties . In thisp-cymene, thymol, and carvacrol [TcTPS02 and α-terpineol synthase gene TcTPS05 were previously validated in T. caespititius [TvTPS2 and three cytochrome P450 genes  were validated in T. vulgaris [Monoterpenoid biosynthesis begins with GPP, which is the precursor of all monoterpenoids, and yields α-terpinyl cations, which are highly unstable intermediates that can then be converted to specific monoterpenoids by certain monoterpene synthases such as γ-terpinene and α-terpineol . GPP alsarvacrol . The synpititius . Similarvulgaris .TPSs were expressed in hybrid thyme progenies, and these results were consistent with the relative contents of the TPS-catalyzed products in thyme. The expression levels of two γ-terpinene synthase genes Tq02G002290.1 and Tq13G005250.1 were verified in the progeny derived from the cross between Tve and Tq, whose chemotypes were thymol- and carvacrol-type, respectively. Similarly, the expression levels of α-terpineol synthase gene Tq03G001560.1 and geraniol synthase gene Tq04G005190.1 were validated in the progeny of Tl and Tvf, whose chemotypes were α-terpineol- and geraniol-type, respectively. Additionally, the relative expression levels of Tq02G002290.1, Tq13G005250.1, Tq03G001560.1, and Tq04G005190.1 were consistent with the relative contents of catalytic products in some F1 lines. Therefore, this method enables only a preliminary screening of gene function and provides a basis for further gene function verification. The above results lay a strong foundation for the creation of new germplasm resources, construction of the genetic linkage maps, mapping of quantitative trait loci (QTLs), and MAS, and provide insight into the mechanism of monoterpenoids biosynthesis in thyme.The results of qRT-PCR analysis showed that the screened 1 populations were obtained, simultaneously, SSR primers were developed based on the whole-genome sequence of T. quinquecostatus to authenticate the F1 hybrids. Based on the chemotype of the parents and their F1 progenies, we examined the expression of genes encoding two γ-terpinene synthases, one α-terpineol synthase, and maybe one geraniol synthase in all genotypes by qRT-PCR. The results of this study provide a strong foundation for the creation of new germplasm resources, construction of the genetic linkage maps, and identification of QTLs responsible for the terpene compositions and essential oil. We screened monoterpene synthases in thyme to gain insight into the mechanism of monoterpenoids biosynthesis in thyme.Thyme is a multi-purpose plant with a wide range of applications in the pharmaceutical, food, and cosmetic industries, owing to its high essential oil content. To obtain thyme essential oil with different terpene compositions, we cross wild Chinese thyme species with European thyme species, and new germplasm resources have developed using the conventional hybridization approach. Two FT. quinquecostatus, T. quinquecostatus var. przewalskii, T. mongolicus, and seven European thyme species, including T. vulgaris ‘Fragrantissimus’, T. vulgaris ‘Elsbeth’, T. guberlinesis, T. serpyllum ‘Aureus’, T. thracicus, T. longicaulis, and T. rotundifolius, were used in this study. T. quinquecostatus plants were collected from the Jinhekou village in the Hebei province of China in 2018. T. quinquecostatus var. przewalskii was collected in 2019 from the Small Wutai Jinhekou Scenic Spot in Yu County, Hebei, China. T. mongolicus was collected in 2018 from Chicheng County, Hebei, China. Three specimens  were obtained from the Chinese National Herbarium, Institute of Botany, Chinese Academy of Sciences. T. vulgaris ‘Fragrantissimus’, T. vulgaris ‘Elsbeth’, T. thracicus, and T. longicaulis were introduced from the Czech Republic . T. guberlinensis and T. serpyllum ‘Aureus’ were introduced from Hungary (introduction no. 002-2016 and 003-2016). T. rotundifolius was introduced from Germany (introduction no. 797–2016). Plants were grown on an experimental farm at the Institute of Botany, Chinese Academy of Sciences (IB-CAS), Beijing, China.Three wild Chinese thyme species, including 1 hybrid combinations of thyme were constructed by cross breeding in 2020. Finally, two F1 populations derived from T. longicaulis (Tl) × T. vulgaris ‘Fragrantissimus’ (Tvf) and T. vulgaris ‘Elsbeth’ (Tve) × T. quinquecostatus (Tq) crosses were selected for further analysis.Male-sterile (without pollen) thyme species were crossed as the female parent with male-fertile (with pollen) thyme species, with the essential oil composition and yield as the main breeding goals. Different cross combinations were designed, and the F1 progenies of Tl × Tvf and Tve × Tq crosses, immediately frozen in liquid nitrogen, and stored at -80 °C. DNA was extracted from the frozen leaf samples using the DNA Secure Plant Kit . DNA concentration and quality were assessed by 1% agarose gel electrophoresis and with a 2.0 Fluorometer .Leaves were collected from the parents and F2O. The thermocycling conditions were as follows: 94 °C for 3 min; 6 cycles at 94 °C for 45 s, 55–65 °C for 1 min, and 72 °C for 1 min; 9 cycles of 94 °C for 45 s, 50–58 °C for 1 min, and 72 °C for 1 min; 19 cycles at 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min; and final extension at 72 °C for 5 min. PCR products were analyzed by electrophoresis on 8.0% (w/v) denaturing polyacrylamide gel in TBE buffer for 1 h on the DYY-6 C electrophoresis apparatus  at a constant voltage of 220 V. DNA fragments were visualized by silver staining  and sized with a 50 bp DNA ladder marker  [PCR was performed on the PCR system  in a 10 µl reaction volume containing 2 µl (20 ng/µl) of genomic DNA, 3 µl of 2× Taq PCR Master Mix II , 2 µl of forward and reverse primer mixture, and 3 µl of ddH, China) . SSR priT. longicaulis, T. vulgaris ‘Fragrantissimus’, T. vulgaris ‘Elsbeth’, T. quinquecostatus, and their F1 lines were frozen in liquid nitrogen and stored at -80 ℃. Total RNA was extracted from the frozen leaves using the Easy Spin RNA extraction kit . The isolated total RNA was treated with DNase I, and then purified with the RNA clean kit . The concentration of each RNA sample was determined using NanoDrop spectrophotometer  and 2.0 Fluorometer . RNA integrity was analyzed using Bioanalyzer 2100 . Then, cDNA was synthesized using the HiScript Reverse Transcriptase Kit , according to the manufacturer’s instructions [Leaves of ructions .T. vulgaris ‘Elsbeth’, T. quinquecostatus, T. longicaulis, T. vulgaris ‘Fragrantissimus’, and their F1 lines was isolated and reverse transcribed, as described above. To amplify four TPS genes , primers were designed using Primer3 (http://primer3.ut.ee) . The qRT-PCR protocol included 40 cycles of 95 ℃ for 30 s, 95 ℃ for 10 s, and 58 ℃ for 30 s, along with melting curve analysis. Primer sequence, amplicon length, amplification efficiency (%), and linear correlation coefficient (R2) corresponding to two internal reference genes (18 S rRNA and β-actin) are shown in Supplementary Table STPS transcripts was performed using the internal reference gene 18 S rRNA. Relative expression levels were calculated using the 2−ΔΔCt method after transcript data normalization. All analyses were performed in triplicate [To analyze the expression patterns of γ-terpinene, α-terpineol, and geraniol synthase genes, the total RNA of The essential oil of 10 thyme species was isolated by steam distillation at 180–200 ℃ for 90 min. Essential oil yield (%) was calculated as volume (ml) of the isolated oil per 100 g of dry plant material. The isolated essential oil was dried using anhydrous sodium sulfate, and stored at 4 ℃ until needed for further analysis .The essential oil composition of 10 thyme species was analyzed by gas chromatography-mass spectrometry (GC-MS) using Agilent 7890 A-7000B gas chromatograph , equipped with Agilent 5975 C MS detector . Using the HP-5MS  capillary column, volatiles were separated using the following temperature program: 5 min at 60 °C; increased to 220 °C at the rate of 4 °C/min; increased to 250 °C at the rate of 60 °C/min; hold at 250 °C for 5 min. The following parameters were used: injector and detector temperature, 250 °C; carrier gas, He; flow rate, 1 m/min; split ratio, 1:10; acquisition range, 50–500 m/z in electron-impact mode; ionization voltage, 70 eV; and injected sample volume, 1 µl. The determination of the content of each compound (%) was based on the normalization of GC peak areas. The identification of essential oil components was based on the comparison of retention indices (RIs), relative to a homologous series of n-alkanes (C7–C40), and mass spectra (MS) from the NIST (v14.0) library and data from scientific literature . RIs werwhere RT(x), RT(z), and RT(z + 1) for the composition, and the number of carbons Z and Z + 1 for the retention time of the normal alkane.T. vulgaris ‘Elsbeth’, T. quinquecostatus, T. longicaulis, T. vulgaris ‘Fragrantissimus’, and their F1 lines were detected via headspace solid-phase microextraction (HS-SPME). Briefly, 0.25 g of fresh leaf powder was weighed and immediately placed into a 20 ml headspace vial  containing 20 µl of internal standard solution . The vials were sealed using crimp-top caps with TFE-silicone headspace septa . Subsequently, each vial was immediately incubated at 40℃ for 30 min. Then, to absorb the volatiles, the headspace of each vial was exposed to 100 μm coating fiber polydimethylsiloxane  for 30 min. All VOCs on the coating fiber were analyzed by GC-MS using Model 7890 A GC instrument and 7000B mass spectrometer  [The leaf VOC profiles of CA, USA) .The GC-MS conditions were as follows: injector temperature, 250 ℃; transfer line temperature, 250 ℃, respectively; column temperature, initially maintained at 50 ℃ for 3 min, gradually increased to 150 ℃ at 4 ℃/min for 2 min, and finally raised to 250 ℃ at 8 ℃/min for 5 min; carrier gas (helium) flow rate, 1 ml/min; injection, splitless mode; ionization voltage, 70 eV; source temperature, 250 ℃; and MS range, 35–500 m/z. Agilent MassHunter 5.0 was used to analyze the chromatograms and MS. VOCs were identified by comparing the retention times of individual peaks with those of authentic standards, and MS were determined based on the NIST v14.0 MS database and the data reported previously [where RT(x), RT(z) and RT(z + 1) for the composition, and the number of carbons Z and Z + 1 for the retention time of the normal alkane.Glandular trichomes were visualized using a stereomicroscope . The number of glandular trichomes within a certain leaf area was counted using the ImageJ software. The average glandular trichome density was calculated based on three plants.p ≤ 0.05).Data were expressed as the mean ± standard deviation of three biological replicates. Statistical analysis, including variance analysis, hierarchical clustering analysis, and correlation analysis, was performed using IBM SPSS Statistics for Windows, version 19.0 . Significant differences among the different genotypes were tested using a one-way analysis of variance (ANOVA), followed by Duncan’s multiple range test at 5% probability level  loci in the Thymus quinquecostatus genome.Additional file 3: Supplementary Table S1. Phenotypic characteristics of the parents of two hybrid thyme populations.Additional file 4: Supplementary Table S2. Primers used for the development of SSR markers.Additional file 5: Supplementary Table S3. SSR primers used for the identification of F1 hybrids in the Tl × Tvf population.Additional file 6: Supplementary Table S4. SSR primers used for the identification of F1 hybrids in the Tve × Tq population.Additional file 7: Supplementary Table S5. Relative contents of volatile organic compounds (VOCs) in the leaves of Tl, Tvf, and their F1 progeny.Additional file 8: Supplementary Table S6. Relative contents of VOCs in the leaves of Tve, Tq, and their F1 progeny.Additional file 9: Supplementary Table S7. Reported amino acid sequences used to construct the TPS phylogenetic tree.Additional file 10: Supplementary Fig. S3. Alignment of the deduced amino acid sequence of Tq04G005190.1 with the reported amino acid sequence of geraniol synthase ObGES.Additional file 11: Supplementary Table S8. Primer sequence, amplification length, amplification efficiency (%), and linear correlation coefficient (R2) of four TPS genes and two reference genes (18 S rRNA and β-actin).Additional file 12: Fig. 3-6 gels and blots.


The electrocatalytic activities of these catalysts were investigated by thin-film RDE technique. The catalyst calcined at 1000 °C exhibits an outstanding bi-functional activity towards the ORR and OER in alkaline electrolyte, and it also exhibits an outstanding performance in primary and rechargeable Zn–air batteries, which is comparable with the commercial noble metals Pt/C and RuO2.Developing low-cost, highly efficient electrocatalysts for the oxygen reduction reaction (ORR) and oxygen evolution reaction (OER) is desirable for rechargeable metal–air batteries. Herein, a series of perovskite structured  and oxygen evolution reaction (OER) is desirable for rechargeable metal–air batteries. However, to achieve a Zn–air battery with high performance and long cycle life, an efficient and highly stable bifunctional air electrode is essential, because the sluggish kinetics of the oxygen reduction reaction (ORR) and the oxygen evolution reaction (OER) during discharge and charge have limited the practical application of Zn–air batteries.8,9 To solve this problem, a variety of highly active ORR and OER electrocatalysts have been investigated, including noble metals,10,11 carbon materials,12–14 transition metal selenide,15 and transition metal oxides.16–18 Noble metals exhibit high performance, but their low abundance and high cost prohibit their particle use. The carbon materials possess larger surface areas and high conductivities, but poor durability.Metal–air batteries have attracted great attention because of their high energy and power densities.19–21 As far as we know, in perovskite catalysts, the A-site deficiency and substitute A-site metal with low valence metals can manipulate the B-site element valence, and which has a critical influence on the catalytic behaviours of the materials. Herein, a series of perovskite structured catalysts are designed with same A-site stoichiometry and same content of doping metals, but calcined at different temperatures. The relationship between the preparation temperature and the oxidation state of B-site metal is investigated; and it obviously affect their catalytic activities towards ORR and OER in alkaline medium.22–24 The selected catalyst can be used as bifunctional electrocatalyst and is comparable with commercial noble metals.Transition metal oxides, especially the perovskite structured oxides have been intensively investigated as bifunctional electrocatalysts in Zn–air batteries, owing to their low-cost, abundant varieties, structural stability and great potentials.2O3. To avoid the effect of La2O3 impurity phase, the sample calcined at 900 °C is not selected for further investigated. To confirm the chemical compositions of the LSF0.95 catalysts, inductively coupled plasma with optical emission spectroscopy (ICP-OES) is used and the results are shown in Table S1.2 g−1 (LSF0.95-1000 °C), 1.070 m2 g−1 (LSF0.95-1100 °C) and 0.816 m2 g−1 (LSF0.95-1200 °C), respectively. These values represent that the higher calcination temperature leads to the smaller specific surface area. Because the particles are easier to agglomerate at higher calcination temperature, resulting in the bigger particle size, which has been discussed in SEM and PSD. The surface element compositions of the LSF catalysts are analyzed by X-ray photoelectron spectroscopy (XPS). The survey XPS spectrum in Fig. S3(a)3/2 and Fe 2p1/2, respectively;25,26 and the weak Fe-2p satellite peak indicates the multiple oxidation states of Fe. High-resolution XPS spectra of Fe 2p3/2 and O 1s are analyzed in 3/2 in 3+ (709–711 eV) and Fe4+ (711–713.6 eV), and the content of Fe4+ increases as the temperature increasing from 1000 °C to 1200 °C. The peaks at 528.5 eV, 529.9 eV and 531.5 eV , and the results are shown in 531.5 eV  in O 1s 2 and N2 saturated KOH. Two obvious cathodic peaks centered at 0.34 V and −0.02 V (vs. RHE) only can be observed in CV curve tested in O2 saturated KOH, indicating the electrocatalytic activity of LSF catalyst towards ORR in KOH. LSV curves of the LSF catalysts with different preparation temperatures measured at 1600 rpm are compared in −1, 65.5 mV dec−1) indicates more favorable ORR kinetics relative to those of LSF0.95-1100 °C  and LSF0.95-1200 °C . Electrocatalytic activities of the LSF0.95 catalysts towards ORR are investigated by thin-film RDE technique. Fig. S4− ORR process, and the reaction steps are shown as follow:31To investigate the ORR mechanisms in these two ORR ranges, electron transfer numbers in these two ORR potential ranges are calculated from the Koutecky-Levich (K-L) plots Fig. S5. In the 2 is reduced to the intermediate HO2− by accepting 2e− in step   measurements are used to investigate the electrochemical stability of LSF0.95/C for the ORR and OER, and the results are shown in Fig. S11.2/C. These results reveal an excellent electrochemical durability of LSF0.95/C. The XRD, XPS and HRTEM results of LSF0.95 in Fig. S12 and S13E = EOER − EORR was used to calculate the overpotential between the OER at the current density of 10 mA cm−2 and the ORR at the current density of −1 mA cm−2, which is used to access the bifunctionality of the catalysts.37,38 The overpotential values of LSF0.95/C are compared with the reported ones in Table S5.E values for LSF0.95/C, 20 wt% Pt/C and RuO2/C are 1.04 V, 1.10 V and 1.12 V, respectively. Obviously, the ΔE of LSF0.95/C is smaller than commercial noble-metal based Pt/C, RuO2/C and some other reported excellent perovskite-based bifunctional catalysts. The results further demonstrate the high bifunctional electrocatalytic activity of LSF0.95 in alkaline electrolyte.By comparing the LSV curves and Tafel plots of LSF0.95-1000 °C/C (20 wt% Vulcan XC-72 carbon) with commercial 20 wt% Pt/C and RuO Fig. S10, the ele−2, the voltage and power density of the battery based on perovskite LSF0.95-1000 °C are very close to those of the commercial Pt/C. But when the current density > 70 mA cm−2, LSF0.95-1000 °C exhibits a higher voltage and power density compared with commercial Pt/C, and the peak power density could be as high as 94 mW cm−2 at 0.63 V, which is superior to the commercial Pt/C. The galvanostatic discharge curves shown in −2, after 10 h discharge, no obvious voltage drop was observed until all of the Zn metal was consumed. The specific discharge capacities at different current density are shown in −2, the specific capacity of LSF0.95-1000 °C based battery normalized to the mass of consumed Zn is 755 mA h gZn−1, corresponding to a gravimetric energy density of 928 W h kg−1. At 30 mA cm−2, the specific capacity normalized to the mass of consumed Zn is ∼732 mA h gZn−1, corresponding to a high gravimetric energy density >768 mA h gZn−1. The rechargeable Zn–air batteries with short cycles (10 min per cycle) are performed to explore the bi-functional properties of LSF0.95-1000 °C. The batteries are running in the air at room temperature, no further oxygen or air is bubbled into the system. At 10 mA cm−2, the charge–discharge voltage gap for LSF0.95-1000 °C is ∼0.74 V, which is very close to voltage gap between the commercial 20 wt% Pt/C and RuO2/C (∼0.66 V), and it can keep this value after 10 h running 0.95FeOδ3− perovskite catalysts are prepared by a solid-state synthesis method at different temperatures. LSF0.95-1000 °C exhibits the highest bifunctional catalytic activity among these LSF0.95 catalysts and a smaller overpotential between ORR and OER. It also performs a stronger durability than commercial noble-metal catalysts. The LSF0.95-1000 °C catalyst is used as the bifunctional electrocatalyst in primary and rechargeable Zn–air batteries. The maximum power density of the primary Zn–air battery using LSF0.95-1000 °C can reach 94 mW cm−2, which is superior to the commercial Pt/C. The performance of the rechargeable Zn–air battery of (La0.65Sr0.3)0.95FeOδ3−-1000 °C catalyst is comparable with combined commercial catalysts Pt/C and RuO2. The outstanding activity can be attributed to the amount of active sites, and the higher content of oxygen vacancy.In summary, a series of (LaThere are no conflicts to declare.RA-011-D1RA07920D-s001


Punica granatum L.) during the 2019 and 2020 seasons. One hundred pomegranate trees were chosen and sprayed three times at the beginning of flowering, full bloom, and 1 month later with the following treatments: water as control, 0.025, 0.05 and 0.1 mg/L Se; 5 mL/L, 7.5 and 10 mL/L Ag NPs, and 0.5, 1 and 2 mg/L K2Si2O5. The results showed that spraying of SE, Ag NPs, and K2Si2O5 ameliorated the shoot length, diameter, leaf chlorophyll content, set of fruiting percentage, and fruit yield per tree and hectare compared to control through studying seasons. Moreover, they improved the fruit weight, length, and diameter, as well as total soluble solids, total, reduced, and non-reduced sugars percent, while they lessened the juice acidity percentage compared to control. The most obvious results were noticed with Se at 0.1 mg/L, Ag NPs at 10 mL/L, and K2Si2O5 at 2 mg/L in both experimental seasons over the other applied treatments. By HPLC analysis, peel extracts showed the presence of several bioactive compounds of catechol, syringic acid, p-coumaric acid, benzoic acid, caffeic acid, pyrogallol, gallic acid, ferulic acid, salicylic acid, cinnamic acid, and ellagic acid. The extracts applied to Melia azedarach wood showed promising antifungal activity against Rhizoctonia solani and were considered wood-biofingicides.The current study was performed on 8 years old "Succary" pomegranate cultivar ( Punica granatum L.) is a deciduous tree, which has been tamed for thousands of years, has a big range from cultivars and supplies opportunities for the consumers2. Its productivity can be boosted and enhanced by using numerous substances and elements. It was found that Se was enhanced greatly the development, yield and fruit quality of some fruit crops5, such as on grape, where it raised its content and the level of its nutrition, while diminishing the concentration of heavy metals particularly in European and American species6. Se exerts useful effects on plant growth and may works as quasi-fundamental micronutrient through change various physiological and biochemical features, therefore the plants differ greatly in their physiological and biochemical response to Se7. The Foliar application of Se at 0.017 g/L 6 times, with interval 10 days increased photosynthesis, yield of in pear, grape, and peach8.Pomegranate , have strong biological activity20. Ag NPs influence plants by different levels22, such as germination promotion23, growth activation24, increasing the accumulation of biomass25, improving shoot growth26, and raising the pigment content27.Using nano-fertilizers is differentiated by the possibility to use in small quantity compared with conventional fertilizers28, increased biotic and abiotic stresses’ tolerance like drought via maintaining the balance of water, improving photosynthesis process and the composition of xylem vessels during the high rates of transpiration33. The application of Si on avocadoes reduced the respiration and the production rate of ethylene and consequently increased its growth34, could adhere to the reverse activity of big quantities from boron35. Moreover, spraying date palm with silicon raised the date development, yield and quality36, “Hindy bisinara” mango trees37, banana38, pomegranates40, and Flame seedless grapevines41, “Washington” navel orange42. Loquat tree  cv. Emanual foliar treated with K2Si2O5 at 1.0 and 2.0%, increased the percentages of fruit set and retention and thus the yield, fruit physical and chemical characteristics, and the concentration of 2% was better than 1%43. Moreover, it was noticed also that the pro harvest foliar spraying of K2Si2O5 minimized the percentages of fruit decomposition, loss, and total acidity.Si implementation improved the employment of water, and dry matter45. Peel extract had markedly higher antioxidant capacity than the pulp extract46. Hydrolyzable tannins such as punicalin, punicalagin, pedunculagin, gallic and ellagic acid were identified in peel extracts47. Other phytochemicals identified from the pomegranate are organic and phenolic acids  (Alfa aesar®) at 0.5, 1, and 2 mg/L. The chosen trees were foliar treated at the first of flowering, full bloom and 1 month later and were received the same horticultural treatments applied at the orchard. The aforementioned treatments were ordered in Randomized Complete Block Design, where each one of them contained ten replicates/trees. The influence of the aforementioned treatments was evaluated by measuring their impacts on the following:One hundred pomegranate trees were chosen and were similar in their growth and size as possible to apply the following treatments: Control (water only), Selenium (Se)  at 0.025, 0.05 and 0.1 mg/L; silver nanoparticles (Ag NPs) at 5, 7.5 and 10 mL/L; Potassium silicate  and the results were expressed in SPAD units. Fruit set percentage was assessed according to this equation:Fruit drop percentage was estimated by accounting dropping fruits number from the middle of June till fruit harvesting time conditions of the experiment, then expressed as a percentage according to this formula:Fruit cracking percent was measured according to the following equation:51Fruit sun burn proportion by accounting the sunburned fruit with the respect of total number of fruits on each tree before the picking timeAt the time of picking up (October), the number and weight of fruits per tree were counted.52. Non-reducing sugars were by the difference between total sugars and reducing sugars. Fruit Titratable acidity percent was determined by AOAC method53, where it was expressed as citric acid in g/100 ml fruit juice. TSS/acid ratio was counted. Vitamin C mg/100 mg juice was by titration with 2,6 dichloro phenol-indo-phenol54 and calculated as mg/100 mL of juice. Anthocyanin content was determined55.At the time of picking up, the sample of six fruits from every tree was picked up by random way to assess the fruit weight (g), tallness, diameter, volume. Fruit firmness was determined by using Magness and Taylor pressure tester . Total soluble solids (TSS) percent was measured by using a hand refractometer . Total and reducing sugars were determined by Nelson arsenate—molybdate colorimetric method2Si2O5 in April 2020 were air-dried at room temperature for 2 weeks. The dried peels from pomegranate fruits were ground to a fine powder using a small laboratory mill. About 50 g from each of the dried powder peels were extracted by the soaking method56 with 100 mL of acetone solvent (99%) with stirring for 6 h at room temperature. After the extraction process was finished, the extract filtered through a cotton plug and then with Whatman No. 1 filter paper. The filtered extract was concentrated by evaporating the acetone solvent to have the dried peel extracts. To prepare the concentrations, the extracts were dissolved in dimethyl sulfoxide (10% DMSO), and the concentrations levels of 2%, 4%, and 6% were obtained57.Peels from treated fruit with Se, Ag NPs, and K58.The phenolic compounds from the acetone extracts of each of the pomegranate peels were identified by HPLC (Agilent 1100) was composed of binary LC pump, a UV/Vis detector, and C18 column . Chromatograms were obtained and analyzed using the Agilent ChemStation. The separation and identification conditions ca be found in the previous workMelia azedarach tree were air-dried and be ready at about 0.5 × 1 × 1 cm. Designed wood samples were autoclaved for 20 min at 121 °C, and then left to cool.Rhizoctonia solani (acc# MH352450) was used for the antifungal bioassay. Three wood samples were utilized for each concentration, as well as for hesta (2.5 g/L), the positive (thiophanate-methyl 70 wp) and negative (10% DMSO) controls. The antifungal action of the extracts wood-treated in terms of the inhibition percentage of fungal linear growth (IPFLG) was measured following our last works62 by utilizing the next formula; IPFLG (%) = [(GC − GT)/GC] × 100, where GC and GT ready the average diameters of the fungal colony of control and treatment, respectively.Wood samples of 63. A least significant difference at 0.05% was used to compare between the means of the treatments and measured with CoHort Software .Obtained data were analyzed by one-way analysis of varianceS and K2Si2O5 with respect to control. Besides, the same measured vegetative growth parameters were greatly improved particularly with Se at 0.1 mg/L, Ag NPs at 10 mL/L and K2Si2O5 at 2 mg/L compared with the other applied concentrations, and K2Si2O5 was the superior treatment in both studying seasons.Table 65, and lessen the oxidation stress in chloroplast66, thus it can enhance the development of plants67. Selenium plays a crucial role in physiological processes and in improving the growth parameters of orange68. reported that the foliar spraying of “Washington” navel orange by selenium at 0, 20, 40, 80 and 160 ppm improved significantly the shoot number, length, secondary shoots, leaf number per shoot and leaf surface area compared with control69. Small dose of Se can induces the growth of plant, increases the process of photosynthesis and helps in the balance of the necessary nutrient elements70. Foliar application of orange trees by Se at 0, 20, 40, 80 and 160 ppm raised the growth, yield, fruit quality attributes, leaf mineral content and enzymes activity, and the concentration of 40 ppm was the best results compared with the rest applied treatments69. The application of selenium at five different concentrations at 0, 10, 20, 40, and 80 mg L−1 on four different olive cultivars: “San Felice”, “Canino”, “Frantoio”, and “Moraiolo”. It was noticed that Se concentration between 10 and 40 mg L−1 increased fresh and dry weight of the explants and shoot lengths71.These results are in parallel with the reported results of a lot of authors, who mentioned that spraying Se is capable of raising the resistance of plants to biotic and abiotic exertion75. Spraying of saffron corms with Ag NPs at 20–60 mg/L increased significantly the number of roots, dry weight of leaves76, and also the growth of plants, the surface area, length of roots and shoots as well as chlorophyll content77. Photosynthesis, chlorophyll content, fresh weight, root and shoot length, as well as developing of seedlings in Brassica juncea seedlings were enhanced by Ag NPs compared with control78. Spraying cucumber with Ag NPs raised the number of leaves per plant and plant height79. Ag NPs raised leaf area, shoots and growth of plants81, content of pigments27, and the accumulation of biomass25 and improved shoot induction and proliferation. Ag NPs-foliar spraying increased plant growth attributes in terms of length of shoot, and root, and leaf area as well as leaf total chlorophyll16. The foliar spraying of onion with Ag NPs at 20 ppm increased plant height, total leaf and leaf content from total chlorophyll15. Additionally, the foliar application of Ag NPs at 10, 12.5, and 15 mL/L. positively increased the diameter of shoots, leaf surface area, leaf total chlorophyll in leaves in ‘Florida prince’ peach cultivar82.With the same trend, it was reported by many authors that Ag NPs had a good influence on growth, root- shoot ratio, and root prolongation86. Spraying 250 ppm K2Si2O5on ‘Flame seedless’ grapevines increased the leaf area and the length of main shoot as well as leaf total chlorophyll as compared to control in the two seasons41. Moreover, the exogenous application of K2Si2O5 at 0.05–0.1% on ‘Grandnaine’ banana increased greatly pseudostem height, and girth, leaf surface area, green leaves number38. Spraying K2Si2O5 at 0.05, 0.1 and 0.2% on ‘Keitte’ mango trees at the start of growth, just after fruit setting and 1 month later increased shoot length, leaf number per shoot, leaf area and total chlorophylls87. The spraying of orange cv. Olinda Valencia with K2Si2O5 increased shoot length, and diameter, leaf number per shoot, leaf area surface, as well as the height, volume and diameter of tree with respect with untreated plants88. Potassium silicate plays an important role in minimizing the production of ethylene and chlorophyll degeneration43. Spraying orange trees with K2Si2O5 at 0.1, 0.2 and 0.3%89, induced an obvious improving of shoot length and thickness, leaf area, leaf nutrient, pigment contents and total chlorophyll and the utilization of 0.2% was the superior treatment compared with control and the rest applied treatments.Silicon plays a great role in increasing photosynthetic rate, cell division, number of pigments, the absorption and transferring of water, and root growth as well as the tolerance against biotic and abiotic exertions2Si2O5, while they lessened obviously the fruit drop proportion with the respect of untreated trees. Besides, spraying 0.1 mg/L Se, 10 mL/L Ag NPs and 2 mg/L K2Si2O5, extremely increased fruit set percentage and fruit number, and minimized the fruit drop percentage compared with the other applied treatments or control in the two seasons. Spraying of Se, Ag NPs and K2Si2O5 reduced statistically the fruit cracking and sunburn proportion, while they increased significantly the fruit yield  which noticed prevention values of 32.96%. Also, it can be shown from Table 2Si2O5, and 0.05 mg/L Se showed inhibition proportions of 45.92, 44.81, and 43.70%, respectively, opposed to the growth of R. solani.Figure p-coumaric acid was observed in acetone extract from peels of fruits treated with 0.1 mg/L Se (9.56 µg/mL), 7.5 mL/L Ag NP (9.55 µg/mL), 0.05 mg/L Se (9.22 µg/mL) and in untreated (6.77 µg/mL). Benzoic acid was identified only in acetone extracts of peels from fruits treated with 5 mL/L Ag NP (9.66 µg/mL), 10 mL/L Ag NP (5.47 µg/mL), 0.5 mg/L Si (5.98 µg/mL) and in untreated fruits (5.98 µg/mL).Data in Table 2Si2O5 (10.77 µg/mL) and 10 mL/L Ag NP (7.88 µg/mL). Pyrogallol compound showed the highest abundant concentrations in acetone extract from peels of fruits collected from trees sprayed with 0.1 mg/L Se (12.39 µg/mL) and 1 mg/L K2Si2O5 (12.69 µg/mL) and in untreated (10.23 µg/mL). Gallic acid with high amount was observed in acetone extract from peels of fruits from trees treated with 10 mL/L Ag NP (12.44 µg/mL) and in untreated (9.44 µg/mL). The highest amount of ferulic acid was identified in acetone extract from peels of fruits treated with 0.025 mg/L Se (13.09 µg/mL), 0.1 mg/L K2Si2O5 (11.97 µg/mL) and 7.5 mg/L Ag NP (6.12 µg/mL). Salicylic acid was found at high quantity in acetone extracts from peels of fruits treated with 0.5 mg/L K2Si2O5 (12.44 µg/mL) and 0.05 mg/L Se (11.45 µg/mL). Cinnamic acid showed the highest amount in acetone extracts from fruit peels of trees treated with 5 mL/L Ag NP (14.33 µg/mL) and untreated trees (19.36 µg/mL). The highest concentration of ellagic acid was observed in acetone extracts from peels of treated trees with 0.05 mg/L Se (18.33 µg/mL) and 0.1 mg/L Se (7.69 µg/mL). The full HPLC chromatograms of the isolated and identified phenolic compounds are shown in Fig. The highest concentration of caffeic acid was seen in acetone extract from peels of fruits treated with 0.025 mg/L Se (12.06 µg/mL), 2 mg/L K109. The predominant compounds were gallic acid, ellagic acid, quercetin, caffeic acid, p-coumaric acid, and vanillic acid were found in peel extract111. Gallic and ellagic acids were presented in the methanolic extract of pomegranate peel112. Peel extracts demonstrated power antifungal activity against Aspergillus niger and Trichoderma reesei113. Peel extract showed markedly antifungal activity against A. parasiticus and A. parasiticus114. Peel extract noticed high activity against A. parasiticus and no activity against A. flavus115. The development levels of Alternaria alternata, Stemphylium botryosum, and Fusarium spp. growth rates were significantly inhibited by the peel extracts116 that were negatively linked to the rates of punicalagins, the major ellagitannins in pomegranate peels. F. oxysporum mycelia development was prevented by 62% (propanol extract) to 78% (water extract) of peel extracts117. The current study is in harmony with our previous works related to wood-biofungicides. Acetone extracts of Acer saccharum var. saccharum inner and outer bark in combination with citric acid when applied to wood showed strong bioactivities against Trichoderma viride, Fusarium subglutinans, and A. niger56, where, phenolic compounds p-hydroxy benzoic acid, gallic acid, salicylic acid, vanillin and o-coumaric acid, and ferulic acid were identified by HPLC. Wood samples of M. azedarach wood treated with flower aqueous extract of Acacia saligna showed good antifungal activity against Penicillium chrysogenum and moderate activity against Fusarium culmorum and Rhizoctonia solani59, and the HPLC showed the presence of benzoic acid, caffeine, and o-coumaric acid as most abundant compounds. F. culmorum and R. solani mycelial growths were inhibited significantly as wood treated with 3% methanolic extract of Musa paradisiaca peel that showed phenolic compounds of ellagic acid and gallic acid118.Therefore, the bioactivities of acetone treated-wood are linked to the existence of bioactive phenolic compounds. Peel extract has been reported to contain more phenolics than seed or pulp extracts2Si2O5 raised the shoot length, diameter, leaf chlorophyll, fruit set proportion, and fruit yield, fruit weight, length and diameter, total soluble solids, total, reduced and non-reduced sugars, while they minimized the juice acidity percent with the respect to control during our study. Fruit cracking and fruit sunburn were lessened markedly by the application of Se, Ag NPs and K2Si2O5 with respect to control. The application of 0.1 Se mg/L, 10 ml/L Ag NPs and 2 mg/L K2Si2O5 was more effective than 0.025 or 0.05 mg/L Se, 5 or 7.5 mL/L Ag NPs and 0.5 or 1 mg/L K2Si2O5 in improving the developing performance, yield and yielding components through studying times. The supreme treatments, which achieved the best results, was the application of 2 mg/L K2Si2O5 over the other applied treatments during our study seasons. Additionally, the extracts from fruit peels identified several bioactive phenolic compounds and the extracts observed good wood-biofungicide.Spraying Se, Ag NPs and K


Li Chang decoction (LCD), a Chinese medicine formula, is commonly used to treat ulcerative colitis (UC) in clinics.  This study aimed to identify the major components in LCD and its prototype and metabolic components in rat biological samples.  The chemical constituents in LCD were identified by establishing a reliable ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF/MS) method. Afterwards, the rats were orally administered with LCD, and the biological samples  were collected for further analyzing the effective compounds in the treatment of UC.  A total of 104 compounds were discriminated in LCD, including 26 flavonoids, 20 organic acids, 20 saponins, 8 amino acids, 5 oligosaccharides, 5 tannins, 3 lignans, 2 alkaloids, and 15 others . About 50 prototype and 94 metabolic components of LCD were identified in biological samples. In total, 29 prototype components and 22 metabolic types were detected in plasma. About 27 prototypes and 96 metabolites were discriminated in urine, and 34 prototypes and 18 metabolites were identified in feces.  The flavonoids, organic acids, and saponins were the major compounds of LCD, and this study promotes the further pharmacokinetic and pharmacological evaluation of LCD. Traditional Chinese medicine (TCM) attracts more attention in the world since it possesses reliable therapeutic efficacy in some complex diseases, especially chronic illness . The cheLi Chang decoction (LCD), a Chinese compound prepared from twelve Chinese medicine including Codonopsis Radix (CR), Notoginseng Radix et Rhizoma (NRR), Bletillae Rhizoma (BR), Sophorae Flos (SF), Glycyrrhizae Radix et Rhizoma (GRR), Cynanchi Paniculati Radix et Rhizoma (CPRR), Typhae Pollen (TP), Chebulae Fructus (CF), Atractylodis Macrocephalae Rhizoma (AMR), Ailanthi Cortex (AC), Coicis Semen (CS), and Halloysitum Rubrum (HR), has been commonly used to treat ulcerative colitis (UC) in clinics for over 20 years. UC is ain vitro, the prototypes in plasma, urine, and feces were further analyzed based on the similarity of mass spectrometry behavior (accurate molecular weight and secondary fragments) and chromatographic behavior (retention time). Metabolites were matched e mass defect filtering (MDF) caused by biotransformation and were further confirmed by MS/MS spectrum analysis.Ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF/MS) provides a rapid and reliable method to identify the component of natural medicine, which promotes the development of natural medicine component analysis and new drug discovery , 10. HerCodonopsis pilosula (Franch.) Nannf.), Atractylodis Macrocephalae Rhizoma , Chebulae Fructus , Halloysitum Rubrum (Lot: 190300991), Sophorae Flos , Typhae Pollen , Ailanthi Cortex  Swingle), Bletillae Rhizoma  Reichb. f), Coicis Semen  Stapf), Notoginseng Radix et Rhizoma  F. H. Chen), Cynanchi Paniculati Radix et Rhizoma  Kitag.), and Glycyrrhizae Radix et Rhizoma  was purchased from Kangmei Pharmaceutical Co., Ltd . Trigonelline, chebulic acid, gallic acid, 6,7-dihydroxycoumarin, corilagin, typhaneoside, rutin, hyperoside, liquiritin, nicotiflorin, lobetyolin, ginsenoside Re, ginsenoside Rg1, quercetin, ginsenoside Rb1, naringenin, 20S-ginsenoside Rh1, isorhamnetin, ginsenoside Rd, and glycyrrhizic acid, a total of 20 reference standards, were purchased from Chengdu Alfa Biotechnology Co., Ltd. The purity of each compound was more than 98% determined by the HPLC analysis. Methanol was of HPLC grade. Ultrapure water was obtained by the filtration of distilled water using a Milli-Q system . LC-MS grade acetonitrile was purchased from Fisher Scientific , and LC-MS grade formic acid was purchased from Sigma-Aldrich .LCD was prepared by the Pharmaceutical Department, Shenzhen Traditional Chinese Medicine Hospital. The Chinese medicine including Codonopsis Radix  were obtained from the Medical Experimental Animal Center of Guangzhou University of Chinese Medicine, China. Rats were housed in specified pathogen-free conditions (23 ± 2°C) under a 12-h light/12-h dark cycle and given free access to food and water. The protocols were approved by the Animal Experimental Ethics Committee of Guangzhou University of Chinese Medicine .The Medicine Codonopsis Radix, Atractylodis Macrocephalae Rhizoma, Chebulae Fructus, Halloysitum Rubrum, Sophorae Flos, Typhae Pollen, Ailanthi Cortex, Bletillae Rhizoma, Coicis Semen, Notoginseng Radix et Rhizoma, Cynanchi Paniculati Radix et Rhizoma, and Glycyrrhizae Radix et Rhizoma were weighed and mixed at a ratio of 6 : 3:3 : 6:3 : 3:6 : 3:6 : 2:6 : 2. The total weight of LCD is 245g, and the mixture was extracted twice by boiling in distilled water, and eight times distilled water (1960 ml) (w/v) was used to boil for 40 min in the first time, which changes to four times distilled water (980 ml) (w/v) in the second time. The two extracts were merged and centrifuged at 3,000 rpm, for 5 min to exclude dregs, and the supernatant was concentrated to 3.185 g/ml under reduced pressure at 55°C.The dose of LCD used in this experiment is 22.05 g/kg, which is the biological equivalent dose of humans. Three rats were fasted for 12 h with free access to drinking water, and then, the rats were orally administered with LCD. LCD was diluted to 2.205 g/ml with distilled water before giving to rat. Then, the blood samples were collected in the heparin anticoagulant tube through retro-orbital plexus at 0.25, 0.5, 1, 2, 4, 6, 8, 10, and 12 h. The plasma samples were obtained by centrifugation at 3000 rpm for 10 min. Samples of the same point were combined and stored at −80°C until use. Feces and urine samples were collected during 0–12 h.μl plasma was mixed with 600 μl acetonitrile (containing 0.2% methanoic acid). After vortexing for 2 min, the samples were centrifuged at 13000 rpm, 4°C, 10 min. Then, 400 μl supernatant was removed, dried under nitrogen gas, and redissolved in 100 μl acetonitrile (50%). Finally, the samples were centrifuged at 13000 rpm, 4°C, 10 min, and a 2 μl aliquot was injected into UPLC-QTOF-MS.For the plasma sample, about 200 μl supernatant was removed, dried under nitrogen gas, and redissolved in 200 μl acetonitrile (50%). Finally, the samples were centrifuged at 13000 rpm, 4°C, 10 min, and a 2 μl aliquot was injected into UPLC-QTOF-MS.For the fecal sample, about 300 mg of feces was weighed and mixed with 1 ml methanol. After the addition of magnetic beads, the samples were homogenized using tissue grinders  and centrifuged at 13000 rpm, 4°C, 10 min. About 200 μl supernatant was transferred and dried under nitrogen gas. The residues were redissolved in 400 μl acetonitrile (50%). Finally, the samples were centrifuged at 13000 rpm, 4°C, 10 min, and a 2 μl aliquot was injected into UPLC-QTOF-MS.For the urine sample, the mixed urine was centrifuged at 4000 rpm for 10 min, and 1 ml supernatant was loaded on pre-activated Sep-Pak Vac C18 columns . After washing with 1 ml ultrapure water and eluting with 1 ml methanol, the elution was collected and centrifuged at 13000 rpm, 4°C, 10 min. About 400 μm). The temperature was set at 35°C, and the flow rate was 0.3 ml/min. The mobile phases were 0.1% formic acid in water (A) and acetonitrile (B), with the optimized gradient as follows: 0–5 min from 3% B to 8% B, 5–11 min from 8% B to 30% B, 11–20 min from 30% B to 80% B, 20–21 min from 80% B to 95% B, 21–25 min was maintained at 95% B, and then back to the initial ratio and re-equilibration for 7 min.The separation equipment for this assay was Sciex Exion LC, and the chromatographic column was Waters Acquity HSS T3 , ±15 V. Sciex OS 1.6.1 was the basal data processing platform, and MetabolitePilot 2.0.4 software was applied for further metabolite fishing.The 5600 QTOF mass spectrometer  equipped with an ESI ion source was operated in positive and negative modes, and the mass range was The base peak chromatograms of LCD in negative and positive ion modes are shown in Generally, the characteristic components of AC were triterpenes, and the CS was characterized by lipids. However, both chemical categories were difficult to extract by water so that only flavonoids and organic acids in AC and CS were still detected and identified. P4 fructose, P6 sucrose, P7 raffinose, P8 stachyose, and P14 verbascose) and alkaloids (P5 trigonelline and P25 codonopsine) were characteristic components [− in the negative ion mode and [M + NH4]+/[M + Na]+in the positive ion mode. The successive neutral loss of hexose (−162 Da) and H2O (−18 Da) was used for identification. The typical fragmentation pattern of P14 verbascose is drawn in P5 trigonelline produced a [M + H]+ ion at m/z of 138.0546 and had fragment ions at m/z of 94, 92, and 78, which correspond to [M - CO2 + H]+, C6H6N+, and C5H4N+, respectively. P25 codonopsine showed [M + H]+ ion at m/z of 268.1546, and the fragment ions at m/z of 161, 88, and 58 were produced by penta-heterocycle cracking. The typical fragmentation pathways of P5 trigonelline are drawn in A total of 17 compounds were identified in CR. Among them, saccharides  − ion at m/z of 845.4899 and [M + H]+ ion at m/z of 801.4983. The characteristic product ions at m/z of 621 [M-Glc-H2O]+, 603 [M-Glc-2H2O]+, 441 [M-2Glc-2H2O]+, 423 [M-2Glc-3H2O]+, and 405 [M-2Glc-4H2O]+ were observed. The typical fragmentation pathways of P59 ginsenoside Rg1 are drawn in The neutral loss of Glc (162 Da) and Rha (146 Da) was characteristically appeared in saponin compounds. P47 dactylorhin A − ion at m/z of 887.3181 and [M + NH4]+ ion at m/z of 906.3601, while it had characteristic fragment ion at [M-Glc-H2O–H]− at m/z of 707, [M-gastrodin-H]− at m/z of 619, [M-gastrodin-Glc- H2O–H]− at m/z of 439, [M-gastrodin-Glc + H]+ at m/z of 459, [gastrodin]+ at m/z of 269, and [gastrodin-Glc]+ at m/z of 107. The typical fragmentation pathways of P47 dactylorhin A are drawn in A total of 5 characteristic compounds were detected in Bletillae Rhizoma. lorhin A , P56 gymlitarine  were strP37 quercetin 3-O-glucosyl-rutinoside [P39 manghaslin [P43 rutin [P45 isoquercitrin [P48 nicotiflorin [P50 narcissin [P70 quercetin [P79 isorhamnetin [P43 rutin was a vital constituent of Sophorae Flos. It had the [M + H]+ ion at m/z of 611.1607 and gave characteristic fragment ions at m/z of 465 and 303 by successive loss of Glc (162 Da) and Rha (146 Da). The typical fragmentation pathways of P43 rutin are drawn in Thirteen compounds were isolated from Sophorae Flos –20, and tinoside , P39 mannghaslin , P43 rut43 rutin –25, P45 ercitrin , P48 nictiflorin , P50 nararcissin , 28, P70uercetin , 29, 30,hamnetin ). In negP44 licuraside/liquiritin apioside, P46 liquiritin, P54 naringenin-7-O-glucoside, P60 violanthin, P67 pallidiflorin, P69 isoliquiritigenin + at m/z of 985.4644) observed fragments ions at [M-GlcA + H]+ at m/z of 809, [M-Glc-GlcA + H]+ at m/z of 647, [M-2GlcA-H2O + H]+ at m/z of 615, [M-Glc-2GlcA + H]+ at m/z of 471, and [M-Glc-2GlcA-H2O + H]+ at m/z of 453. The fragmentation pathways were similar to P59 drawn in A total of 23 compounds were discriminated in Glycyrrhizae Radix et Rhizoma, and 14 of them were flavonoids . DifferP89) + at m/z of 771.2327) was a flavonol, and fragment ions were observed after successive loss of Rha (146 Da) and Glc (162 Da). The fragmentation pathways were similar to P43 drawn in 2 (-44 Da) was the most usual fragments.Typhaneoside (P42), [M-H]P13 chebulic acid pyran-6-one + at m/z of 231, [M-H2CO2+H]+ at m/z of 185, [M-C3H4O + H]+ at m/z of 175, C10H10O2+ at m/z of 163, and C6H7+ at m/z of 79. The typical fragmentation pathways of P92 atractylenolide III are drawn in The characteristic compound in Atractylodis Macrocephalae Rhizoma was lactone . LaP9 L-malic acid + ion at m/z of 443.3881 and gave fragment ions at m/z of 425 by neutral loss of H2O (18 Da). The crack of C ring formed ions at m/z of 221 and 207. The typical fragmentation pathways of P104 are drawn in In Ailanthi Cortex, 4 compounds were attributed: briefly, 2 flavonoids , 1 carboic acid ), and 1 P59 ginsenoside Rg1 is taken as example, as shown in the XIC of LCD  of ginsenoside Rg1 in LCD  and retention time of chromatography were adopted to analyze the components in plasma, urine, and feces. C of LCD  and multC of LCD , and a p1 in LCD  and bio-1 in LCD  were simP43, P46, and P50) and saponins (P55 and P72) deserved higher attention as the five components were observed in all three bio-samples besides that were common to organisms . In addition, 12 compounds were just observed in the fecal sample, mainly including some alkaloids (P25 and P65), flavonoids , saponin (P74), and other small molecules . These compounds may not be absorbed into the blood, but are still effective in regulating gut microbiota. The detailed information about the distribution of components in plasma, urine, and feces is summarized in Based on the above principles, a total of 50 components were matched in biological samples, and these components would play a key role in explaining the mechanism of LCD in the future. In particular, flavonoids  + ketone formation (phase I).” Furthermore, neutral loss of glycosides and methylene was both observed in the MS/MS spectra of P50 and M70/71, which implied the similar skeleton. That was to say, these compounds were structurally related, and M70/71 could be the metabolites of P50. As a result, a total of 107 metabolites were matched with 25 prototypes in plasma, urine, or feces. The network of prototype-metabolite matching is drawn as in Furtherly, the phase I and phase II metabolic regularity, as well as the similarity of secondary mass spectrum profile, was used to identify the metabolite. Those metabolites were annotated through automatic matching with prototype components by MetabolitePilot Software. Briefly, MetabolitePilot operated prototype-metabolite matching through mass defect filter (MDF), characteristic product ion filter (PIF), and neutral loss filter (NLF). As shown in P2 arginine [P5 trigonelline [P59 ginsenoside Rg1 [P69 isoliquiritigenin [P82 ginsenoside Rd [P84 glycyrrhizic acid [P15 isoleucine [P17 uridine [P21 guanosine [P23 gallic acid [P43 rutin [P51 vanillic acid [P70 quercetin [P72 ginsenoside Rb1 [P81 betulin [κB pathway. P8 stachyose increased beneficial microbiota and bacterial diversity to alleviate colitis mice [P45 hyperoside ameliorates ulcer colitis mice through MKRN1-mediated regulation of PPARγ signaling and Th17/Treg balance [arginine –56, P5 tonelline , P59 ginside Rg1 , P69 isooside Rd , 61, andzic acid –64 wouldoleucine , P17 uri uridine , 66, P21uanosine , P23 gallic acid , 69, P4343 rutin , 71, P51lic acid , P70 queuercetin , 74, P72side Rb1 , and P81 betulin were conftis mice . P45 hyp balance . The eff


As most porous organic cages are soluble, solution processability can be a possible way to regulate the porosity of such materials. Herein, a triptycene-based cage (TC) is demonstrated to be stable in acid, base or boiling water. Meanwhile, its porosity can be tuned by adjusting the solution-state assembly processes. TC molecules crystallized slowly from solution exhibit nearly no porosity to nitrogen (off-state). While, after rapid precipitating from methanol/dichloromethane solution, the obtained TC (TC-rp) is in a porous state and exhibit a high BET surface area of 653 m Here, a kind of triptycene-based cage is demonstrated to have good chemical stability in acid, base and boiling water. Moreover, its porosity can be tuned by varying the solution-state assembly processes. Different from network polymers and frameworks, organic cages contain “extrinsic” and “intrinsic” pores, which refer to the pores located between molecules or within molecules, respectively.10 In principle, the organic cages are porous to some guests when their intrinsic and extrinsic pores are interconnected, which is deeply influenced by the assembly patterns.1 Such characteristic offers an opportunity to control the porosity of organic cages which is not available for insoluble organic and inorganic frameworks.Porous organic cages,11–15 However, most POCs exhibit non-porosity to the guest molecules because the internal cavities of the cages are blocked by their window-to-arene assembly patterns.16,17 To tune the POCs' porosity from off to on, coupling POCs into frameworks could be a fashionable method, while such cage-based polymeric frameworks were generally constructed by covalent synthesis or coordination chemistry, which would lead to the sacrifice of their solution processability.18–22 Other methods were also developed, for example, Cooper and co-workers realized guest-induced “on–off” porosity transformation by virtue of POCs' noncovalent intermolecular packing.23,24 Doonan et al. controlled the porosity of POC by kinetic methods.25 In a recent study of Banerjee et al., non-porous organic cage could be converted to a porous polymorph by treatment with DMF.26 But still, the porosity tuning is still a challenge for POCs.Versatile POCs have been successfully synthesized by diverse building blocks and synthetic methods and applied in different fields with varying porosity.27–30 and supramolecular hosts.31–34 Mastalerz's group synthesized a series of remarkable triptycene-based cages through reversible reactions of imine chemistry or boronic acid condensation.35–39 The cages based on reversible reactions were always obtained with high yields because of the self-correcting mechanism. Meanwhile, they usually lacked good chemical stability. Since organic cages with better chemical stability have more widespread applications, recently, Mastalerz's group also obtained several chemically stable organic cages based on previously reported imine cages by the conversion of imine groups to more stable amide groups or carbamate groups.40,41 But such stable triptycene-based cages were rarely reported. Herein, we found the ethynylene-linking triptycene-based cage (TC) from a Glaser coupling reaction had excellent stability in acid, base or boiling water. Moreover, the porosity of TCs were tunable by controlling their assembly processes.Triptycene, with rigid three-dimensional structure, is an attractive building block for porous materials42 As shown in the spectrum of Proton nuclear magnetic resonance (1H NMR)  spectrum of TC showed a peak at 2214 cm−1 corresponding to the acetylene signal  Å, b = 20.1018(8) Å, c = 21.0854(8) Å, α = 113.054(4)°, β = 97.737(3)°, γ = 114.431(4)°) contained two cage molecules and twelve 1,3,5-trimethylbenzene molecules. The solvent molecules were distributed disorderly around cage molecules.The triptycene-based cage (TC)  was synt) Fig. S1, there wl Fig. S2, which w43–45  surface area of only 7 m2 g−1 (Langmuir surface area was 12 m2 g−1)  Fig. S4.1H NMR and FT-IR spectra of TC-rp were almost the same as that of TC  , suggesting the existence of abundant micropores. The pore size distribution calculated using DFT method also confirmed the presence of micropore structure  Fig. S8. It seemtructure . The CO2at 298 K , respector TC-rp  and S9†.1H NMR spectra, the FT-IR spectra and the PXRD patterns of TC before and after treatment were basically the same  synthesized by the copper-mediated modified Eglinton–Glaser oxidative coupling reaction was demonstrated to have excellent chemical stability in acid, base or boiling water. The single crystal structure of TC showed a window-to-arene fashion which blocked its porosity, after rapid precipitation in a methanol/dichloromethane solution, TC-rp was porous to N42 the mixture of CuCl , Cu(OAc)2  and dry pyridine (15 mL) was stirred under argon at 60 °C for 20 min. Then a solution of 2,7,14-tri[(4-ethynylphenyl)ethynyl]triptycene  in dry pyridine (3 mL) was added dropwise, and the mixture was stirred at the same temperature for another 10 h. After cooling down to room temperature, the solvents were removed under reduced pressure, and the residue was dissolved in CH2Cl2 (50 mL) and washed with 1 M aqueous HCl (3 × 30 mL). The aqueous solution was extracted with CH2Cl2 (2 × 30 mL), the organic phase was combined and dried with Na2SO4. TC was purified by chromatography with silica gel  to give a white solid .TC was synthesized following the literature:154H118): Mr = 1968.64, triclinic, space group P1̄, a = 18.4087(7) Å, b = 20.1018(8) Å, c = 21.0854(8) Å, α = 113.054(4)°, β = 97.737(3)°, γ = 114.431(4)°, V = 6113.3(5) Å3, Z = 2, ρcalcd. = 1.047 g cm−3, μ = 0.447 mm−1, reflections collected 40 251, data/restraints/parameters 23 863/146/1345, GOF on F2 1.317, final R1 = 0.1355, wR2 = 0.3514, R indices : R1 = 0.1755, wR2 = 0.3947, largest diff. peak and hole: 1.51 and −0.76 e Å−3. Which is similar to the literature:42 C136H94: Mr = 1728.11, triclinic, space group P1̄, a = 18.417(3) Å, b = 20.223(3) Å, c = 21.134(3) Å, α = 101.472(4)°, β = 109.674(2)°, γ = 114.6330(10)°, V = 6185.7(17) Å3.Crystallographic data for TC (CThere are no conflicts to declare.RA-010-D0RA00128G-s001