Ligaria cuneifolia (R. et P.) Tiegh. (Loranthaceae) is a South American hemiparasitic species with antioxidant, antitumoral, antimicrobial, and antilipidemic activities attributed to its polyphenolic content. We studied the polyphenolic pattern of L. cuneifolia during different phenological stages: flowering, fruiting, and post-fruiting. The highest total phenolic content was found in stems at post-fruiting  and fruiting , followed by post-fruiting leaves . Flavonoids accumulated at higher levels in leaves and hydroxycinnamic acids in leaves at flowering and post-fruiting. The polyphenolic pattern was similar between organs from wild plants and in vitro cultures, although at a significantly lower level in the latter ones. The performance of calli growing under a 16 h photoperiod in a modified White medium with 1-naphthalene acetic acid (2.50 μM) and Kinetin (9.20 μM) was better than in the dark. When calli grew in media only with auxins , its growth and polyphenolic content improved. Cell suspensions with 2.50 µM NAA and 9.20 µM KIN grew slowly and produced very small amounts of polyphenols. As for the antioxidant activity, it was detected in all samples (approximately 1000 µmol trolox eq·g−1 DW) except fruits, where a lower value was found (328 µmol trolox eq·g−1 DW). In vitro cultures have the lowest antioxidant activity when compared to methanolic extracts from organs of wild specimens. Finally, antimutagenic or mutagenic activity in wild plants and in vitro culture extracts was not detected by the Ames test. Ligaria is represented by L. teretifolia (Rizzini) Kuijt, endemic to Brazil, and L. cuneifolia (Ruiz & Pav.) Tiegh, from Uruguay, Brazil, Perú, Chile, and Argentina [L. cuneifolia is a hemiparasite species that, due to its morphological similarities and growth behavior, was used as a substitute for the European mistletoe  by the first European immigrants [L. cuneifolia use as antihemorrhagic, abortive, emmenagogue, and oxytocic and against cephalgia, gastralgias, sore throat, and hypothermia [L. cuneifolia extracts decreased cholesterol and lipid blood levels in rats [L. cuneifolia use for the treatment of cardiovascular diseases and cancer is a promising alternative [L. cuneifolia specimens growing on different hosts and coming from different regions. QE could be found free or as a 3-O-glycoside derivative with glucose, xylose, rhamnose, or arabinose. Leucoanthocyanidins, catechin-4-ß-ol, and proanthocyanidins (PA) as polymers, oligomers, and dimers that produced cyanidin after hydrolysis were also reported [O- rhamnoside, QE-3-O- rhamnoside, QE-3-O--arabinofuranoside, and QE-3-O--arabinopyranoside. Production of secondary metabolites, including plant polyphenolics, depends on numerous factors, such as growth conditions , plant phenological stage, and organ. Changes in the polyphenolic content are related to variations in the expression of the genes encoding the activity of enzymes involved in their biosynthesis [L. cuneifolia and to harvest it at the highest levels of bioactive compounds. In this work, we studied the dynamics of polyphenolic production in different organs of L. cuneifolia wild specimens in different phenological stages.Mistletoes are plant species from the Loranthaceae and Santalaceae families, spread worldwide. Among the approximately 1000 Loranthaceae species, 300 are endemic to America. The genus rgentina . L. cuneothermia . On the  in rats ,5, had a in rats , produce in rats , had a b in rats , and dis in rats ,10,11. Ternative ,13. Phytreported . Dobreckreported  has alsoynthesis . ConsequL. cuneifolia in vitro cultures as a source of plant material appears as an environmentally friendly strategy to avoid the excessive exploitation of the species. In addition, these cultures have the advantage of providing plant material of uniform quality produced under controlled environmental conditions free from pests and diseases. There are only a few reports about the establishment of in vitro cultures from Loranthaceae species [L. cuneifolia on White medium with 500 mg L−1 casein hydrolysate, 100 mg L−1 myo-inositol, B5 vitamins, 4% (w/v) sucrose, 2.50 µM NAA, and 9.20 µM KIN as plant growth regulators (PGRs) and a 16 h photoperiod [On the other hand, the establishment of  species ,18. We htoperiod . The behtoperiod ,22,23. Atoperiod . We measFinally, we tested the antioxidant, mutagenic, and antimutagenic activities of wild specimens and in vitro culture extracts.p < 0.05) among organs  in the three studied phenological stages . The highest values corresponded to stems at post-fruiting (214 ± 12.1 mg GA eq·g−1 DW) and fruiting (209 ± 13.7 mg GA eq·g−1 DW), followed by post-fruiting leaves . The lowest values were found in fruits , the highest content was found in leaves in the three analyzed phenological stages .p < 0.05) were found between leaves and stems; however, no significant differences (p > 0.05) were found among phenological stages. The highest values corresponded to leaves harvested at the flowering (3.02 ± 0.62 mg CA eq·g−1 DW) and post-fruiting stages , significant differences  content were found among the different organs and phenological stages  were found at lower levels (−1 DW), followed by leaves (19.64 ± 0.24 mg g−1 DW) in the post-fruiting stage. The lowest values corresponded to fruits (4.32 ± 0.04 mg g−1 DW).Catechin was the main compound found in wild plants. QE glycosides . However, the calli morphology differed, being green and compact under light, or whitish and friable in the dark  were found between cultures maintained under illumination or in darkness. Maximum yields for calli grown under light were 7.84 ± 0.01 mg GA eq·g−1 DW at 4th week, 1.86 ± 0.16 mg QE eq·g−1 DW and 0.26 ± 0.07 mg CA eq·g−1 DW at the 2nd week of culture, respectively. For calli grown in darkness, maximum yields were 6.62 ± 0.01 mg GA eq·g−1 DW and 1.11 ± 0.01 mg QE eq·g−1 DW at the 2nd week, and 0.12 ± 0.06 mg CA eq·g−1 DW at the 1st week of culture, respectively. PA was not detected by spectrophotometry in any case. On the other hand, TLC revealed more intense spots in extracts from calli grown under 16 h photoperiod than in darkness. The PA(+) catechin was only detected by this method (−1) than in darkness (0.40 ± 0.02 mg g−1) methanolic extracts  or 2,4-D , while GI and µ were lower with NAA . The doubling times (dt) of calli in media with 2.5 µM 2,4-D, IAA, or NAA as PGR were 30.27 d, 42.27 d and 68.35 d, and the maximal biomass was 513 ± 73 mg FW at the 7th week, 455 ± 49 mg FW at the 8th week, and 453 ± 47 mg FW at the 6th week, respectively.All PGRs at the lowest concentration (2.5 µM) with inoculum sizes between 250 and 500 mg FW per tube were effective in inducing growth . On the The spectrophotometric analysis has determined that the higher TPC corresponded to treatments with IAA and NAA. As for FL, the higher amounts corresponded to calli growing in media with IAA and 2,4-D. No significant differences were found in HCA content among treatments. Finally, the highest amount of PA corresponded to calli growing in the presence of IAA and NAA .Mono-dimensional and bi-dimensional TLC confirmed that calli have the same pattern of polyphenolic compounds a,c as th−1) was not effective in inducing growth. On the other hand, with a higher inoculum size (25 mg mL−1) the growth curve from cell suspensions was sigmoidal with a lag phase of 7 days, followed by an exponential phase that extended up to the 28th day, when the culture entered the stationary phase . Evans blue and FDA tests proved that all cultures were viable  except in the case of fruits that showed a lower value  than in darkness (23 ± 7.54 µmol trolox eq·g−1 DW) with the PGR ratio 2.50 µM NAA and 9.20 µM KIN. In calli cultures growing with auxins, the highest value was obtained in CWM with 2.5 µM IAA as PGR (137 ± 7.92 µmol trolox eq·g−1 DW). As for cell suspension cultures , their antioxidant activity was 44.1 ± 9.84 µmol trolox eq·g−1 DW . The results of the positive controls  for both strains gave values of CFU (colony forming units) greater than 1000 without metabolic activation, while when metabolic activation was performed, CFU values were 704.7 ± 55 for TA 100 and 919.67 ± 43.02 for TA 98.As can be seen in p > 0.05), the results were compared using the Kruskal–Wallis test, and no significant differences were observed (p > 0.05) between the positive controls and the extracts tested. This indicates that the extracts obtained from wild specimens of L. cuneifolia did not show an antimutagenic effect.Once the assumption of homogeneity of variances was confirmed by the Bartlett test  Mol (Fabaceae), a common host of L. cuneifolia. Previous reports from our group also evaluated specimens from Barreal , in the province of San Juan, which is also part of the Cuyo geographical region, the Neotropical phytogeographical region and the Chaqueño domain. In that study, samples growing on Prosopis chilensis (Molina) Stuntz, Prosopis flexuosa D.C., and Geoffroea decorticans (Gillies ex Hook. & Arn.) Burkart, also from the Fabaceae family, were collected during the post-bloom stage. For comparative purposes, specimens from the Catamarca province  developing on different hosts  Griseb. (Zygophyllaceae), Geoffroea decorticans (Gillies ex Hook. & Arn.) Burkart, and Prosopis flexuosa D.C.) were also included in our prior study [L. cuneifolia FL fingerprint is highly conserved among geographical regions, climate conditions, and host families.Phenolic compounds are redox-active species widely distributed in the plant kingdom. Among them, we focused our analysis on flavonoids (FL), hydroxycinnamic acids (HCA), and proanthocyanidins (PA) that, besides lectins, betulin, and betulinic acid, were the major components found in tivities . Althougtivities ,26. In t summers . Our resor study . This arO-glycosides and galloyl glycosides were also present at lower values. Our results are in agreement with those found in V. album, in which leaf extracts showed higher concentrations of total phenolics and flavonoids compared to fruit and seed extracts [C. palirus leaves. Several authors attributed the increase in phenolics content to higher intensity of solar radiation [Vaccinium myrtillus exposed to sunlight [L. cuneifolia where the post-fruiting stage showed the highest polyphenolic values, coinciding with the summer season when solar radiation reaches its maximum. HCA were higher in leaves, and were not affected by the phenological stage. These results correlate with those found in Geoffroea decorticans extracts, in which leaves showed higher levels of HCA derivatives when compared to stems [Calluna vulgaris (L.) Hull, reported that the higher amounts of polyphenolic compounds, including HCA, were found in stems and leaves at all phenological stages except during flowering, while no significant differences in PA content were observed among different organs and phenological stages. As in L. cuneifolia, PA were also found in adult plants of the Mexican mistletoes Phoradendron bollanum and Viscum album subsp. austriacum [O-glycosides and galloyl glycosides were also present but at lower values.With regard to polyphenolic content, stems and leaves displayed the highest values, especially in the fruiting and post-fruiting stage. From a climatological point of view, temperature variation is not significant but the average precipitation is at its peak, so this period could be considered the “humid season.” This particular set of conditions favors plant metabolism, which results in a polyphenolic increase. Aerial parts are largely exposed to environmental conditions and different stressors, so a rapid turnover is generally observed. HCAs are prevalent in leaves, followed by stems, and are not significantly affected by the phenological stage. Polyphenols, and particularly FL, are secondary or “specialized” metabolites that provide protection and are involved in defense mechanisms. In terms of concentration, leaves showed the highest levels, especially at the post-fruiting stage where catechin is the predominant compound. This is consistent with the role of tannins as defense compounds. QE-3-extracts . Similaradiation ,30,31. Tsunlight . These rto stems . Chapel to stems , workingstriacum . HPLC reMicrolaena stipoides, Eurycoma longifolia, and Themeda triandra, which suggests the presence of condensed tannins and their precursors. The resulting pink coloration could indicate the existence of flavan-4-ols that produce anthocyanidins in the presence of concentrated HCl [Arabidopsis thaliana seedlings in three main zones: the cotyledonary node, the hypocotyl, and the root apical end [L. cuneifolia embryos.A selective spatial distribution was also seen in histochemical analysis, where flavonols and PA were located near the leaf surface, mostly in the epidermis and first parenchyma layers, which is consistent with the results reported in other plant species. Positive results were obtained in the reaction with vanillin/HCl, in ated HCl . Ellis [ated HCl  found taical end , which iViscum album calli [L. cuneifolia calli presented a similar pattern of polyphenolic compounds as the adult plant regardless of the tested conditions. Illumination (16 h photoperiod) seemed to be a fundamental requisite for long-term maintenance and to polyphenolic production of calli cultures in L. cuneifolia. The positive effect of light on polyphenolic compounds accumulation could be related to the induction of some enzymes that participate in their biosynthetic pathway [Tecoma stans and Kumar [Basella rubra.As far as we know, there are few reports about the establishment of in vitro cultures from hemiparasitic plants for plant secondary metabolite production, e.g., chlorogenic acid production by um calli . Most ofum calli ,39,40, wum calli ,41,42. T pathway . Shipilo pathway , working pathway  working nd Kumar  working Considering the modest performance of calli in the previous assays and as PGRs are culture media components with utmost relevance in in vitro culture growth and biosynthetic capacity, we tested the influence on growth and polyphenolic content of calli growing on media with different auxins  at three concentrations . IAA (2.5 μM) appeared as the most favorable plant growth regulator for producing polyphenolic compounds. However, their amounts were low in comparison with those from organs of wild plants, except for PA. This group of polyphenols is rarely studied for in vitro production by plant cells ,48,49.−1), medium (100–160 g PF L−1), and high (>200 g PF L−1). Álvarez [S. elaeagnifolium starting from 20% (v/v) inoculum density (GI = 4) in MS medium with 50 µM NAA and 0.25 µM KIN. In the case of Tilia americana, batch suspensions  were started from 6% (v/v) inoculum density in MS medium supplemented with 2 mg L−1 2,4-D and 0.5 mg L−1 KIN [Capsicum baccatum, the maximum GI (3.11) was achieved when using modified MS with the addition of 2,4-D (1.14 mg L−1) and BAP (0.23 mg L−1), with 12.5 g L−1 inoculum size [Acer pseudoplatanus, 48 h for N. tabacum, 36 h for Rosa sp., and 24 h for Phaseolus vulgaris, among others [L. cuneifolia suspension cultures, under the conditions tested here, showed limited growth when compared with those reported for other plant species. It is necessary to continue optimizing other variables, such as the carbon source, the base culture medium, or the addition of other combinations of growth regulators to achieve better biomass yields for its subsequent scaling up to the bioreactor.As for cell suspension cultures, the presence of starch in cells was previously reported for calli cultures of Loranthaceae, as well as in suspension cultures of other plant species ,51,52. A Álvarez  reached  L−1 KIN . In Capslum size . Biomassg others . It is nL. cuneifolia [V. album, which was attributed to the presence of phenolic compounds in earlier studies [Regarding antioxidant activity, we found higher values in wild plant extracts, where polyphenol contents were also higher when compared to in vitro cultures. These in vitro results correlate with the in vivo and ex vivo experimental designs previously performed in neifolia . In acco studies ,60.The Ames test is used to evaluate the mutagenic activity of a given chemical. In addition, it can be adapted for the detection of “protectants” or substances that decrease mutagenic action. We did not detect mutagenic or anti-mutagenic activity in any case.L. cuneifolia (2–4 years old) growing on Vachellia caven (Mol.) Mol. was collected from Villa de Merlo, San Luis . A specimen was deposited, under the name BAF 9018, in the herbarium of the “Juan Aníbal Domínguez” Pharmacobotany Museum at the Faculty of Pharmacy and Biochemistry, University of Buenos Aires. In 2018, branches of healthy specimens of L. cuneifolia were cut during the flowering, fruiting, and post-fruiting seasons . Flowering corresponded to the months of March and April , fruiting corresponded to the months of November , and post-fruiting to December . Finally, they were dried at room temperature in a dark and aerated area for approximately one week and were stored in a desiccator until they were used for polyphenol extraction. For histochemical analysis, fresh material was used. For that, medium- to large-size branches of L. cuneifolia-containing fruits were cut and moistened paper towels were placed around the cutting site. These were placed in sealable plastic bags and transferred to Ciudad de Buenos Aires in camping refrigerators or polystyrene boxes containing cooling gels, to keep the material as fresh as possible until its arrival at the laboratory, where it was kept at 4 °C until processing.Plant material of v/v) methanol, and finally with 50% (v/v) methanol. Each stage was performed at room temperature for 24 h. The supernatants were pooled and stored at −20 °C for further analysis.Dry leaves, stems, and flowers from adult wild specimens were separately ground in a rotary knife grinder (IKA). In both cases, a three-stage extraction was performed, first with pure methanol, then with 80% . The results were expressed in mg GA eq·g−1 DW. All experiments were performed in triplicate.Total phenolic content was determined using a modified Makkar  procedurTotal flavonoid contentw/v) aluminum trichloride (AlCl3), 10 µL of 1 M potassium acetate, and 120 µL of distilled water were added to each well of a 96-well plate. For plant extracts, 25 µL of extract, 45 µL methanol, 10 µL of 10% (w/v) AlCl3, 10 µL of 1 M potassium acetate, and 120 µL of distilled water were added to each well of a 96-well plate. Then, the mixture was homogenized and incubated for about 30 min in the dark. The absorbance of the solution was measured at a wavelength of 415 nm in a microplate reader . Total flavonoid content was calculated with a QE calibration curve (stock solution 1 mg QE mL−1 in methanol) and the results were expressed as mg QE eq·g−1 DW. All experiments were performed in triplicate.Total flavonoid content was determined using a modified Rafi  protocolHydroxycinnamic acid content−1 in absolute ethanol stock solution). The results were expressed as mg chlorogenic acid eq·g−1 DW. All experiments were performed in triplicate.HCA content was determined using a modified Ricco  procedurProanthocyanidin contentw/v) vanillin and 75 µL 32% (v/v) hydrochloric acid were added to each well of a 96-well plate. Then, the mixture was homogenized and incubated for about 15 min in the dark. Absorbance was measured at 500 nm in a microplate reader . Total PA content was calculated with a calibration curve prepared with various concentrations of (+)-catechin. Results were expressed as mg catechin eq·g−1 DW.PA content was determined by a modified Horszwald and Andlauer  method. p < 0.05 were considered significant.Statistical analysis: ANOVA and when corresponding a post hoc Tukey’s analysis were performed. Jamovi  and R 66 statistv/v) methanolic solution of the NP reagent and PA with a 5% (w/v) vanillin:HCl ethanolic solution [The qualitative analysis of polyphenols by thin-layer chromatography (TLC) was performed using Silica Gel 60 (Merck) as stationary phase and ethyl acetate:formic acid:acetic acid:water (100:11:11:23) as mobile phase. Flavonoids and HCA derivatives were revealed with a 1% .Considering the results obtained by spectrophotometric methods, extracts from calli, cell suspensions, and adult specimens were selected for HPLC-UV analysis . Each anCross sections of leaves, primary stems, and embryos were made with a sliding microtome. The sections were stained with 1% AEDBE methanolic solution for the detection of flavonoids and HCA derivatives, or with 5% vanillin:hydrochloric acid for the detection of PA . All sec−1 casein hydrolysate, 100 mg L−1 myo-inositol, B5 vitamins, and 4% (w/v) sucrose) supplemented with 2.50 µM NAA, 9.20 µM KIN, and 6.5 g L−1 of agar (micropropagation grade); pH was adjusted to 5.6–5.8. One half of the glass tubes were cultured in a chamber with a 16/8 h (light/darkness) photoperiod given by fluorescent lamps  and the other half in a chamber in darkness. Cultures were maintained at 24 ± 2 °C. After 60 days, the frequency of calli production (%) was estimated. Then, the established calli were transferred to identical fresh media and samples were taken once a week for 16 weeks to determine fresh weight (FW) and dry weight (DW) and for polyphenol qualitative and quantitative analysis as described in Plant material for in vitro cultures was collected, preserved, and shipped to the lab as described in n = 29 for each treatment). Cultures were maintained in a growing chamber at 24 ± 2 °C, under a 16 h light photoperiod using fluorescent daylight lamps as previously detailed. Samples were taken every seven days during 8 weeks to measure FW and DW and for polyphenol qualitative and quantitative analysis as described in td) were calculated according to Equations (1)–(3), respectively. Statistical analysis was performed by ANOVA once the assumptions were confirmed. In case of significance in ANOVA, a Tuckey’s post hoc test was carried out. If lack of homoscedasticity was detected, a Welch’s heteroscedastic F-test [Calli were transferred to glass tubes containing CWM with the addition of 1-naphthalene acetic acid (NAA), 3-indole acetic acid (IAA), or 2,4-dichloro phenoxy acetic acid  at 2.5, 5.0 or 10.0 µM. After four weeks of adaptation to the new media, the growth curve was initiated in the identical fresh medium using “low” inocula (100 to 250 mg FW) or “high” inocula sizes . x: biomass at time t; x0: initial biomassEquation (1): formula for calculating the growth index : formula for calculating the specific growth rate (µ). td). µ: specific growth rateEquation (3): formula for calculating the doubling time  and 750 mg/flask (high). The flasks were transferred to an orbital shaker at 110 rpm, 24 ± 2 °C, and maintained in the dark. Samples were taken on days 7, 14, 21, and 28 to determine viability (Evans blue and fluorescein diacetate tests ), FW, anin vitro culture extracts were obtained as described in −1 DW and descriptive statistical analysis was performedWild plant and L. cuneifolia wild plants and in vitro cultures. Salmonella typhimurium TA98 and TA100 strains were used in order to detect frameshift mutations and base-pair exchanges, respectively [−1 of the selected control strains (TA98 or TA100), and 0.5 mL of S9 mix for the assay with metabolic activation or phosphate buffer (pH 7.4) in the assays without metabolic activation. Then, 50 µL of the dilution of the wild plant or calli extract was added. This mixture was gently homogenized with a vortex and plated with a minimal medium for its incubation at 37 °C for 48 h. Then, the revertant His+ colonies were counted. In all the tests, negative controls were included to know the rate of appearance of spontaneous revertant colonies. As positive controls, B1 aflatoxin (2 µg/plate) and 2-aminoanthracene (5 µg/plate) were used for the assays with metabolic activation in both strains, and sodium azide (5 µg/plate) and 2-aminofluorene (10 µg/plate) for assays without metabolic activation, in TA 100 and TA 98, respectively. Results were expressed as the reversion coefficient or C.R. = No. of revertant colonies per assayed plate/No. of revertant colonies per control-spontaneous-plate.The Ames test was carried out to evaluate the possible mutagenicity of the extracts of ectively . Tests wA volume of 0.5 mL of S9 mix, 50 µL of B1 aflatoxin, and 50 µL of tester strain suspension was placed in each tube and brought to a final volume of 2 mL with water. 50 µL of water were added to the positive control, and 50 µL of wild plant extract to the unknown sample. A blank without aflatoxin was also performed in which 100 µL of water and an extract blank without B1 aflatoxin and with 50 µL of water were used. The results were statistically analyzed using the Bartlett test to determine the homogeneity of variances and then compared using the Kruskal–Wallis test.® Methanol was supplied by Merck . Formic acid was purchased from Baker . Ultrapure water was generated with a Barnstead Thermo Scientific™ . Quercetin-3-rhamnoside (Q-3-O-Rh) was from Extrasynthese ; catechin (C), quercetin-3-O-glucoside (Q-3-O-G), quercetin-3-O-xyloside (Q-3-O-X), quercetin-3-O-arabinofuranoside (Q-3-O-AF), quercetin-3-O-arabinopyranoside (Q-3-OAP), and chlorogenic acid (CA) were from Sigma . The other chemicals, standards, and solvents were purchased from Sigma-Aldrich .White medium , sucroseL. cuneifolia appeared to be a very challenging species to be introduced in in vitro culture. Different PGR ratios, light conditions, and inoculum sizes were tested, and only a slight improvement in growth and polyphenolic content was achieved. We established that, among the conditions tested, calli grew and produced FL, HCA, and PA in CWM with 2.5 µM IAA and 16 h photoperiod, but at low levels compared to wild plants. As for cell suspension cultures, the conditions tested were not successful either for growth or for polyphenolic production. Other media components such as carbon and nitrogen source and PGR ratios must be assayed to establish the optimal conditions to obtain cultures with better growth. Then, other strategies such as elicitation or two-step cultures (culturing cell suspensions in a medium that promotes growth and then transferring them to a medium that favors polyphenolic production) could be examined to obtain improved yields. No mutagenic or antimutagenic activity was detected in wild plants or in in vitro culture methanolic extracts.Regarding wild plants, the post-fruiting stage proved to be the best time of harvesting to obtain the maximum amounts of polyphenolic compounds. Further experiments will be performed to continue analyzing the influence of different host plants on the polyphenolic profile.


Arthrospira platensis (A. platensis)—a genus of nonheterocystous filamentous cyanobacteria—is used in industrial applications and as a food supply. The current research work aims to study the physicochemical characteristics of A. platensis indigenous to the Moroccan Atlantic coast at Laayoune (Foum El Oued lagoon). The contents of proteins, carbohydrates, vitamins, lipids, minerals, heavy metals, energy value, humidity, ash, pigments, and tannins in A. platensis were investigated using protocols as described in the earlier literature. The values of protein, carbohydrate, and lipid contents in A. platensis were 58.9 ± 0.07, 14.67, and 45.54% respectively. The values of vitamins B2 and B3 dosed in A. platensis were 1.31 ± 0.19 and 30.8 ± 0.001 mg/kg, respectively. The values of heavy metals including lead and chromium were 70 ± 4.5 and 5 ± 0.5 PPB (parts-per-billion), respectively; however, no trace concerning cadmium was detected. The values of energy value, humidity, and ash content were 346.48 ± 0.21, 11.6 ± 0.17%, and 9.1 ± 0.21% kcal/100 g, respectively. The results of pigment content showed the presence of chlorophyll b, chlorophyll a, and carotenoids of 37.506 ± 3.38, 26.066 ± 3.08, and 9.52 ± 0.22 mg/g, respectively. The results obtained revealed that A. platensis indigenous to the Moroccan Atlantic coast at Laayoune was found to be very rich in proteins, carbohydrates, vitamins, minerals, ash, and pigments and lower in heavy metals and saturated fats when compared with species investigated in the literature. Thus, A. platensis indigenous to the Moroccan Atlantic coast at Laayoune fulfills the requirements for being used as dietary supplements.The cyanobacterium  Microalgae comprising large photosynthetic plants whose vegetative system is called “thallus.” They have variable shapes and dimensions. Some of them are microscopic, and others are macroscopic, but they share structural and genetic similarities . OverallNowadays, microalgae seem to be one of the best solutions for producing high-quality food supplements . MicroalArthrospira is a genus belonging to nonheterocystous filamentous cyanobacteria that live in an alkaline environment [Arthrospira species were classified in the genus Spirulina, and some of them are still being named under this name [Arthrospira is a distinct genus including more than 30 different species [In recent times, the use of photosynthetic microorganisms has progressively increased. They have been used in different fields, including food dyes, cosmetics, dietetics, and biotechnology . Cyanobaironment . Even thhis name . Anyway, species .Arthrospira species are rich in nutrients like essential fatty acids, minerals, vitamins, and pigments [Arthrospira species have also the requirement for being introduced to serve heath by exhibiting interesting pharmacological activities like anti-inflammatory, antioxidant, and immunomodulating ([Arthrospira platensis (A. platensis) is recommended for being applied in environmental sectors for wastewater treatment  [pigments . Thus, tpigments . It has pigments . In addiulating  has not yet been investigated. It is thus fitting that the present research work aimed to achieve this goal by studying the physicochemical criteria of A. platensis collected from this local cultivar.It was reported that the Moroccan Arthrospira platensis was obtained from the culture collection at the Moroccan Foundation for Advanced Science, Innovation and Research, which was originally isolated from the Atlantic coast at Laayoune (Foum El Oued lagoon)—south of Morocco (2 s−1 of cool-white fluorescent light (12-h:12-h light:dark cycle) and aerated with ambient air (360 ppmv CO2). Samples in the exponential growth phase were used to perform the analysis  than in February (16.3–19.5°C) .Salinity showed an increasing gradient from downstream to upstream. Salinity gradually increased inside the lagoon with values close to those of the ocean (34–35 PSU). Salinity was higher in September than in February as reported in earlier works .−1). A strong concentration gradient was noted from downstream to upstream in the lagoon [Dissolved oxygen concentrations were variable according to stations (6.9–8.5 mg le lagoon .µg l−1 (H4) to 9.9 µg l−1 [.The lagoon was found to be generally richer in nitrates in February than in September. The concentration of nitrates decreased from downstream to upstream in the lagoon with values ranging from 80 9 µg l−1 .µg l−1, and the water was generally richer in phosphates in February than in September as reported elsewhere [.The spatial distribution of phosphates showed two trends depending on the tide and the season. This distribution was more homogeneous across the lagoon with 97 lsewhere .A. platensis dried sample was introduced into a flask containing 7.5 g of catalyst  and 15 mL of sulfuric acid H2SO4 (0.1 N). The assay was carried out in duplicate. The mixture was subjected to mineralization using a mineralization ramp apparatus (Büchi) for 4 hours until reaching a maximum production of ammonium sulfate (NH4)2SO4. After cooling, the volume of the mineralized sample was mixed with 50 mL of distilled water. Afterward, 85% NaOH solution (65 mL) was added to the mineralized sample before being distilled, and then the solution was trapped in boric acid . Next, the ammoniacal distillate was titrated with sulfuric acid (0.1 N) to perform analysis [A zero-point seventy-five gram of analysis ., F: conversion factor (6.25), VV: volume of the sulfuric acid solution, and PE: test portion.The total protein content was calculated using the following formula:A. platensis was carried out using high-performance liquid chromatography (HPLC). Briefly, 5 g of A. platensis were dried in an oven set at 40°C for 24 h before being added to 40 mL of sulfuric acid H2SO4 (2N). After maceration for 3 hours, the extract obtained was filtered before being analyzed using HPLC (Agilent 1100) ; column: C18; volume injection: 20 µL; UV detection at a wavelength of 333 nm; flow rate: 0.8 mL/min) [The determination of the amino acid composition  of  mL/min) .The concentration of amino acids was calculated using the following formula:A. platensis was calculated using the formula:Total carbohydrate content contained in the test portion of Carbohydrate content = 100 −  .A. platensis sample was extracted with 50 mL of demineralized water for 2 hours. Afterward, the carbohydrate concentration of the filtrate obtained was dosed using HPLC ; flow rate: 1 mL/min) [Five grams of  mL/min) .A. platensis sample was mixed with 31.5 mL of hydrochloric acid supplemented with 125 mL of distilled water for 2 hours (12 N). After filtration, the residues obtained were placed in an oven set at 105°C overnight. Next, the residues obtained were extracted again with 300 mL petroleum ether using a Soxhlet for 4 hours [Five grams of  4 hours .T1: the weight of empty flasks, T2: the weight of flasks containing fat, and PE: test portion.After removing the solvent under reduced pressure, the measures were performed using the following formula:The fatty substance was esterified with methanol. Next, the fatty acid methyl esters were separated through a polar column using gas chromatography (GC) (Annex 5). Briefly, 2 mL of isooctane and 0.1 mL of methyl KOH (2N) were added to 0.5 g of the extracted fat to prepare methyl esters. Afterward, the mixture was stirred for one minute before adding 2 mL of NaCl (40 grams/100 mL). Next, one gram of sodium bisulfate was added to the recovered supernatant before proceeding with the gravimetric analysis .2O3 (50g/L) to the mother solution. Potassium (K) determination was conducted by adding 2 mL of cesium chloride. Next, the mineral content was measured using atomic absorption spectrophotometry with flame (Varian SpectrAA 220FS Spectrometer FLAME AA with Varian SIPS-10 Sample Introduction Pump System with Varian SPS-5 Sample Preparation System) [Concentrations of calcium (Ca) and magnesium (Mg) were determined by adding 2 mL of lanthanum chloride La System) .L: reading, B: blank, VR: recovery volume, PE: test portion, and FD: dilution factor (g).One milliliter of mother solution was mixed with 10 mL of the monovanado-molybdic reagent. The analysis was conducted using a UV spectrophotometer at 430 nm . The min2 (riboflavin) and B3 (nicotinamide) contained in A. platensis were determined by using HPLC (NM 08.1.264 (2009). Briefly, 2 g of A. platensis sample was added to 40 mL of sulfuric acid (0.1 mol/L) before being stirred for 15 min. Afterward, the mixture was completed with sulfuric acid to reach 100 mL as a final volume. The concentration of vitamins was measured with HPLC (mobile phase: mixture of 970 mL of n-octane sulfonic acid 7 mmol/L (pH = 3), 30 mL of acetonitrile; stationary phase: Hypersil HyPURITY C18 5 µm 250∗4.6 mm; injection volume: 50 µL; flow rate: 1 mL/min; fluorimetric detection: 375nm–525 nm; UV detection: 261 nm; gradient in min: 0-9-9.1-22-22.1 and 35).Vitamins B2 and B3 was calculated using the following formula:The concentration of vitamins B for B3) .A. platensis energy value was based on the calorific value of different components  [The evaluation of ydrates) .The energy value was calculated according to the following formula:A. platensis were placed in previously weighed glass capsules before being introduced into an oven set at 105°C for 4 h. Next, the humidity percentage was calculated according to the following formula:T1: the weight of the empty capsule, T2: the weight of capsule containing the fresh sample, and T3: the weight of capsule containing the dry sample [Two grams of y sample .A. platensis sample was introduced into a capsule before being placed in a muffle oven set at 550°C for six hours [T1: the weight of empty capsule, T2: the weight of capsule containing the fresh sample, T3: the weight of capsule containing the dry sample, and PE: test sample = T2 − T1.Three grams of ix hours . After cA. platensis sample was extracted with 10 mL of acetone under ultrasound  for 15 min. After filtration, the mixture was centrifuged at 3000 rpm for 10 min. The pigment content  was determined according to the following formula:The content determination of carotenoids, chlorophyll a, and chlorophyll b was done according to the earlier reported data . BrieflyThe content of tannins was determined according to the previously reported method .t-test. The Mann–Whitney test was used as a post doc test to perform the comparison. Statistically, a significant difference was considered at P < 0.05.Data were expressed as means of triplicate assays ± SD (standard deviation). The significant differences were investigated using the A. platensis was 58.9 ± 0.07%. Besides, this species was quantitatively rich in essential amino acids as reported in our findings . This species contains protein twice as much as soybeans and thrice as much as meat [A. platensis was 43.71 ± 0.68 mg/kg. Therefore, we can confirm that A. platensis collected from the Moroccan Atlantic coast at Laayoune belongs to the rare cyanobacteria that contain a high amount of methionine. These results agree with those reported elsewhere since it was stated that Arthrospira constitutes a promising source of protein not only because of its high rate in the dry biomass but also due to the composition of amino acids and high digestibility. It is worth reporting that Arthrospira biomass possesses all 8 exogenic and 12 endogenic amino acids [In the current research work, we investigated  as meat , 30. A pno acids .A. platensis was 14.67 ± 0.001%. The characterization of A. platensis total carbohydrate content showed the presence of a significant amount of glucose (3.17 ± 1.01 g/L), unlike fructose, sucrose, and maltose, which were detected in very little quantity.High-performance liquid chromatography analysis showed that the value of carbohydrate content in dry A. platensis has a low content of carbohydrates (14.67 ± 0.001%) with a very little amount of simple carbohydrates (glucose and fructose). These findings were in agreement with those reported in earlier works [Arthrospira was described by the presence of two specific polysaccharides including sodium spirulan and calcium spirulan, which are involved in antiviral, anticoagulant, and immunostimulatory activities of A. platensis as reported elsewhere [The present results showed that er works . Arthroslsewhere , 33.A. platensis was determined at 5.8 ± 0.21% using gravimetric analysis. The characterization of A. platensis total lipid content revealed the presence of monounsaturated and saturated fatty acids with 53 ± 0.003% and 45.54 ± 0.15%, respectively. The total lipid content of the presently studied species was majorly constituted of palmitoleic acid (45.52 ± 0.01%), palmitic acid (37.06 ± 0.502%), and oleic acid (7 ± 0.003%)  .A. platensis, the results obtained showed that this species was majorly constituted of saturated and unsaturated fatty acids with a total percentage of 5.8 ± 0.25%. A. platensis possessed both monounsaturated and saturated fatty acids with values of 53 ± 0.003% and 45.54 ± 0.15% respectively. Our findings are in accordance with those reported in earlier works, which showed that lipid in Arthrospira varies from 1.5 to 12% of dry mass [Regarding the total lipid content in the local dry mass .A. platensis collected from the South Atlantic Coast of Morocco have important mineral elements . Statically, there was a significant difference between the content of heavy metals detected in A. platensis and WHO threshold values (P < 0.05). Moreover, A. platensis was free of cadmium as shown in The mineral composition analysis with flame atomic absorption spectroscopy revealed that elements  and veryelements . The valArthrospira largely meets the nutritional requirements for the body since it is rich in essential mineral elements. The present findings showed that this species has important mineral elements   helps prevent hypertension and improve bone health, whereas phosphorus (P) is required for skeletal mineralization [, Na, P) . Therefo, Na, P) . Accordilization . Furtherlization , and irolization .µg/kg/week; lead, 25 µg/kg/week; cadmium 7 µg/kg/week).Lead and chromium were found to be present in the local species with values of 70 ± 4.5 PPB and 5 ± 0.5 PPB, respectively; however, no trace was detected for cadmium. These values are lower than those of the World Health Organization threshold , A. platensis could cover the requirements of vitamins, which the body is unable to synthesize. Thus, these results were in agreement with those reported in earlier literature [Since it contains significant levels of fat-soluble vitamins  and water-soluble vitamins . The remarkable value of total lipid content (5.8 ± 0.25%) found in A. platensis could be responsible for its high energetic value . In this sense, arachidonic as derived acid from palmitic acid plays a key role in the synthesis of prostaglandins and leukotrienes [The findings of energetic values assessed in the current research work showed that otrienes . Moreoveotrienes , 39.A. platensis was 11.6% ± 0.17. Therefore, these results were partially in agreement with those stated in earlier works, which showed that the moisture content in A. platensis was 12.5% corresponding to 56% relative humidity [The findings obtained in the current research showed that the value of moisture content determined in the studied organism was 11.6% ± 0.17. Moisture content is defined as a quantity of water that exists in the biomass. Moisture plays a key role in food storage due to its either direct or indirect effect on microorganism development. In the present work, the moisture content defined in humidity .A. platensis was estimated at 9.1% ± 0.21. The ash is a measure of mineral content in biomass. In food, ash content is an important part of food quality analysis. Herein, A. platensis was also investigated in terms of ash content. As reported in the current research, the ash content was estimated at 9.1% ± 0.21. This finding was supported by the earlier found data, which reported that Arthrospira grown in Zarrouk's medium acquired the highest percentage of ash [The results reported in the present study showed that the ash content determined in e of ash .A. platensis showed the presence of chlorophyll a, chlorophyll B, and carotenoid with values of 26.066 ± 3.08 mg/g, 37.506 ± 3.38 mg/g, and 9.52 ± 0.22 mg/g, respectively. Regarding the pigment production, the analyzed sample evidenced the presence of chlorophyll b, chlorophyll a, and carotenoids with values 37.506 ± 3.38 mg/g, 26.066 ± 3.08 mg/g, and 9.52 ± 0.22 mg/g, respectively. Thus, these findings were in accordance with those reported in earlier works, which showed that the values of chlorophyll and carotenoid content in the genus Arthrospira were 26 mg/g and 3 mg/g DM, respectively. Arthrospira cells possessing carotenoids in different forms including α-carotene, β-carotene, cryptoxanthin, zeaxanthin, xanthophylls, echinenone, and lutein as reported elsewhere [The analysis of pigment content in lsewhere . Therefolsewhere .A. platensis extracts revealed a low tannin content. Gallic tannins were also present in little amount. Tannins are belonging to the secondary metabolites synthesized by plants and microorganisms to accomplish ecological functions. Our results showed that our organism has no important amount of tannins. Hence, these results were in contrast with the previously reported literature, which revealed the presence of promising tannin content in the genus Arthrospira [Qualitative analysis of hrospira .A. platensis indigenous to the Moroccan Atlantic coast at Laayoune possess unique features in terms of physicochemical contents when compared with the same species collected from different collection areas as reported in earlier works [2, vitamin B3, ash, Mn, Zn, Ca, Mg, K, Na, and P when compared with the same species indigenous to other areas. Our species was also found to be lower in heavy metals  and saturated fats as nonrequired parameters in foods , which act as coenzymes for mitochondrial enzymes and play important roles in cell metabolism and energy production according to prior research [Our results are in accordance with those reported by Jourdan, who showed that orophyll . In thisresearch .A. platensis possesses an interesting protein family that is recognized by its activities such as antioxidant, anticoagulant, antihypertensive, immunomodulatory, and antimicrobial [A. platensis indigenous to the Moroccan Atlantic coast at Laayoune have unique features in terms of physicochemical contents when compared with the same species investigated elsewhere. The obtained results showed that our studied species were higher in proteins, polyunsaturated and monounsaturated fats, minerals, vitamins , ash, and pigment contents when compared with species studied by Bensehaila, (2015). Moreover, our species was lower in heavy metals and saturated fats when compared with those studied by Bensehaila, (2015), and Falquet, (2012). Therefore, we could confirm that A. platensis indigenous to the Moroccan Atlantic coast at Laayoune is a promising source of food supplement due to their high values concerning proteins, unsaturated fats, carbohydrates, minerals, vitamins, and pigments and their low values regarding heavy metals and saturated fats. A. platensis is reported to be of high nutritional value and pharmacological and biological potentials so that they can be used for medicinal purposes for humans or animals [A. platensis has a biofuel production potential, and therefore, it can significantly contribute to economic performance [icrobial . Species animals . In addiformance .A. platensis cannot be free of toxins. In this sense, the rate of trace elements  alongside toxic metals  was detected in A. platensis samples. The highest element concentration was detected in the powder format, except for Li. When element levels in A. platensis exceed the tolerable weekly intake (TWI), the consumer would place his health at risk. The consumption of spirulina contributes largely to the Al intake by a value higher than TWI determined at 1 mg/kg bw/w (body weight/week), followed by Cd exceeding its TWI set at 2.5 μg/kg bw/w was reported in previous works [µg/kg/week; lead, 25 µg/kg/week; cadmium 7 µg/kg/week). Therefore, A. platensis indigenous to the Moroccan Atlantic coast at Laayoune (Foum El Oued lagoon) can be considered safe for being ingested.us works . Pb intaA. platensis collected from the Moroccan Atlantic coast at Laayoune. The obtained results showed that A. platensis indigenous to the Moroccan Atlantic coast at Laayoune was found to be very rich in proteins, carbohydrates, vitamins, minerals, ash, and pigments and lower in heavy metals and saturated fats when compared with species investigated in the literature. Therefore, we could confirm that A. platensis indigenous of the Moroccan Atlantic coast at Laayoune can be a very promising source of dietary supplements. Overall, A. platensis should be optimized further, and processing strategies based on the optimization approaches can be developed.The present research work aims to assess the nutritional value of


The appearance of a surface is a crucial characteristic of a part or component. Laser-based micromachining gets increasingly important in generating tailored surface topographies. A novel structuring technique for surface engineering is surface structuring by laser remelting (WaveShape), in which surface features are created without material loss. In this study, we investigated the evolution of surface topographies on Ti6Al4V for a laser beam diameter of 50 µm and scan speeds larger than 100 mm/s. Surface features with aspect ratios (ratio of height to width) of almost 1:1 were achieved using the WaveShape process. Furthermore, wavelengths smaller than 500 µm could be effectively structured using scan speeds of up to 500 mm/s. The experimental results showed further that the efficiency of the WaveShape process in terms of achieved structure height per unit time significantly increases for high scan speeds. Surface structuring processes have long been established for industrial manufacturing processes. As a result of the manufacturing processes, surfaces are often designed and produced according to specific customer requirements. The development of structuring techniques requires, among other things, miniaturization of surface structures in the respective field of application. In general, mechanical tools are often used for surface processing. In the field of micro- and nanomachining of surfaces, however, the use of tools is only possible to a limited extent. Therefore, contactless, laser-based surface structuring techniques utilizing small interaction areas are becoming increasingly important.Surface structuring in laser beam-based processes is often achieved by partial vaporization of the surface. Chichkov et al.  laid a b2/s (or approx. 167 s/m2) are already feasible on stainless steel. Overall, DLIP is a promising surface texturing technique, but also has its limitations, e.g., in terms of achievable dimensions of the generated surface features.Direct laser interference patterning (DLIP) results from two or more coherent laser beams interfering at a workpiece surface   is a novel surface structuring process used for structuring metallic surfaces in the micro- and millimeter range by power modulation of a laser beam source. In contrast to other laser structuring processes, surface features are not generated by localized ablation but created by redistribution of molten material at the surface ,21. An aLd = 50 µm) and high processing speeds (scanv ≥ 100 mm/s) on Ti6Al4V. The foci of this study are to achieve significantly smaller wavelengths, to reduce processing time, and to increase process efficiency. Furthermore, high aspect ratios shall be achieved by high-speed multi-processing. The titanium alloy Ti6Al4V was chosen since it has a wide range of industrial applications, particularly for aviation, aerospace, and medical engineering. Additionally, Ti6Al4V proved to be well suited for laser remelting processes in general and for WaveShape in particular [This study significantly extends previous investigations on the WaveShape process for small laser beam diameters (rticular .emiλt = 1062 ± 3 nm).The “FluidStruc” laboratory system a was useM2 = 2.8–3.6. In this study, laser radiation was used in continuous wave (cw) mode with a maximum laser output power of approx. LP = 36 W at the workpiece surface. The laser beam was coupled into the optical setup  via an optical fiber and collimation with a collimated raw beam diameter of Laserr = 5.5 mm. Using various combinations of fused silica lenses and f-theta objectives, enables a wide range of different laser beam diameters on the workpiece for laser processing. In this setup, the lenses were used as a relay system in an 1:1 imaging configuration and an f-theta objective with a focal length of Tf = 163 mm  = 0.19 mm, beam divergence fΘ = 87 mrad, and laser beam diameter Ld = 50.0 (±2.4) µm (86% energy inclusion).The measured beam characteristics  were as follows: The material investigated in this work was the titanium alloy Ti6Al4V  which has very high strength and corrosion resistance. Ti6Al4V is categorized as light metal due to the low density and is often used in aerospace and medical applications. Sampled of the round samples is 80 ± 1.0 mm, while the thickness Samplet is 18 ± 0.5 mm. The initial surface was remelted, milled, and ground to a homogenous surface roughness of Sa = 0.31 ± 0.04 µm.Cylindrical round specimens made of Ti6Al4V and titanium are used to perform the experimental investigations. The diameter The active principle of surface structuring by laser remelting (WaveShape) is based on a remelting process of a surface boundary layer of a metal surface, while simultaneously modulating laser power in a controlled manner. Since the laser power is limited by melting and ablation thresholds, typically no material is removed during the process b. The comaxP) and a lower limit (minP), which span the process window between which the laser power was modulated during surface structuring (Equation (1) and In the WaveShape process, structuring is carried out by means of a remelting process that avoids material evaporation. The material is in a molten state during structuring. In a preliminary investigation, suitable process windows were determined for each combination of laser beam diameter and scan speed. This process window consists of an upper limit , which corresponds to the length of a single track. The individual tracks generated in a processing field were each processed unidirectionally.dy is the distance between the individual tracks. To avoid interfering heat effects of neighboring tracks, the track offset was set to 0.5 mm. The Scan speed scanv was investigated between 100 and 500 mm/s. The wavelength of laser power modulation was investigated between two times (2·Ld) and eight times (8·Ld) the laser beam diameter Ld, since the most significant effects were expected in this range. The number of passes n were mainly investigated in the range from 1 to 32, since more repetitions lead to structure characteristics (height to width ratio), which made it impossible to be comprehensively measured by WLI.An overview of the essential process parameters and their range of investigation is shown in λ and the amplitude corresponds to structure height h. At least four single tracks of 5 mm length were used to determine the structure height. The specifics of this method are described in detail in various publications [The surface analysis is based on white light interferometry (WLI) images of the remelted tracks. Based on a longitudinal section along the middle of a remelted track a fast Fourier transformation was conducted to calculate the dominant spatial frequency and the corresponding amplitude. The dominant spatial frequency corresponds to the structuring wavelength ications ,24,25,28dy = 1 mm were structured and analyzed by microscopy .Previous work of Bordatchev et al.  and Temmh increases linearly as function of laser power amplitude. For example, the structure height obtained at a wavelength λ = 0.1 mm was approx. 0.19 µm at AP = 1.15 W and increased almost linearly to approx. 0.96 µm at AP = 5.75 W. Compared to the structures generated at other wavelengths, the structure height obtained was greatest at a wavelength λ = 0.2 mm (four times the laser beam diameter). Temmler et al. [scanv = 200 mm/s and wavelength λ = 0.2 mm (four times the laser beam diameter) are shown in r et al.  showcaseAP = 5.75 W and led to an additional deviation from a sinusoidal longitudinal section. The shape deviation shown in AP = 5.75 W occurred since the laser power fell below the minimum laser power limit minP. In this case, no material was molten for a short time. Subsequently, the melt pool was formed again when the laser power was within the process limits again. However, this led to a significant shape deviation of the structure profile from an ideal sinusoidal form. Furthermore, this shape deviation was only observed at larger wavelengths (λ ≥ 0.2 mm). In addition, a pronounced high-frequency structural noise was observed on the resulting surface topography. Due to the small laser beam diameter, a comparatively small amount of material was remelted during processing. Thus, the initial surface roughness had a larger influence on the resulting surface topography.Continuous remelting no longer occurred at a laser power amplitude of AP and wavelength λ determine the maximal laser power gradient. At constant laser power amplitude, the wavelength has a significant influence on the local laser power gradient and hence temperature gradient and was therefore investigated in detail. The wavelength was investigated as multiples of the laser beam diameter  and scan speeds scanv . The achieved structure heights as function of wavelength for different scan speeds are shown in The laser power amplitude scanv = 100 mm/s, the maximum structure height h was approx. 1.23 µm at a wavelength of λ = 0.15 mm. The respective structure height achieved at twice and three times the laser beam diameter was approx. 0.84 and 0.9 µm, respectively. At scanv = 200 mm/s, the maximum structure height was h = 1.1 µm, also at a wavelength of λ = 0.15 mm. The structure height is h = 0.92 µm at λ = 0.1 mm and h = 1.03 µm at λ = 0.2 mm. When using even larger scan speeds from scanv = 300 to 500 mm/s, structuring at small wavelengths (λ = 0.1 mm or 0.15 mm) only occurred with a reduced laser power amplitude, since technical limits in terms of the maximal modulation frequency of laser beam source or control card were exceeded. The maximum structure height at a scan speed of scanv = 300 mm/s was generated at the wavelength λ = 0.15 mm and is approx. 0.87 µm. The maximum structure height is achieved at wavelength λ = 0.2 mm for scan speeds of scanv = 400 mm/s (h = 0.79 µm) and scanv = 500 mm/s (and h = 0.69 µm). In general, the achieved structure height became smaller as the scan speed increased, even though the laser power was adapted to the scan speed and laser beam diameter . Thus, the shape was deviating from the ideal sinusoidal shape along a longitudinal section.λ = 0.15–0.25 mm for scan speeds from 100 to 500 mm/s. The maximum structure height maxh = 1.23 µm was generated at scanv = 100 mm/s and λ = 0.15 mm. However, previous studies often showed that a significant increase in structure height was obtained by multiple processing.The investigations to this point have been carried out exclusively on the basis of single processing. A consequence is that only comparatively small structure heights were achieved so far . Maximum structure heights in single track processing were obtained at wavelengths in the range n of a processing step on the achieved structure height h was investigated. A single track was unidirectionally remelted n times with the same modulated laser power signal. The number of passes n was investigated in the range from one to thirty-two passes in six steps. Thereby, the number of repetitions was doubled stepwise in each case. The generated structure height after sixty-four passes is already so large that it can no longer be measured using WLI. The dependence of the generated structure height on the number of passes n was investigated exemplarily for the wavelength range λ = 0.1 mm to λ = 0.4 mm in four steps, while the scan speed was investigated in the range from scanv = 100 mm/s to scanv = 500 mm/s in five equidistant steps. The process limit was adapted based on the process parameter combination used and the maximum laser power amplitude was used. The dependence of the obtained structure height h on the number of passes/repetitions n is exemplarily shown for wavelengths λ = 0.1–0.4 mm and scan speeds of scanv = 100 mm/s and scanv = 400 mm/s . At n = 1 and scanv = 100 mm/s, the structure height h was approx. 0.86 µm. Processing twice (n = 2) increased the structure height to h ≈ 1.47 µm. This corresponds to a magnification factor of approx. 1.79. However, at n = 32, the structure height was only increased to approx. 2.69 µm. The magnification factor with respect to n = 1 is thus only approx. 3.1. At the wavelength λ = 0.2 mm, a significant increase in the structure height was observed from n = 1 to n = 16. After single processing (n = 1), the structure height was approx. 0.94 µm. The structure height h at n = 16 was approx. 7.48 µm and the magnification factor corresponds to approx. 8. The structure height increased further at n = 32, but the magnification factor in comparison to n ≤ 16 decreases. The achieved structure height was approx. 8.36 µm at a magnification factor of approx. 8.9. When structuring of wavelengths λ > 0.2 mm, the achieved structure height was further increased when the number of passes was increased (n ≥ 16). For example, at λ = 0.4 mm, the structure height h ≈ 0.63 µm at n = 1 was increased by a factor of approx. 21 to h ≈ 13.29 µm at n = 32.A significant increase of structure height λ = 0.1 mm is comparatively small, while the largest structure height is obtained at the largest investigated wavelength λ = 0.4 mm and the most repetitions n = 32 (h ≈ 12.57 µm). For wavelengths of λ > 0.2 mm, the structure height became larger when the number of repetitions was increased at wavelengths λ = 0.1 mm or λ = 0.4 mm. While the maximum structure height was achieved in the range between λ = 0.15 mm and λ = 0.25 mm for single processing, the preferred working range for multiple laser processing is at a comparatively large wavelength  and a large number of repetitions .The increase of the structure height at small wavelength n is shown in scanv = 400 mm/s and λ = 0.2 mm .An undesirable micro-roughness often remains after single processing on the structured single track. Due to the small laser beam diameter, only a comparatively small amount of material was remelted, so that the initial roughness was relatively large compared to the generated structure height. A reduction of the high frequency structural noise (micro-roughness) was achieved by multiple processing. A change of the structural cross section for an increasing number of repetitions Bt was conducted for the range of scan speeds from scanv = 100 mm/s to scanv = 500 mm/s. The investigated wavelengths were λ = 0.1 mm, λ = 0.2 mm, λ = 0.3 mm, and λ = 0.4 mm. Depending on the scan speed and the number of passes, the processing time for each single track is calculated as follows: B,single trackt = SVL · scanv−1 · n . For example, the generation of a 5 mm long single track with scanv = 100 mm/s requires B,single trackt = 50 ms. At a scan speed of scanv = 500 mm/s, the number of repetitions was increased to n = 5, so that the processing time remained the same. This was done analogously for all scan speeds.An investigation of the obtained structure height at constant processing time h are plotted as function of the wavelength λ for different scan speeds scanv with a corresponding number of repetitions n. A maximum structure height of approx. 3.17 µm was achieved at a scan speed of scanv = 500 mm/s and a wavelength of λ = 0.3 mm. The structuring of small wavelengths λ = 0.1 mm and large scan speeds scanv = 400 mm/s and scanv = 500 mm/s was not meaningful due to the technical limitations of the system technology. A significant increase in structure height was achieved at large wavelengths and high scan speeds. The largest structure height was produced at scanv = 500 mm/s and λ = 0.3 mm (h = 3.07 µm).In h was calculated as the quotient of structure height h  and structure height at scanv = 100 mm/s and n = 1 according to Equation (2):The magnification factor Δh as a function of the examined scan speeds at different wavelengths and for the same processing time. The largest increase in magnification factor occurred at a larger wavelength λ = 0.4 mm and was approx. 3.87 at scanv = 500 mm/s. No significant increase in the magnification factor occurred at a larger scan speed with a corresponding number of repetitions for the wavelength λ = 0.1 mm. Overall, the magnification factor significantly decreases for smaller wavelengths.λ = 0.1 mm). This suggests that structuring using high scan speeds has a significant advantage due to the corresponding multiple processing, and thus greater structure heights will be achieved in the same processing time. Additionally, a smoother structure profile was produced at the same time due to multiple processing.For structuring at high scan speed and the corresponding number of repetitions, larger structure heights were achieved ).In this section, structuring at a constant processing time scanv = 100 to 500 mm/s in five equidistant steps. The track offset is set to dy = 12 µm at the scan speed scanv = 100 mm/s, so that a significant track overlap is ensured even at the laser power minimum , the structure height is increased by a factor of 3 to 4 with respect to single processing. For multiple processing n = 16, a maximum of approx. 14 times the structure height was produced compared to structuring for n = 1. The maximum structure height was generated at scanv = 500 mm/s, dy = 2.4 µm, and n = 4 (h ≈ 29 µm at λ = 0.3 mm). Furthermore, the maximum structure height at n = 16 was approx. 90 µm .In the case of quadruple processing . Since this study aimed to investigate high scan speeds and small wavelengths, comparatively high modulation frequencies were reached. In general, a laser beam source requires considerable times for the rise and fall of the laser power. These rise and fall times limit the maximum modulation frequency of the SPI fiber laser to approx. 3 kHz, which is already significantly higher than in previous studies on the WaveShape process [scanv = 400 mm/s, λ = 0.1 mm), it led to an undesirable reduction of the achieved structure height-additionally to the existing limitation due to the size of the laser beam diameter. Exceeding the maximum modulation frequency only occurred for few combinations of scan speed and wavelength. Thus, using a laser beam diameter of approx. 50 µm, structures with small wavelengths such as λ = 0.1 mm, could be generated and investigated.The minimum wavelength is limited due to two main effects. Firstly, the minimum wavelength could not be smaller than the doubled laser beam diameter, since the melt pool surface needs considerable space to be deformed . Secondl process ,23,28. NOverall, the achieved structure heights achieved in this study are significantly smaller than the structure heights achieved with higher laser power and larger laser beam diameters on Ti6Al4V . NonetheThe investigations showed that multiple processing with the same set of process parameters leads to a significant increase of structure height, which is also in good agreement with previous studies for larger laser beam diameters . Additionally, the generated surface structures show a shape deviation from an ideal sinusoidal profile. These shape deviations are similar to those reported and discussed by Oreshkin et al. [In areal processing, the track distance n speeds ,26,27,28n et al. . Dependin et al. .λ = 0.2 mm. This is a new dimension for the WaveShape process, since surface features with an aspect ratio close to 1:1 (height:width) were generated. Typical for the WaveShape process are structure heights of approx. 50–100 µm at a wavelength of λ ≈ 1.0 mm [2. However, if structure height increases even further for higher scan speeds (as the results of this study indicate), a significant decrease in processing time can be expected. Moreover, if smaller aspect ratios are required, e.g., 1:5 or 1:10 as for light guiding surfaces [2 and might already be applicable for industrial processes such as structuring of tool inserts.Finally, ≈ 1.0 mm ,23,24. Asurfaces , the pro3 m/s. In this case, the maximum modulation frequency would be on the order of approx. 1 GHz. However, as studies for heat accumulation in pulsed laser processing show [6–107 K/s for Inconel 718 (T > 1.500 K), while Nüsser [8 K/s (T > 1500 K) in laser micro polishing of Ti6Al4V. This indicates that a physical limit regarding modulation frequencies might be in the range between hundreds of kHz and several tens of MHz. Currently available fiber lasers from renown laser companies achieve maximum modulation frequencies of approx. 50 kHz. These systems or laser beam sources with even higher capabilities in terms of modulation frequency  are specifically worthwhile for further investigations to increase processing speed and structure height. However, not only the available modulation frequency of the laser beam source might be a bottleneck, but also control and command frequencies of the auxiliary equipment necessary to achieve a controlled, reproducible, and space-resolved control of the laser power signal. High modulation frequencies as well as control and command frequencies might be achieved by external modulators, e.g., acousto-optical or electro-optical modulators. In sum, further investigations on the significance of thermophysical properties such as thermal diffusivity on usable modulation frequencies in the WaveShape process are highly relevant for future applied and scientific studies.Physical limitations of the Waveshape process presumably result from the time required for thermalization and for heat conduction of the thermalized energy. Usually laser radiation is thermalized on the order of femtoseconds and melting due to heat conduction typically requires up to several picoseconds . In the ing show ,42, manying show , for exae Nüsser  found coscan,maxv = 500 mm/s) and small laser beam diameters (Ld = 50 µm) was investigated for the material Ti6Al4V. The objectives were to achieve larger structure heights in a shorter processing time for small wavelengths. In general, some fundamental characteristics of the WaveShape have been confirmed for smaller laser beam diameters, high scan speeds, and small wavelengths. Firstly, the largest structure heights were achieved at a wavelength of approximately three to four times the laser beam diameter (n = 1). Secondly, structure height was significantly increased by multiple processing (single tracks) or choosing adapted track offsets . Thirdly, structuring of wavelengths smaller or equal than two times the laser beam diameter is not efficiently possible.In this study, surface structuring by laser remelting (WaveShape) using a SPI fiber laser at high scan speeds .Nonetheless, the study also came to some surprising results. Firstly, structure height is almost the same regardless of the scan speed used. Particularly, when multiple processing steps were used at high scan speed, significantly larger structure heights were achieved. Secondly, the interdependency of track offset λ = 0.5 mm were generated and structure heights larger than a few micrometers were achieved. Furthermore, scan speeds higher than scanv = 100 mm/s have been systematically investigated and it has been shown that the processing time can be significantly reduced using high scan speeds, adapted laser power modulation, and an adapted number of repetitions. In addition, a structure height to wavelength ratio of approx. 1:1 was achieved for a wavelength of λ = 0.2 mm with a processing time of less than 30 s/mm2  towards a broad variety of potential applications.


Tetrastigma hemsleyanum Diels & Gilg (T. hemsleyanum) has attracted much attention due to its ability on pneumonia, bronchitis, and immune-related diseases, while its functional components and underlying mechanism of action on pneumonia have not been fully elucidated. Herein, we used a systematic network pharmacology approach to explore the action mechanism of T. hemsleyanum leaf in the treatment of pneumonia. In this study, the results of network pharmacology demonstrated that there were 34 active components and 80 drug–disease targets in T. hemsleyanum leaf, which were strongly in connection with signal transduction, inflammatory response, and the oxidation–reduction process. Subsequently, a mouse model of pneumonia induced by Pseudomonas aeruginosa (P. aeruginosa) was established to validate the predicted results of network pharmacology. In the animal experiments, aqueous extract of T. hemsleyanum leaf (EFT) significantly attenuated the histopathological changes of lung tissue in P. aeruginosa–induced mice and reduced the number of bacterial colonies in BALFs by 96.84% (p < 0.01). Moreover, EFT treatment suppressed the increase of pro-inflammatory cytokines IL-17, IL-6, and TNF-α in lung tissues triggered by P. aeruginosa, which led to the increase of Th17 cells (p < 0.05). High concentration of EFT treatment (2.0 g/kg) obviously increased the anti-inflammatory cytokine levels, accompanied by the enhancement of Treg proportion in a dose-dependent manner and a notable reversal of transcription factor RORγt expression. These findings demonstrated that network pharmacology was a useful tool for TCM research, and the anti-inflammatory effect of EFT was achieved by maintaining Th17/Treg immune homeostasis and thereby suppressing the inflammatory immune response. Pseudomonas aeruginosa (P. aeruginosa), an omnipresent environmental Gram-negative microorganism, is one of the major pathogenic bacteria of hospital-acquired pneumonia (HAP)  . HAP chiia (HAP) . Similarpatients . Charactfatality . The train China . TherefoTetrastigma hemsleyanum Diels & Gilg (T. hemsleyanum), known as “San Ye Qing,” belongs to the grape family Vitaceae and is traditionally used to treat pneumonia, hepatitis, children with high fever, and immune-related diseases  was used to confirm its molecular structure, and then it was saved in MDL molfile format.The chemical composition of http://www.swisstargetprediction.ch/) and UniProt database (https://www.uniprot.org/) were used to identify the relevant targets of the compound based on chemical similarities and pharmacophore models , GeneCards (https://www.genecards.org/), and OMIM (http://www.ncbi.nlm.nih.gov/omim) databases. The standard gene names of pneumonia-related targets were also acquired from UniprotKB database. Finally, the targets of the three databases were merged, and the duplicate targets were deleted to keep the item unique; the results were the pneumonia-related target.The acknowledged pneumonia-related targets were collected from DigSee (T. hemsleyanum and pneumonia-related targets were uploaded to the online Venn diagram package (http://bioinfogp.cnb.csic.es/tools/venny/index.html) for mapping; that is, the intersection of the two was taken to obtain the potential target of T. hemsleyanum in the treatment of pneumonia.The targets of active components in http://string-db.org/), which provides known and predicted interaction information on the basis of systematic co-expression analysis, detection of shared selective signals across genomes, and automated text-mining of the scientific literature . The central network analysis was performed by the topological method. Degree centrality, betweenness centrality, and closeness centrality, three topological parameters which indicated the importance of network node topology in the networks, were also analyzed using Cytoscape software.The protein–protein interactions (PPI) of each target were generated by the String database (terature . The spehttps://david.ncifcrf.gov/). GO terms were associated with biological processes (BP), cellular components (CC), and molecular functions (MF). GO terms and KEGG pathways with p value < 0.01 were considered to be statistically significant. Pathways with p-value less than 0.01 and target numbers greater than three were selected. Finally, Cytoscape3.6.0 was used to build the drug–compound–target–disease network.The gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were performed using the online functional annotation and enrichment tool DAVID  was prepared in our laboratory. Briefly, 20 g of the powdered T. hemsleyanum leaf was weighed and soaked in 200 ml ultrapure water for 30 min, and then ultrasonically extracted for 45 min at 40°C; the ultrasonic extraction was performed using water refluxing for 60 min at 55°C. The refluxed extraction solution was centrifuged at 3,000 rpm for 10 min to collect the supernatant, and the insoluble residue was repeated for two times as described above. The total extract was concentrated to 100 ml using a rotary vacuum evaporator  at 50°C. The highest concentration of the extraction is 0.2 g/ml, the stock solution was diluted to 0.1 g/ml and 0.05 g/ml with ultrapure water, and the latter was centrifuged at 3,000 rpm for 10 min and filtered with a 0.45-μm filter paper and preserved at -20°C for further use.The fresh leaves of 2O (solvent B). The gradient elution program was performed as follows: 0–30 min, 10–25% A; 30–40 min, 25–95% A; 40–45 min, 95% A; and 45–60 min, 95–100% A. The flow rate was 0.8 ml/min, and the monitoring wavelength was set at 320 nm. The compounds of EFT were identified and quantified based on their spectra and standards. All experiments were repeated three times.The chemical profile analysis of EFT was conducted by UPLC. Briefly, 10 μl of EFT and standards were, respectively, injected into the UPLC system equipped with a DAD detector , and separated on Agilent SB-C18  column at 30°C. The mobile phase was acetonitrile (solvent A) and 0.1% (v/v) phosphoric acid in HP. aeruginosa–infected model group; low, medium, and high EFT treatment groups ; and amikacin (25 mg/kg) treatment. Mice in the sham group were intratracheally injected with aseptic PBS solution. Mice in other groups were intratracheally injected with a dose of 5 × 106 of the planktonic P. aeruginosa strain PAO1 (ATCC 27853) as described by Simone , weighing 22 ± 2 g, were purchased from Zhejiang Laboratory Animal Center . All mice were housed at 25 ± 2°C with an alternating 12 h light and dark cycle. Mice were given free access to standard laboratory food and water. All animal experiments were approved by the Ethics Committee of Zhejiang Pharmaceutical College. The mice were randomly divided into six groups of 22 mice each as follows: sham control group; y Simone , which wThe upper page of right lung tissue was isolated from the mice and inflated with 10% formalin for 10 min, and then ligated and removed. Inflated lungs were fixed for 24 h and then embedded in paraffin. Tissue sections (5 μm thick) were stained with hematoxylin–eosin (HE) and observed under an Olympus microscope . Continuous midsagittal sections were taken for morphological and histological analyses, including the pathological changes of the alveolar structure in mice, pulmonary necrosis, and inflammatory in lung tissue.Blood samples of the orbital venous plexus and BALFs were collected under mild pentobarbital anesthesia, centrifuged at 3,000 rpm for 10 min at room temperature, and then were stored at -80°C for enzyme-linked immunosorbent assay (ELISA). According to the manufacturer’s instructions, ELISA kits were used to detect the serum levels of interleukin (IL)-17, IL-6, IL-10, transforming growth factor (TGF)-β1, and tumor necrosis factor (TNF)-α in blood samples and BALFs.8 cfu/ml).BALFs were collected by lavaging the upper page of left lung tissue with 2 × 0.5 ml saline as previously reported . In shor−ΔΔCt, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were used to apply to all samples for normalization. Three technical replicates were performed for each sample. The primer sequences used in this study were as follows:Total RNA was extracted from the second page of left lung tissue with Trizol reagent  according to the manufacturer’s instructions. For reverse transcription, 2.0 μg of total RNA was reverse-transcribed using a HiScript™ Q RT SuperMix for qPCR (+gDNA wiper). Quantitative real-time PCR was performed with SYBR Green Ⅰ using a Thermal Cycler Dice™ Real Time System  according to the manufacturer’s instructions. A relative mRNA level was calculated by the 2Foxp3: 5′-ACCCAGGAAAGACAGCAACC-3′ (forward)5′-CTCGAAGACCTTCTCACAACCA (reverse);RORγt: 5′-CCATTGACCGAACCAGCC-3′ (forward)5′-TCTGCTTCTTGGACATTCGG-3′ (reverse);GAPDH: 5′-TGTGTCCGTCGTGGATCTGA-3′ (forward)5′-TCTGCTTCTTGGACATTCGG-3′ (reverse).As described previously , lung sip < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS statistical software version 13.0.Quantitative data were presented as mean ± standard deviation (SD). The differences among the groups were assessed by one-way ANOVA. Correlation coefficients were calculated using Pearson’s rank correlation test. T. hemsleyanum leaf were collected in the present study according to the screening conditions, and 124 compound-related targets with 324 frequencies were found after eliminating the duplicates. These findings revealed that the effectiveness of T. hemsleyanum leaf against complex diseases depended on the synergy between multiple compounds and their targets. Although the target number of each component was varied, there was significant overlap among the 34 components. As for disease target identification, we retrieved collected 4,779 pneumonia-related targets from the OMIM, Gene Cards, and DigSee databases after eliminating overlapping targets. Moreover, a total of 80 genes were identified with Venn diagrams, which were related to both T. hemsleyanum leaf and pneumonia, and the result is exhibited in T. hemsleyanum leaf were inextricably linked with 80 pneumonia-related targets. In order to clarify the relationship among the disease, active compounds, and potential targets, a disease–compound–target network was constructed with 13 compounds and their corresponding targets, and is presented in T. hemsleyanum leaf for pneumonia treatment.A total of 34 effective components of T. hemsleyanum, and Cytoscape was used to carry out the visual composition. The nodes and edges in the network indicated target proteins and protein–protein associations. After eliminating disconnected nodes, there were 60 nodes and 128 edges in the network , including 83 for BP, 13 for CC, and 34 for MF. The results showed that the drug–disease targets were mainly relevant to protein kinase activity, protein phosphorylation, signal transduction, ATP binding, oxidation–reduction process, and inflammatory response (p < 0.01), and many target genes were strongly in connection with the Rap1 signaling pathway, PI3K–Akt signaling pathway, and Ras signaling pathway. The active component–target–pathway network of T. hemsleyanum leaf was visualized by using the software of Cytoscape and depicted in T. hemsleyanum.To elucidate the mechanism of response . MoreoveThe HPLC chromatograms of reference standards and EFT sample were exhibited in P. aeruginosa ; the bacterial burdens were remarkably decreased in the EFT and amikacin groups compared with the model group (p < 0.01), and the bacterial load in the model group was more than 30 times that in the EFT (2.0 g/kg) group. Moreover, the antibacterial effect of EFT was in a dose-dependent manner considering that the bacterial burden in the EFT (2.0 g/kg) group was only 6.4% of that in the EFT (0.5 g/kg) group (p < 0.01).To confirm the successful establishment of the T. hemsleyanum against pneumonia was remarkably associated with the inflammatory response. Therefore, the levels of pro-inflammatory  and anti-inflammatory cytokines (TGF-β1 and IL-10) in serum and BALFs were determined by ELISA. There were no significant differences in the levels of TGF-β and IL-10 in serum between the groups. But the levels of TGF-β and IL-10 in BALFs decreased in the model group compared to the control group (p < 0.05). In contrast, the levels of IL-17, IL-6, and TNF-α in serum and BALFs of the EFT group were lower than those in the model group. The downregulation of IL-17 in serum of EFT groups  was 36.82, 51.68, and 61.95%, respectively. Meanwhile, its production in BALFs of mice treated with 0.5, 1.0, and 2.0 g/kg EFT was decreased by 33.47, 36.32, and 40.41%, respectively. The reduction ratios of IL-6 in serum of three EFT groups were 5.25, 6.50, and 11.16%, and those of TNF-α were 10.36, 15.70, and 20.09%, respectively. Meanwhile, the changes of IL-17 in BALFs of EFT groups  were accompanied by the attenuation of IL-6 and TNF-α; the former were 4.97, 5.26, and 7.18%, and the latter were 6.03, 7.47, and 25.98%, respectively.As indicated in the network pharmacology analysis, the underlying pathway of ol group . The levT. hemsleyanum on pneumonia were related to the immune system, the frequencies of splenic Treg cells and Th17 cells were detected by flow cytometry. The results demonstrated that the percentages of Treg cells in the model group were dramatically lower than percentages of those in the sham group (p < 0.05). On the contrary, the numbers of Th17 cell in the model group showed a significant uptrend compared with the control group. Dramatically, the increasing trend was significantly reversed by 46.62 and 57.90% in EFT  groups, respectively. Ultimately, the ratio of Th17/Treg in EFT groups  was declined by 60.58 and 76.32% compared with the model group (p < 0.05), respectively.To verify that the suppressive effects of am group , whereas+, IL-17+ (Th17), CD8+, and IL-17+ (Tc17) cells, the mRNA expressions of retinoid-related orphan nuclear receptor-γt (RORγt) and forkhead box protein3 (Foxp3) were detected using the RT-PCR method. As shown in P. aeruginosa–infected mice showed a noteworthy upregulation tendency compared with sham control (p < 0.05), respectively, which revealed that EFT had a dose-dependent inhibition effect on the expression of RORγt.As the vital transcription factors participated in the development and function of CD4 control . TreatmeP. aeruginosa lung infection is a typical manifestation of persistent pulmonary diseases, such as cystic fibrosis and chronic obstructive pulmonary disease, characterized by pulmonary inflammation, high levels of cytokine and chemokine production, and massive recruitment of neutrophils. Currently, there are no specific drugs for use in the treatment of P. aeruginosa lung infection due to the lack of novel classes of antibiotics as well as the constant increase of multidrug resistance, and most available treatments are symptomatic and supportive. With valuable experience for clinical practice and basic medical research, the application of TCM has become increasingly popular, thanks to its outstanding advantages such as more targets and fewer side effects. Previous studies have shown that TCM not only targeted the etiology of P. aeruginosa lung infection but also effectively alleviated a series of complications caused by P. aeruginosa lung infection , and the expression of the transcription factor RORγt were conspicuously increased in the model group, compared with the control group. EFT could significantly reverse those increasing tendency, similar to the positive drug amikacin. Moreover, there was a positive correlation between the reversal effect and the concentration of EFT.It has been recognized that T lymphocytes, especially CD4nd IL-22 . Althougnd IL-22 , tubercund IL-22 , lung cand IL-22 , and chrnd IL-22 , there wT. hemsleyanum might also be attributed to its ability of maintaining Treg/Th17 balance in activated lymphocytes and suppressing the expression of pro-inflammatory cytokines.Treg/Th17 cells kept a dynamic balance in normal physiological conditions. Once the balance between Th17 and Treg cells is broken, it may lead to a series of immune inflammatory diseases and suppression of the immune response. Clinical studies have shown that the balance between Th17 and Treg played a vital role in the pathogenesis of allergic airway inflammation . The Th1T. hemsleyanum leaves on pneumonia using the network pharmacology approach, simultaneously verifying the curative effect of T. hemsleyanum leaf on the lung injury in vivo. The results of animal experiments have shown for the first time that EFT might contribute to relieve pneumonia induced by P. aeruginosa treatment, mainly through regulating the Treg/Th17 cell balance and suppressing the expression of pro-inflammatory cytokine levels. This new finding not only extended our understanding of the underlying mechanisms of the anti-inflammatory activity of T. hemsleyanum on the cellular but also laid the foundation for the future application of this herb in regulating T-cell homeostasis that contributed to immune-mediated diseases.In summary, our study explored drug–disease target interactions and multi-target mechanism of P. aeruginosa lung infection via in vivo experiment. Third, the protective mechanism of EFT against P. aeruginosa lung infection needed more deeply experimental confirmation.However, there were some limitations which still require further evaluation and exploration. First, due to the characteristics of network pharmacology, the results of network pharmacology analysis in this article depended on the discovery and mining of more chemical components. Second, multiple signaling pathways were identified through network pharmacological analysis; we only selected inflammatory and immune factors to further analyze and confirm the mechanism of


Serum lipid concentrations are affected by long-term high-fat diets; thus, we hypothesize that lipid levels increase after the Spring Festival in China.In total, 20,192 individuals  were enrolled in this retrospective cross-sectional study based on clinical data from the Laboratory Information System (LIS) and Hospital Information System (HIS) in Peking Union Medical College Hospital from 2014 to 2018. Total cholesterol (TC), triglycerides (TGs), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were analyzed.The serum TC [male vs. female: (4.71 ± 0.90 vs. 4.56 ± 0.85) mmol/L], TG [male vs. female: (1.71 ± 1.56 vs. 1.02 ± 0.68) mmol/L], and LDL-C [male vs. female: (3.01 ± 0.77 vs. 2.73 ± 0.74) mmol/L] levels were significantly higher in males than in females (P < 0.001); serum HDL-C [male vs. female: (1.18 ± 0.28 vs. 1.50 ± 0.34) mmol/L] was significantly lower in males (P < 0.001). In February, the TC, TG, and LDL-C levels were 8.4%, 16.3%, and 9.3% higher than the lowest levels recorded, respectively. The prevalence of dyslipidemia of the two weeks before the Spring festival was significantly lower than that of the first week after the Spring festival (43.6% (168/385) vs. 54.1% (126/233), P=0.007). Additionally, the prevalence of dyslipidemia was statistically higher in the first week after the Spring Festival than in May–January.Higher TC, TG, and LDL-C in winter could be associated with high-fat diets during the Spring Festival. The Spring Festival was immediately followed by a higher lipid concentrations. Thus, we don't recommend lipid assessment or physical examination immediately after the holiday especially Spring festival.The online version contains supplementary material available at 10.1186/s12872-021-02328-4. Lipid profiles, including TC, TG, HDL-C, and LCL-C, are primary analytes for the diagnosis of dyslipidemia in clinical settings. A seasonal variation in lipid profiles has been reported by several studies in various countries –7, with Similarly, our previous study reported seasonal variation in lipid profiles, with peak values during the winter and decreasing levels in summer . AlthougAccordingly, in this study, we hypothesize that the TC, TG, and LDL-C levels increase along with higher rate of dyslipidemia, while HDL-C decreases during the first week after the Spring Festival, based on clinical laboratory big data.A total of 280,206 records were derived from the Laboratory Information System (LIS) and Hospital Information System (HIS) of Peking Union Medical College Hospital from 2014 to 2018. The inclusion and exclusion criterion schematic is described in our previous study [Serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), total protein (TP), albumin (Alb), total bilirubin (TBil), direct bilirubin (DBil), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cr), uric acid (UA), and glucose (Glu) were measured with a Roche C8000 automatic analyzer .th according to the lunar calendar. Thus, the timing of the Spring Festival differs from year to year. The dates of the Spring Festival during the study period are shown in Table According to the traditional festival in China, the first day of the lunar calendar marks the beginning of the Spring Festival. In China, seven days are allocated to the celebration of this festival from the last day of December to January 6The four seasons were defined as follows: spring , summer , autumn , and winter .Dyslipidemia was defined as TC ≥ 5.2 mmol/L, TGs ≥ 1.7 mmol/L, HDL-C < 1.0 mmol/L or LDL-C ≥ 3.4 mmol/L . The nonData were analyzed using SPSS 20.0 software , Excel 2010  and GraphPad Prism for Windows . A Kolomogorov-Smirnov analysis was used to evaluate the distribution of data. Normally distributed data are presented as the mean ± standard deviation, while nonnormally distributed data are expressed as the median and quartiles. Kruskal–Wallis or Mann–Whitney U tests were used to compare the differences. A chi-squared test was used to analyze the differences in the prevalence of dyslipidemia by month. P values < 0.05 were considered statistically significant.2, 118 mmHg, and 73 mmHg, respectively. The basic characteristics of the enrolled participants by season are shown in Table In total, 20,192 individuals, including 10,108 males and 10,084 females, were enrolled in this study. The average age, BMI, SBP, and DBP were 39.4 years, 23.7 kg/mThe serum TC, TG, HDL-C, and LDL-C distributions by month and sex are shown in Fig. The distribution of lipid profiles between the first week after the Spring Festival and other days in the year is shown in Fig. The overall prevalence of dyslipidemia was 44.7%, and the prevalence was significantly higher in males than in females . Moreover, the prevalence of dyslipidemia in spring, summer, autumn, and winter was 48.3%, 42.5%, 42.3% and 47.7%, respectively. After stratifying by sex, the prevalence of dyslipidemia in spring, summer, autumn, and winter was 61.5% and 32.8%, 56.3% and 28.1%, 59.7% and 28.7%, and 61.6% and 32.2% for males and females, respectively. The prevalence of dyslipidemia by month is shown in Fig. Seasonal variations in lipid levels have been confirmed by many studies –4, 9, soIncreased LDL-C levels have been described to be associated with cardiovascular disease. In fact, reduced LDL-C levels are often prescribed as an effective method to prevent cardiovascular diseases . FurtherIn this study, we confirmed the association between increased lipid levels and high-fat diet intake during the Spring Festival, which was consistent with a Denmark study enrolling a total of 25,764 participants from the general population . This obThere are several strengths to emphasize in this study. Firstly, we used clinical laboratory big data downloaded from LIS and HIS to conduct this study, which is not only cost-effective but also efficient. Secondly, we only included information from the initial reports generated for individuals who visited the Department of Health Medicine to ensure the consistency of all data. Additionally, to avoid having sex as a confounding variable when examining the effect of Spring Festival and prevalence of dyslipidemia, we performed individual analyses for variation and prevalence by sex. Last but not least, this study was the first to report an association between increased lipid levels and the Spring Festival in China.However, certain limitations were also noted in this study. Although we used clinical data to analyze the association between increased lipid levels and the Spring Festival, we did not collect dietary intake information. However, it is well known that during the Spring Festival, a large proportion of the population gathers to celebrate the holiday with more high-fat meals and reduced levels of exercise . MoreoveIn conclusion, we confirmed that higher lipid levels are associated with a high-fat diet during the Spring Festival, with notable differences observed between males and females. However, the prevalence of dyslipidemia during the first week after spring was higher than in any other month for both males and females. Thus, to accurately diagnose dyslipidemia immediately after the Spring Festival, it is necessary to consider the effect of high-fat diet during Spring Festival. We don't recommend lipid assessment or physical examination immediately after holiday especially Spring Festival.Additional file 1. Supplemental Figure 1. Prevalence of dyslipidemia by sex and monthGroup A represent the first week after Spring Festival.