Objective: To develop an accurate and rapid ultra-performance liquid chromatography (UPLC) coupled with a photodiode array (PDA) method for the simultaneous determination of artemisinin (Art), arteannuin B (Art B), arteannuin C (Art C), dihydroartemisinic acid (DHAA) and artemisinic acid (AA) in Artemisia annua L. Methodology: Chromatography separation was performed on an ACQUITY UPLC BEH C18 Column with isocratic elution; the mobile phase was 0.1% formic acid aqueous solution (A) and acetonitrile (B) . Data were recorded at an ultraviolet (UV) wavelength of 191 nm for Art, Art C, DHAA and AA, and 206 nm for Art B. Results: The calibration curves of the five sesquiterpene components were all linear with correlation coefficients more than 0.9990. The linear ranges were 31.44–1572 μg/mL, 25.48–1274 μg/mL, 40.56–2028 μg/mL, 31.44–1572 μg/mL and 26.88–1396 μg/mL for Art, Art B, Art C, DHAA and AA, respectively. The precision ranged from 0.08% to 2.88%, the stability was from 0.96% to 1.66%, and the repeatability was all within 2.42% and had a mean extraction recovery of 96.5% to 100.6%. Conclusion: The established UPLC–PDA method would be valuable for improving the quantitative analysis of sesquiterpene components in Artemisia annua L. Artemisia annua L. (A. annua), is widely used as a famous traditional Chinese medicine (TCM). The major biological effects attributed to the constituents of A. annua include treating heat, malaria In the earliest stages of the study, separation effects were investigated using different mobile phase systems, such as methanol–water, acetonitrile–water, acetonitrile–0.1% acetic acid water, 0.1% acetonitrile–0.1% acetic acid water and acetonitrile–0.1% formic acid water. The effects of different organic fractions, specifically from 20% to 80%, on the separation efficiency were optimized under the conditions of isocratic elution and gradient elution, respectively. The results showed that the mobile phase system of acetonitrile–0.1% formic acid water was the best under the condition of isocratic elution. These five sesquiterpene compounds contain acidic compounds, and the addition of an acid to the mobile phase can reduce the degree of dissociation and have good retention properties. Acid addition is a good modifier.The five compounds were simultaneously determined by an HPLC method in our laboratory before. However, using a common HPLC column, the separation effect is not good, and the separation is better with a smaller particle size of the UPLC column. Therefore, the UPLC method was used to simultaneously quantify these five compounds. In order to verify the accuracy of the UPLC method, the quantitative results of Art and Art B were compared with the quantitative results of a single compound measured by HPLC. The results showed that the UPLC method was 90%–110% of the HPLC results, indicating that the UPLC method was accurate and reliable.A. annua has no areas with the best yields, this study determined extracts of A. annua from 15 regions in China. The preliminary results showed that the contents of five compounds in A. annua were quite different, indicating that the contents of sesquiterpenes in A. annua in different regions were dissimilar. In the preliminary studies of various components, a peroxide bridge in Art structure was found, which made it the main component in the treatment of malaria. In recent years, more and more researchers—including our team—have paid attention to the therapeutic activities of A. annua multi-components in the treatment of malaria [Single factor experiments provide a basis for further accurate orthogonal experiments to extract the materials. Since  malaria . A recen malaria ,24,25. TA. annua. Most of the existing research only measures up to four components in A. annua, and no references have been published to simultaneously determine these five sesquiterpenes. Therefore, it is not easy to accurately grasp the transformation and dynamic equilibrium of sesquiterpenes in A. annua biosynthesis. The UV or ELSD method is not sensitive enough, but UPLC uses a smaller particle size column for better resolution and sharper peaks, which improves sensitivity [A. annua. The validation results of the method showed good linearity, repeatability, and intra-day and inter-day precision, stability and recovery, indicating that the method was successfully applied to simultaneous analysis of these five compounds, which provided an analytical means for further quality control of A. annua.In conclusion, the UPLC method established by the current research was used to quantitatively analyze five compounds—Art, Art B, Art C, DHAA and AA—in the extract of sitivity . In mostA. annua medicinal materials used in the experiment were all commercial samples purchased from 15 places in China, including Burqin County People’s Hospital, Altay region, Xinjiang Uygur Autonomous Region (N47°50′44.30″E88°08′24.83″) (Xinjiang); Ezhou Hospital of Traditional Chinese Medicine, Hubei Province (N30°23′27.06″ E114°53′41.82″) (Hubei); Quzhou Nankong Chinese Medicine Co., LTD., Zhejiang Province (N28°56′9.31″ E118°52′27.08″) (Zhejiang1); Heze City, Shandong Province (N35°14′1.07″ E115°28′52.14″) (Shandong1); Shengzhou City, Zhejiang Province (N29°35′18.56″ E120°49′18.12″) (Zhejiang2); Anguo Baicao Medicinal Material Shop, Hebei Province (N38°25′6.42″ E115°19′35.90″) (Hebei); Yan’an City, Shaanxi Province (N36°35′7.04″ E109°29′23.21″) (Shaanxi); Meitan County, Zunyi City, Guizhou Province (N27°44′56.00″ E107°27′55.84″) (Guizhou); Xinning County, Shaoyang City, Hunan Province (N26°26′0.46″ E110°51′24.26″) (Hunan1); Capital Medical University Chinese Medicine Clinic-Beijing Herborist Chinese Herbal Pieces Co., Ltd., Beijing City (N39°54′16.88″ E116°24′25.81″) (Beijing1); Jinan Hebao Chinese Medicine Co., Ltd., Shandong Province (N36°33′14.50″ E116°45′7.16″) (Shandong2); Self-collection, Beijing City (N39°54′16.88″ E116°24′25.81″) (Beijing2); Longshan County Medicinal Materials Market in Xiangxi Autonomous Prefecture, Hunan Province (N28°18′44.00″ E109°44′20.15″) (Hunan2); Changtu County, Tieling City, Liaoning Province (N42°47′10.07″ E124°06′39.31″) (Liaoning); Medicinal materials provided by the pharmaceutical company, Hunan Province (N26°25′13.22″ E111°36′44.10″) (Hunan3). They were all identified by Professor Manyuan Wang  as the dry ground part of Artemisia annua L. and stored at room temperature and protected from light.The A. annua in our laboratory  [1H NMR, 13C NMR and HR–ESI–MS techniques. Their purities were also evaluated to be all greater than 99.0% by the HPLC–DAD method at UV 210 nm using the area normalization procedure. The conditions for mass spectrometry detection were as follows. The mass spectrometric analysis was performed on an LC–ESI–TOF equipped with an ESI source . All acquisitions were performed under positive ionization mode with a capillary voltage of +1000 V. The source temperature was 120 °C, and nitrogen was used as nebulizer and desolvation gas, as well as the drying gas at 800 L·h−1 at a desolvation temperature of 450 °C. The cone gas was 50 L·h−1; the sampling cone was 40 V and the collision energy (CE) was 4 V. 8 V and 12 V was selected as the trap collision energy. The acquisition mass range was from m/z 50 to m/z 1000. The total run time for analysis was 10 min  ,22. Theiv/v).The injection volume was 1 μL. Data were recorded at a UV wavelength of 191 nm for Art, Art C, DHAA and AA, and 206 nm for Art B.An Agilent 1290 UPLC system  equipped with a pump, degasser, column oven, auto sample injector, and PDA detector  was used for liquid chromatography analysis. The chromatographic separation of the five marker compounds was performed at 45°C using an ACQUITY UPLC BEH C18 Column  and ACQUITY UPLC C18 Van Guard Pre-column  with an isocratic elution at a flow rate of 0.20 mL/min, using a mobile phase consisting of 0.1% formic acid (A) and acetonitrile (B)  of Art, Art B, Art C, DHAA and AA was accurately weighed and dissolved in HPLC grade acetonitrile to make a stock solution. Calibration standards were prepared by diluting the stock solution with acetonitrile in appropriate quantities.The extraction method (A), extraction solvent (B), ratio of material to liquid (C), extraction time (D), and cycle of extraction (E) were taken as the influencing factors, while the respective content and total content of Art, Art B, Art C, DHAA and AA were taken as the comprehensive evaluation indicators. Single factor experiments were carried out to optimize the extraction process. The level of the extraction process factors was shown in A. annua) were used as the index, and the L (33) orthogonal table design test plan was used to determine the optimum extraction conditions of the A. annua. The optimum process conditions for extracting are shown in The ratio of material to liquid, extraction time and number of extractions were selected as the investigation factors. Referring to the single factor experiment results, two types of content . The filtrate was concentrated in vacuum by a rotary evaporator  system to evaporate the sample and prepare a paste extract (0.1405 g). The yield of Artemisia annua L. ether extract was 14.1%. The extract was accurately weighed, and the volume of acetonitrile was adjusted to 5 mL to obtain a final extract of A. annua. The sample solution was then filtered through a syringe filter (0.22 μm) and used for UPLC analysis.The five reference standard solutions were diluted 2, 5, 10, 20, and 50 times with acetonitrile, filtered through a 0.22 μm microporous membrane, and injected sequentially. The calibration curves were drawn with the sample concentration (μg/mL) as the abscissa and the peak area as the longitudinal coordinate. The concentration ranges of the reference substances were as follows: Art (1572–31.44 μg/mL), Art B (25.48–1274 μg/mL), Art C (40.56–2028 μg/mL), DHAA (31.44–1572 μg/mL) and AA (26.88–1396 μg/mL). LLOQ was defined as the lowest concentration on the calibration curve with an acceptable accuracy within ± 5% and precision less than 5% [A. annua sample were prepared to act as the sample solutions, according to the method described in A. annua with known content were accurately weighed and a certain concentration of standard solution added into the sample. This was prepared in accordance with the method of The precision of the UPLC method was evaluated by using a mixed standard solution of marker compounds at low, medium and high levels to measure intra-day and inter-day variations . The mix


Aegiceras corniculatum, Avicennia marina, Bruguiera gymnorrhiza, and Kandelia candel in Guangxi, China. The four tree species had similar overall colonisation rates of endophytic fungi (24–33%). The colonisation rates of endophytic fungi were higher in twigs (30–58%) than in leaves (6–25%) in the four plant species. A total of 36 endophytic fungal taxa were identified based on morphological characteristics and molecular data, including 35 Ascomycota and 1 Basidiomycota, dominated by Phomopsis, Phyllosticta, Xylaria, Leptosphaerulina, and Pestalotiopsis. The diversity of endophytic fungi was higher in twigs than in leaves in the four plant species. Some endophytic fungi showed host and tissue preference. The endophytic fungal community composition was different among four mangrove species and between leaf and twig tissues.Mangrove forests play an important role in subtropical and tropical coastal ecosystems. Endophytic fungi are widely distributed in various ecosystems and have great contribution to global biodiversity. In order to better understand the effects of mangrove species and tissue types on endophytic fungal community, we investigated cultivable endophytic fungi in leaves and twigs of four mangroves  A total of 10 mature individuals of each plant species  were randomly chosen and the individuals of one plant species were 50 m away from each other. One twig (ca. 0.8 cm in diameter) with attached leaves was collected from each individual tree and immediately placed in plastic bags, labelled, and transported to laboratory. Samples were stored at 4°C and processed within 4 days.The study was carried out in the Beilun Estuary National Reserve of Guangxi, South China . This site has a mean annual temperature of 22.2°C, mean annual precipitation of 2500–2700 mm and soil salinity of 0.5–1.2%. In late April 2014, we selected four mangrove species The sampling regime was designed with the intention of isolating as many endophytic species as possible from the samples. One twig of each individual tree was cut into 5-mm long segments (ca. 0.8 cm in diameter) and five leaves were removed from one twig and cut into discs (5 mm in diameter). A total of 10 twig segments and 10 leaf discs were randomly selected from each sample. In total, 780 segments were used in this study.Surface sterilisation followed the method of Guo et al. . SegmentMycelia sterilia, which were divided into different “morphotypes” according to cultural characteristics such as colony colour, texture, and extension rate on MEA  were scraped from the surface of the agar plate and transferred into a 1.5 mL microcentrifuge tube with 700 µL of preheated (65°C) 2 × CTAB extraction buffer , and c. 0.2 g sterilised quartz sand. The mycelium was ground using a glass pestle and then incubated in a 65°C water bath for 30 min with occasional gentle swirling. About 500 mL of phenol:chloroform (1:1) was added into each tube and mixed thoroughly to form an emulsion. The mixture was spun at 12,000 g for 15 min at room temperature in a microcentrifuge and the aqueous phase was transferred into a fresh 1.5 mL tube. The aqueous phase containing DNA was re-extracted with chloroform:isoamyl (24:1) until no interface was visible. About 30 mL of 5 M KOAc was added into the aqueous phase followed by 200 µL of isopropanol and inverted gently to mix. The genomic DNA was precipitated at 9200 g for 2 min in a microcentrifuge. The DNA pellet was washed twice with 70% ethanol and dried using SpeedVad  for 10 min or until dry.Genomic DNA was extracted from fresh cultures following the protocol of Guo et al. . Fresh f4)2SO4, 2 mM MgCl2, 20 mM Tris-HCl, pH 8.4), 200 µm of each deoxyribonucleotide triphosphate, 15 pmols of each primer, c. 100 ng template DNA, and 2.5 units of Taq polymerase . The thermal cycling programme was as follows: 3 min initial denaturation at 94°C, followed by 35 cycles of 30 s denaturation at 94°C, 30 s annealing at 52°C, and 1 min extension at 72°C; and a final 10 min extension at 72°C. A negative control using water instead of template DNA was included in the amplification process. About 4 mL of PCR products from each PCR reaction were examined by electrophoresis at 80 V for 30 min in a 1% (w/v) agarose gel in 1 × TAE buffer  and visualised under ultraviolet light after staining with ethidium bromide (0.5 mg/mL). PCR products were directly sequenced with primer pairs as mentioned above in the ABI 3730-XL DNA sequencer .The internal transcribed spacer (ITS) region of rDNA was amplified using primer pairs ITS4 and ITS1F  of endophytic fungi was calculated according to the formula: H′ = −k is the total number of fungal species, and ip is the proportion of individuals that species i contributes to the total (Pielou Cs) was employed and calculated according to the following formula: Cs = 2j/(a + b), where j is the number of endophytic fungal species recovered from both plant species, a is the total number of endophytic fungal species from one plant species, and b is the total number of endophytic fungal species from the other plant species , 45 from A. marina (5 from leaves and 40 from twigs), 83 from B. gymnorrhiza (7 from leaves and 76 from twigs), and 63 from K. candel (21 from leaves and 42 from twigs)  > B. gymnorrhiza (44%) > A. marina (40%) > K. candel (30%) in twigs, and A. corniculatum (25%) > K. candel (20%) > B. gymnorrhiza (7.78%) > A. marina (6%) in leaves  . The dif. candel . The funn leaves .10.1080/Coprinopsis atramentaria), and 35 were members of Ascomycota (A. corniculatum (12 from leaves and 17 from twigs), 16 from A. marina (2 from leaves and 16 from twigs), 18 from B. gymnorrhiza (6 from leaves and 16 from twigs), and 16 from K. candel (6 from leaves and 12 from twigs). The Shannon diversity index (H′) of endophytic fungi from high to low was A. corniculatum (2.69) > A. marina (2.51) > B. gymnorrhiza (2.45) > K. candel (2.31) in whole plant, A. marina (2.46) > A. corniculatum (2.38) > B. gymnorrhiza (2.32) > K. candel (2.09) in twigs, and A. corniculatum (2.23) > B. gymnorrhiza (1.75) > K. candel (1.13) > A. marina (0.67) in leaves (Cs: 0.38–0.59) among the four mangrove species  and 23 rare taxa (IV < 5) . Phomopsnt study . More ennt study .10.1080/Pestalotiopsis humus, Leptosphaerulina chartarum, Phomopsis sp.4, Hortaea werneckii, and Cladosporium perangustum were abundant in plant A. corniculatum and were mainly recovered from twigs, while L. chartarum distributed evenly in twigs and leaves  in this study. This result is in consistence with previous studies on mangrove endophytes (Gilbert et al. s et al.  reporteds et al. .Phomopsis, Phyllosticta, Xylaria, Leptosphaerulina, and Pestalotiopsis were dominant in the four mangroves, as reported in some previous studies (Bayman et al. Phomopsis fungi were widely isolated from mangroves (Suryanarayanan et al. Pestalotiopsis as generalist fungi were common mangrove endophytes and widely occurred on plants across Palmae, Rhizophoraceae, Planchonellae, and Podocarpaceae in China (Liu et al. Xylaria, as endophytic fungi were dominant in subtropical and tropical ecosystems (Bayman et al. Phyllosticta taxa were the most frequently isolated fungi of 10 mangrove plants in Thailand (Chaeprasert et al. (L. chartarum as the predominant species was first reported in mangrove species in this study.We found that t et al. . In addiH. werneckii and C. perangustum were obtained only from plant A. corniculatum, P. azadirachtae, and Pyronema sp. from plant A. marina, and F. striatum and X. feejeensis from plant B. gymnorrhiza. In addition, some abundant fungi were found in two plant species in this study. For example, P. humus was obtained from A. corniculatum and K. candel. Phomopsis sp.1 was recovered from A. marina and B. gymnorrhiza. Similarly, previous studies have shown that some endophytic fungi were abundant in certain mangroves (Pang et al. Pestalotiopsis was dominant in A. corniculatum in Guangxi Province, China. Xing and Guo (Pestalotiopsis along with Phomopsis were the most frequent endophytes in four Rhizophoraceae mangrove species on the south coast of China. Chaeprasert et al. (Phyllosticta was the most frequently isolated fungus from Avicennia alba. Pang et al. (Xylaria sp. was dominant endophytic species in K. candel. These results suggested that some endophytic fungi show certain host preference.The community composition of endophytic fungi was different in the four mangroves in our study, which was consistent with previous studies that endophytic fungal community composition was conspicuously affected by mangrove species (Suryanarayanan et al.  and Guo  found tht et al.  suggesteg et al.  reportedK. candel in Hong Kong. De Souza Sebastianes et al. (R. mangle, A. schaueriana, and L. racemosa in Brazil.We found that the diversity of endophytic fungi was higher in twigs than in leaves in the four plant species. Similar results were reported in previous studies (Maria and Sridhar s et al.  reportedPhomopsis fungi were much more abundant in twigs than in leaves in this study. Similarly, Xing et al. (Phomopsis species more frequently distributed in stems than in leaves in Sonneratia apetala, S. hainanensis and S. paracaseolaris on southern coast of China. Pestalotiopsis was much abundant in twigs rather than in leaves in this study, which was consistent with previous studies in mangrove plants (Pang et al. Phyllosticta fungi were abundant in leaves of the four mangrove trees, as reported in previous mangrove studies (Suryanarayanan et al. L. chartarum and X. feejeensis were isolated only from twigs of A. marina and B. gymnorrhiza, respectively. These results suggested that some endophytic fungi showed certain tissue preference (Guo et al. We found that the community composition of endophytic fungi was different between twigs and leaves in the four mangroves. Similar results have been shown by previous studies (Ananda and Sridhar g et al.  found thPhomopsis, Phyllosticta, Xylaria, Leptosphaerulina, and Pestalotiopsis. The diversity of endophytic fungi was higher in twigs than in leaves among the four mangrove species. Some endophytic fungi showed certain host and tissue preference. The community composition of endophytic fungi was different among host species and between tissue types.Our study investigated the endophytic fungal community associated with twigs and leaves among four mangrove species in Southern China. Although the overall colonisation rate of endophytic fungi was similar among four investigated mangrove plants, the colonisation rate of endophytic fungi was higher in twigs than in leaves. A total of 36 endophytic fungal taxa were identified according to morphological characteristics and ITS sequences, dominated by


As a natural and healthy beverage, tea is widely enjoyed; however, the pesticide residues in tea leaves affect the quality and food safety. To develop a highly selective and efficient method for the facile removal of pesticide residues, the subcritical butane extraction (SBE) technique was employed, and three variables involving temperature, time and extraction cycles were studied. The optimum SBE conditions were found to be as follows: extraction temperature 45 °C, extraction time 30 min, number of extraction cycles 1, and in such a condition that the extraction efficiency reached as high as 92%. Further, the catechins, theanine, caffeine and aroma components, which determine the quality of the tea, fluctuated after SBE treatment. Compared with the uncrushed leaves, pesticide residues can more easily be removed from crushed leaves, and the practical extraction efficiency was 97%. These results indicate that SBE is a useful method to efficiently remove the bifenthrin, and as appearance is not relevant in the production process, tea leaves should first be crushed and then extracted in order that residual pesticides are thoroughly removed. Tea is of great economic importance , not onl2 catalytic treatment [Several studies on pesticide removal from a variety of plants, including vegetable, fruits and crops, have been reported ,7,8. As reatment , and ultreatment ,18 have reatment . It is wreatment ,21,22. Hreatment ,24.Subcritical fluid extraction is a relatively simple, rapid and environmentally friendly technology, which is mostly used in extraction of flavors , specialChina is one of the main producers of tea, but, unfortunately, the export of tea has been declining gradually, and one of the main causes is pesticide residues . HoweverY) for different experimental combinations. The results suggested that the extraction efficiency of bifenthrin is significantly changed depending on the extraction conditions , according to the Box-Behnken design (BBD). Based on the results of the single-factor experiments, the range and center point values of three independent variables, including (A) temperature, (B) time, and (C) number of extraction cycles, were determined. nditions .Y to bifenthrin extract content can be obtained by the second-order regression equation (in terms of coded factors): Y = 2.88 + 0.88 × A + 0.076 × B − 0.013 × C − 0.08 × A × B − 0.46 × A × C + 0.15 × B × C + 0.46 × A2 + 0.18 × B2 + 0.0005 × C2. To ensure the reliability of the model, the determination coefficient (R2) was calculated and came to 0.9859, which indicated an agreement between the experimental and predicted values. The analysis of variance was shown in p > 0.05), suggesting that the model equation was adequate for predicting the bifenthrin extract content in any combination of the variables’ values. In total, the ANOVA demonstrated the Fisher’s F-test for the model had a high F-value (54.53), and a quite low p-value (<0.0001), so the model was significant.The multiple regression analysis was employed based on the experimental data, and the predicted response 2 (the quadratic of extraction temperature) and AC (the interaction of extraction temperature and the number of extraction cycles) were quite significant (p < 0.01), and B2 (the quadratic of extraction time) was significant (p < 0.05). Therefore, we can make such a conclusion: the impact factor of extract content of bifenthrin had a scope trend from extraction temperature (important), extraction time, to the number of extraction cycles (negligible). The three-dimensional (3D) response surface and two-dimensional (2D) contour plots based on the regression equation model are shown in The ANOVA of the linear, quadratic and interaction terms of the model are shown in The crushed tea leaves can be used in instant tea and food additives. To make further enhancements in the extraction efficiency of bifenthrin, we studied the effect of pulverization on the extraction efficiency. Crushed ones (average size of tea particles is 0.3 mm) were divided into two groups and extracted on the basis of the conditions 3 and 9 in Y) versus the corresponding concentration , and the correlation coefficients (R2) of all calibration curves were greater than 0.9921, which indicated that these were sufficiently reliable for the analysis of such substances  via HPLC, aiming at the determination of the quality changes of tea, before and after the experiment. In chromatographic analysis, calibration curves were constructed by plotting peak area (bstances . Based oE)-hexenal, however, the gradient of relative content for β-ionone reached to 64.49% . In experimental processes, the bifenthrin concentration  was adjusted to twice the suggested value. Three days after spraying the pesticide, tea leaves at the top were picked and processed into black tea, then transferred to a laboratory within the same day, at a temperature of 4 °C.The butane was purchased from Puyang Petrochemical Limited Company , and the purity was higher than 99%. Bifenthrin (≥95%), phenethyl acetate (≥99%) and triphenyl phosphate (TPP) (≥95%) were used as internal standard and purchased from Dr. Ehrenstorfer Company . Acetonitrile, methanol, acetone, methylbenzene, ethyl acetate, butane, hexane, etc. were of chromatographic grade and purchased from J. T. Baker . Anhydrous magnesium sulfate, sodium chloride, sodium citrate dibasic sesquihydrate, and citric acid was of an analytical grade and purchased from North Reagent Company . Primary secondary amine (PSA) sorbent was purchased from Supelco . Six kinds of catechins, theanine and caffeine were purchased from Aladdin Company in Shanghai (≥95%). Other reagents were of analytical grade . The Chinese medicine pulverizer was manufactured in Shanghai Dianjiu by traditional Chinese Medicine Machinery Manufacturing Limited Company .The tea leaves with bifenthrin were treated by the SBE method, and the schematic diagram of the adopted apparatus  appears in Y) of the bifenthrin peak area to the TPP peak area was plotted against the concentration (X) of bifenthrin. Linear regression analysis revealed a standard curve with the following equation: Y = 0.00347065 + 0.00188399*X (R2 = 0.9998).The standard stock solution (10 μg/mL) and internal solution (20 μg/mL) were prepared by dissolving bifenthrin and TPP in acetonitrile, respectively. The intermediate standard solutions were obtained by serial dilution of standard stock solutions with acetonitrile, with 100 μL TPP in every one as an internal standard. The final concentration of bifenthrin in the above solutions was 2, 5, 10, 20, 50, 100, and 200 μg/mL. Then, GC-MS/MS  analysis was performed on each solution, and the ratio  crushed tea leaves (2.0 g) were mixed with 10 mL ultrapure water in a 50 mL centrifuge tube, which was then soaked for 10 min; (2) 100 µL internal standard solution TPP (20.0 mg/L), 10 mL acetonitrile and 5 mL methylbenzene were added into the centrifuge tube and shaken for 1 min at 2000 rpm, and was then stored at 4 °C for 10 min; (3) A salting-out reagent kit was put into mixed solution, and was then immediately vibrated for 2 min at 2000 rpm on the vortex mix and centrifuged for 3 min at 9000 rpm; (4) An extract liquid of 1.5 mL was acquired and placed into a 2 mL centrifuge tube, containing 150 mg anhydrous magnesium sulfate, 40 mg PSA adsorbent, and 40 mg C18E adsorbent. The extract liquid was continuously vibrated for 2 min at 2000 rpm and centrifuged for 3 min at 9000 rpm; (5) The extraction solution (1.0 mL) was filtered through a 0.22 µm nylon filter prior to the GC-MS/MS analysis. All tests were conducted in triplicates.In this study, a trace GC ultra with TR-pesticide column (30 m × 0.25 mm × 0.25 µm) was applied in the GC-MS/MS system. The carrier gas was helium, with a constant flow rate of 1 mL per minute. The injector temperature was set as 250 °C. The oven temperature was firstly arranged at isothermal 50 °C for 1 min, and increased to 150 °C at a rate of 25 °C/min, then sequentially raised to 260 °C at 5 °C/min, finally increased to 280 °C at 10 °C/min, and remained isothermal at 280 °C for 20 min.Simultaneous distillation-extraction (SDE) was used for extracting the aroma components from tea . Before The GC-MS system used in this study was Agilent 7890-5790, with DB-5MS column (30 m × 0.25 mm × 0.25 µm). In the typical system, helium was the carrier gas, with a constant flow rate of 1 mL/min. The injector and ion source temperatures were 200 °C and 250 °C, respectively. The oven temperature was isothermal at 40 °C for 2 min, then increased to 260 °C at 10 °C/min, and remained at 260 °C for 20 min. The mass spectrometer conditions were as follows: ionization mode, EI; electron energy, 70 eV; ion source temperature, 250 °C; quadrupole temperature, 150 °C; mass scan range, 33–500 amu; and solvent delay, 3 min.Six kinds of catechins, theanine, caffeine contents were determined by HPLC following the ISO 14502-2:2005, ISO/DIS 19563, ISO 10727:2002, respectively. Fenugreek seed was purchased from a regional pharmacy, located in Zhengzhou, China. Samples were ground into powder with a Wiley Mill  and passed through different sieve sizes according to experimental design. The seed powder was air-dried for 24 h at 80 °C, and then stored at 4 °C for further use. Butane was purchased from Puyang Longyu Chemical Co., Ltd. . Other chemicals and solvents used for this study were of either analytical or chromatographic grade, and all purchased from either Fisher Scientific Chemical , or Sigma Aldrich .SBE technology was firstly applied to remove bifenthrin pesticide residues from tea leaves, and different SBE conditions were surveyed. The optimum SBE condition was as follows: extraction temperature 45 °C, extraction time 30 min, number of extraction cycles 1, and the extraction efficiency reached 92%. Furthermore, the extraction efficiency increased to 97% if the tea leaves were grounded. Meanwhile, the results indicated the quality of tea fluctuated little after SBE treatment, especially for intact samples. Thus, the SBE is a highly efficient cleaning technology for the removal of residual pesticides from tea, and if the appearance of tea is not important in the production process, tea leaves should first be crushed and then extracted using the SBE, in order to wipe out residual pesticides thoroughly.


Terminalia catappa leaves are used in managing both diabetes mellitus and its complications in Southwest Nigeria. However, its inhibitory activity on enzymes implicated in diabetes is not very clear. This study investigated the in vitro inhibitory properties and mode of inhibition of T. catappa leaf extracts on enzymes associated with diabetes. The study also identified some bioactive compounds as well as their molecular interaction in the binding pocket of these enzymes. Standard enzyme inhibition and kinetics assays were performed to determine the inhibitory effects of aqueous extract (TCA) and ethanol extract (TCE) of T. catappa leaves on α-glucosidase and α-amylase activities. The phytoconstituents of TCA and TCE were determined using GC-MS. Molecular docking of the phytocompounds was performed using Autodock Vina. TCA and TCE were the most potent inhibitors of α-glucosidase (IC50 = 3.28 ± 0.47 mg/mL) and α-amylase (IC50 = 0.24 ± 0.08 mg/mL), respectively. Both extracts displayed a mixed mode of inhibition on α-amylase activity, while mixed and noncompetitive modes of inhibition were demonstrated by TCA and TCE, respectively, on α-glucosidase activity. The GC-MS analytic chromatogram revealed the presence of 24 and 22 compounds in TCE and TCA, respectively, which were identified mainly as phenolic compounds, terpenes/terpenoids, fatty acids, and other phytochemicals. The selected compounds exhibited favourable interactions with the enzymes compared with acarbose. Overall, the inhibitory effect of T. catappa on α-amylase and α-glucosidase may be ascribed to the synergistic action of its rich phenolic and terpene composition giving credence to the hypoglycaemic nature of T. catappa leaves. Diabetes mellitus (DM) is an endocrine, chronic, noncommunicable disease plaguing the world populace with a rapid increase. A reported 425 million individuals were globally affected by DM, while 629 million people have been projected to be affected by 2045 . DM is cTerminalia catappa Linn, commonly known as Indian almond, belongs to the Combretaceae family and grows in the tropics of Asia, Africa, and Australia [T. catappa extracts such as hepatoprotective effects, anticancer property, antimutagenic activity, and antiaging property [T. catappa methanolic leaf extract on diabetic-linked enzymes. Despite this antidiabetic claim by the locals, the elaborate antidiabetic mechanism is far from clear. This study assessed the inhibitory properties of T. catappa leaf extracts on α-glucosidase and α-amylase, the mode of enzyme inhibition, as well as identified phytocompounds present and proposed the molecular mechanism of binding in the active sites of the enzymes.ustralia . In urbaustralia . Severalproperty . Divya aproperty  have alsα-Glucosidase, α-amylase enzymes, and their substrates were acquired from Solarbio Life Sciences, Beijing, China. Other chemicals were products of Sigma-Aldrich, St. Louis, USA.T. catappa leaves were sourced between October and December 2016, from Covenant University compound. They were identified by Dr. J. O. Popoola of Biological Sciences Department and voucher specimen deposited at Biological Sciences Department herbarium, Covenant University, Ota, Ogun State, with herbarium number TC/CUBio/H809. Aqueous T. catappa (TCA) and ethanol T. catappa (TCE) leaf extracts were prepared as reported by Iheagwam et al. [Mature α-Glucosidase inhibitory activity of the extracts was evaluated according to the method described by Ibrahim and Islam [μL) were incubated at 37°C for 15 min with α-glucosidase solution . ρ-Nitrophenyl-α-D-glucopyranoside (pNPG) solution  was thereafter added, and the resulting mixture was incubated for 20 min at 37°C. The reaction was terminated by adding Na2CO3 , and absorbance was measured at 405 nm. Phosphate buffer (100 mM) was used as control in place of inhibitors. Inhibitory activity was calculated using the following equation:As = absorbance in the presence of sample and Ac = absorbance of control. All solutions were prepared in 0.1 M phosphate buffer (pH 6.8).nd Islam  with sliα-glucosidase inhibitory kinetics. Extract  was preincubated with α-glucosidase solution  for 10 min at 25°C. Varying pNPG concentrations  were added and incubated for 10 min at 25°C to both sets of reaction mixtures to start the reaction. Thereafter, Na2CO3  was added to stop the reaction. For the control kinetic reaction, 100 mM phosphate buffer  was used in place of the extract. Reaction rates (v) were calculated, and double reciprocal plots of α-glucosidase inhibition kinetics were determined.The method of Sabiu and Ashafa  was adopα-Amylase inhibitory activity of the extracts was evaluated by adopting the method described by Ibrahim and Islam [μL) and acarbose were incubated at 37°C for 20 min with amylase solution . Starch solution  was later added to the reaction mixture and incubated at 37°C for 1 h. Dinitrosalicylic acid (DNS) colour reagent (1 mL) was added to stop the reaction. The resulting mixture was boiled for 10 min, and absorbance was measured at 540 nm. Phosphate buffer (100 mM) was used as control in place of inhibitors. The α-amylase inhibitory activity was calculated using the following formula:As = absorbance in the presence of sample and Ac = absorbance of control. All solutions were prepared in 100 mM phosphate buffer (pH 6.8).nd Islam  with sliα-amylase inhibitory kinetics. In brief, extract  was incubated with α-amylase  for 10 min, before the addition of various substrate concentrations . The reaction proceeded as highlighted for α-glucosidase. α-Amylase inhibition kinetics was determined from the Lineweaver–Burk double reciprocal plot.The method of Sabiu and Ashafa  was adopT. catappa extracts was carried out using GCMS-QP2010SE SHIMADZU, Japan, fused with the Optima 5 ms capillary column (30 × 0.25 mm) of 0.25 μm film thickness following the described method of Ajiboye et al. [μL in the splitless mode with a split ratio of 1 : 1 and injector temperature set at 200°C. Mass spectrophotometer conditions were as follows: ion source temperature (230°C), interface temperature (250°C), solvent delay at 4.5 min, and acquisition in a scan range of 50–700 amu. Electron ionization mode and multiplier voltage were set at 70 eV and 1859 V, respectively. Retention time, fragmentation pattern, and mass spectral data of the unknown components in the extracts were compared with those in Wiley and National Institute of Standards and Technology (NIST) libraries for compound identification.The GC-MS analysis of e et al.  with sliα-glucosidase and α-amylase was modelled using the crystal structures with PDB codes 5kzw and 1b2y, respectively, obtained from RCSB protein data bank as templates in SWISS-MODEL [The structures of the GC-MS identified compounds with ≥5% abundance were prepared as reported by Iheagwam et al. . The 3D SS-MODEL .α-glucosidase and α-amylase, respectively. Inhibition constant (Ki) of docked ligands were calculated by using the following formula:Virtual screening of selected identified ligands, analysis of drug-likeness using the rule of five (RO5), and molecular docking were carried out according to the methodology of Iheagwam et al. . Howeverp < 0.05.Data were analysed using SPSS version 25  and subjected to one-way analysis of variance (ANOVA) using the Duncan multiple range post hoc test. Values were reported as mean ± standard deviation (SD) of three (3) replicates and considered significantly different at α-glucosidase inhibitory activity of TCA and TCE as shown in p < 0.05) lower inhibition by the extracts was observed at all concentrations relative to control. TCA exhibited a significantly (p < 0.05) higher inhibition of α-glucosidase activity compared to TCE. Nonetheless, at lower concentrations (1–3 mg/mL), there was no difference between the inhibitory activities of TCA and TCE. These were further supported by a lower IC50 value (2.23 ± 0.21 mg/mL) for acarbose when compared with TCA (3.28 ± 0.47 mg/mL) and TCE  higher than TCA and acarbose at all concentrations. TCA elicited inhibitory effects that competed favourably with the standard drug (acarbose). These results were supported with an IC50 of 0.24 ± 0.08, 0.75 ± 0.14, and 0.85 ± 0.18 mg/mL recorded for TCE, TCA, and acarbose, respectively - were higher in TCE. It was also observed that there was no much difference in the abundance of vitamin E in both extracts.The identified phytochemicals present in TCE and TCA are shown in Tables For TCE, 9, 26, 13, 30, and 25% of the identified compounds were classified as carbohydrates, fatty acids, hydrocarbons, phenolics, and terpenes/terpenoids, respectively , while fα-amylase. However, only vitamin E (−82.91) and ethyl-α-D-glucopyranoside (−78.11) were relatively comparable with the standard  and vitamin E (−89.64), n-hexadecanoic acid (−81.89) and phytol (−80.87) binding affinities were also comparable with acarbose (−115.55).As illustrated in When the hit compounds were screened for their drug-likeness, they all obeyed Lipinski's RO5. However, phytol and vitamin E, on the one hand, violated only the octanol-water partition coefficient due to higher values than the RO5 threshold as presented in α-amylase and α-glucosidase, respectively. These values though lower were comparable with acarbose where −8.3 was recorded for α-amylase and −7.4 for α-glucosidase. Concomitantly, 1.39 to 40.51 μM was the α-amylase inhibition constant (Ki) recorded for the compounds compared to 0.84 μM for acarbose, while 47.95 to 184.70 μM was the α-glucosidase Ki recorded for the compounds compared to 3.83 μM for acarbose.The binding affinity of the selected compounds as shown in in vitro results as the majority of the ligands favoured active site binding compared to the allosteric site. Hydrogen, van der Waals, and π bonds were the common interactions displayed between the compounds and amino acids present in the binding sites of the enzymes. Trp 73, Trp 74, Tyr 77, Tyr 166, and Ile 250 were common amino acids stabilising the binding of vitamin E and acarbose in the binding pocket of α-amylase, while in the α-glucosidase binding pocket, Ala 284, Asp 616, and Trp 481 were common amino acids stabilising phytol, vitamin E, and acarbose  leaf extracts were higher than our extracts except for A. lanata aqueous leaf extract α-amylase inhibitory activity which was reported to be lower than ours [T. catappa leaf extracts was higher on α-amylase than on α-glucosidase at the varied concentrations and may be attributed to the different mechanism of action on these enzymes. This was further buttressed by the kinetic studies, where the TC extracts exhibited a mixed mode of inhibition on α-amylase, while mixed and uncompetitive inhibition mechanisms were observed for TCA and TCE, respectively, on α-glucosidase. The mixed mechanisms exhibited by TCA and TCE may suggest the bioactives present in the extracts may bind in the active site of these enzymes thereby reducing the affinity of the substrate [α-glucosidase active site affecting catalysis without having an effect on substrate binding in the active site [Smilax aristolochiifolia root extract and its compounds where the number of hydrogen bonds did not affect binding affinity. Vitamin E had the lowest free energy and Ki in amylase and glucosidase binding pockets which was comparable to acarbose. Consequently, it exhibited a more stable affinity with only a small concentration required to inhibit these enzymes [in vitro inhibitory mechanisms as more identified compounds bound to the active site than the allosteric site signifying a preference for the (E) to elicit their potential pharmacological action [lycaemia , 16. Reslycaemia . Promisit manner . Nonetheacarbose , 19. It extracts , 21. Thehan ours , 22. Conhan ours  and Wan han ours , the inhubstrate , 26. Binubstrate , 26. Theive site . The obsive site . Compounive site , 30, varive site , hexadecive site  have beeive site , 32, 33.ive site . In leadive site , 36. Comive site . Since aive site  reported enzymes . Molecull action . The coml action –41.T. catappa leaf extracts on glucosidase and amylase is being reported, making it an effective agent in managing postprandial hyperglycaemia. These extracts preferably bind to the active site of these enzymes where their various identified compounds synergistically illicit their inhibitory action. From the different GC-MS identified compounds, vitamin E was the most potent ligand that qualified as a potential drug candidate after docking studies. These plants can be leveraged upon as a natural source of not only vitamin E but other antidiabetic compounds for drug formulation. On the other hand, isolation and characterisation of these identified phytocompounds in addition to in vivo studies are still required to confirm these findings.This is the first time, to the best of our knowledge, the inhibitory mechanism of


The level of variation in lipids and their fatty acids was determined in the grains of 10 popular durum wheat cultivars commercially grown in Central and Southern Italy. Samples were harvested for two consecutive years to account for differences due to changes in climatic conditions. Total fat content was determined by means of the International Association of Cereal Science and Technology (ICC) Standard Method No. 136, whereas the fatty acid profile was determined by gas chromatography. Total lipid content ranged from 2.97% to 3.54% dry basis (d.b.) in the year 2010 and from 3.10% to 3.50% d.b. in the year 2011, and the average value was 3.22% d.b. considering both years together. Six main fatty acids were detected in all samples in order of decreasing amounts: linoleic (C18:2) > palmitic (C16:0) ≈ oleic (C18:1) > linolenic (C18:3) > stearic (C18:0) > palmitoleic (C16:1). Significant variations in the levels of single acids between two years were observed for three samples. These results will be very useful in the updating of food composition databases in general and will help authorities to set proper quality standards for wholegrain flours and products where the germ should be preserved, considering also the recent interest of industry and consumers for these kinds of products. Triticum durum Desf.) kernels contain about 2.4–3.8% dry basis (d.b.) of lipids [Durum wheat  were assessed. For this reason, we selected 10 cultivars amongst the most commonly grown for pasta making. Samples were collected in several locations of Central and Southern Italy to account, at least partially, for differences due to different pedoclimatic environments; Southern Italy is characterized by milder winters and warmer springs and summers with respect to Central Italy, however both areas are considered highly suitable for durum wheat cultivation. Moreover, crops from two consecutive years were collected from the same fields.The knowledge generated by this research will be very useful in the updating of food composition databases in general and will help authorities in setting proper quality standards for wholegrain flours and products where the germ should be preserved, considering also the recent interest of industry and consumers for these kinds of products and the lack, in several cases, of specific legislation.Representative samples of durum wheat grains, belonging to 10 cultivars selected amongst the most frequently grown in Italy, were collected at harvest for two consecutive years (2010–2011) in 10 different locations of Central and Southern Italy . Eight sDurum wheat in Italy is grown under rain-fed production: it is planted in late autumn or early winter and harvested in early summer, which often leads to limited rainfall and high temperatures, resulting in water stress during grain filling. Crop rotation and balanced nutrient management  are practiced to ensure that the crop produces the greatest possible high-quality yield with the moisture that is available. The main climate factors influencing durum wheat crop quality are rainfall and temperature during the growing season. Data on these two factors of the years 2009–2011 in Central and Southern Italy can be found in the reports by the Italian High Institute for Environmental Protection and Research  ,11.Fifty grams of each cleaned sample were milled by means of a Cyclotec laboratory mill  equipped with a 0.5 mm screen, to obtain wholemeal flours that were used for the subsequent analyses.w/w), methanol, n-hexane, formic acid (99% w/w) hydrochloric acid (37% w/w) and anhydrous sodium sulphate were of analytical grade and were purchased from Carlo Erba . Boron trifluoride (approximately 10% w/w in methanol for gas chromatography (GC) derivatization) was purchased from Sigma-Aldrich . Fatty acid standards  were also purchased from Sigma-Aldrich.Chloroform, ethyl alcohol  was added and the tube caps were loosely screwed. The tubes were put onto a heating plate in a water bath and left to gently reflux at 72 °C for 30 min. Following this, the reaction was quenched with 2 mL of water, then the mixture was cooled to room temperature and extracted three times with 3 mL of n-hexane. The hexane extracts were reunited into a vial and finally the hexane was evaporated by nitrogen flux. The vial was stored under nitrogen at −18 °C for a few days. Immediately prior to GC analysis, the contents of the vial were re-dissolved in 300 μL of hexane and 2 μL were injected. The GC instrument was an HP 5890 equipped with a Supelco  SPB®-PUFA (poly unsaturated fatty acids) column of 30 m length and a flame ionization detector (F.I.D.). The instrumental analysis was run according to Finotti et al. [The fatty acid profile was determined by gas chromatography (GC). About 5 g of wholemeal flour (in duplicate) was introduced in a Corning tube and suspended in 10 mL of chloroform–methanol 2:1 acidulated with 6 N HCl. A magnetic bar was added, and the tube was left to extract overnight at room temperature on a magnetic stirrer. The mixture was filtered through Whatman Grade 1 (1–11 μm) filter paper into an oven dried flask, then the solvent was evaporated by nitrogen flux followed by oven drying at 30 °C. The contents of the flask were re-dissolved in chloroform–methanol 2:1 to a volume of exactly 10 mL, then an aliquot was derivatized according to Zweig and Sherma  as folloi et al. : 50 °C fF-test for homogeneity of variance, Student’s t-test and Friedman test followed by Wilcoxon pairwise comparisons were performed by means of the PAleontological STatistics (PAST) statistical package [The Shapiro–Wilks normality test,  package . Two-wayp < 0.01) and to a minor extent on the growing year (p < 0.05), whereas the interaction cv/site × year was not a statistically significant factor of variation. In any case, differences were very small: up to 0.57 between samples of different cultivars and up to 0.18 between years for samples of a same cv/site  > palmitic (C16:0) ≈ oleic (C18:1) > linolenic (C18:3) > stearic (C18:0) > palmitoleic (C16:1). This can be clearly seen from the box plot elaboration reported for each separate year and for the two years together . This diLinoleic acid (C18:2) was present in amounts ranging from 0.50–1.14 g/100 g d.b. throughout all samples, with a mean of 0.68 and a standard deviation (SD) of 0.16 . For comPalmitic (C16:0) and oleic (C18:1) acids were detected in equal amounts. Palmitic acid ranged from 0.17–0.36 g/100 g d.b., mean 0.24 (SD 0.04) and oleic acid ranged from 0.17–0.43 g/100 g d.b., mean 0.24 (SD 0.07). The USDA reports 0.51 g/100 g d.b. for palmitic acid and 0.40 g/100 g d.b. for oleic acid, whereas the INRAN database reports 0.47 g/100 g d.b. and 0.38 g/100 g d.b., respectively .Linolenic acid (C18:3) ranged from 0.06–0.14 g/100 g d.b., mean 0.08 (SD 0.02). The USDA and the INRAN databases report 0.05 g/100 g d.b. and 0.11 g/100 g d.b., respectively. Stearic acid (C18:0) ranged from 0.01–0.03 g/100 g d.b., mean 0.02 (SD 0.005). The USDA and the INRAN databases report, for this acid, 0.03 g/100 g d.b. and 0.02 g/100g d.b. respectively. Finally, palmitoleic acid (C16:1) was detected in very small amounts, ranging from 0.004–0.007 g/100 g d.b., mean 0.005 (SD 0.001). Both the USDA and INRAN databases report 0.01 g/100 g d.m. for this acid.t-tests, performed for each fatty acid on each pair of samples from the same cv/site between the two growing years, showed a significant difference between the years 2010 and 2011 in a few cases only, namely: all acids except C16:1 varied in Ancomarzio SI and Iride AG; only the acids C18:1, C18:2 and C18:3 varied in Ciccio EN , covered from 0.18–0.44 g/100 g d.b. and from 0.19–0.39 g/100 g d.b., respectively. The unsaturated/saturated ratio ranged from 3.15–4.44 g/100 g d.b. considering all samples, with a mean of 3.83 (SD 0.38). This mean is higher than that reported by USDA (3.0) and INRAN (3.5). A series of t-tests, performed on each pair of samples from the same cv/site grown in different years, showed a significant difference for the unsaturated/saturated ratio between years in only three cases   .Total lipids were in line with the values reported by the USDA and the INRAN databases  and it was not possible to detect any difference between the geographical areas of Central and Northern Italy.In regard to fatty acid composition, even if Bottari et al. in 1999  observedActually, the USDA database (but not the INRAN one) and other works also report small amounts of C14:0 in durum wheat kernels . We did not detect this acid, as it was at the limit of detection of our method. There are publications reporting other fatty acids as well , some in kernels  and the specification of all the elements that allow for conversion of results into different units of measure  make this data very useful in the compilation of databases and easy to compare with other data. Moreover, updated data on lipids are needed to set proper quality standards for products such as wheat wholegrain flours and foods where the presence of germ is desirable.


AbstractTylencholaimushelanensissp. n., extracted from the rhizosphere soil of unidentified grasses from Helan Mountain, Inner Mongolia, China was identified. The new species is characterized by having a body length of 0.93–1.07 mm with the lip region approximately one-quarter of the body diameter at the posterior end of the neck region wide; female didelphic-amphidelphic; pars proximalis vaginae violin-shaped. Males were not found. SEM observations of the new species were made and a phylogenetic analysis of both the 18S rDNA and the D2-D3 region of 28S rDNA is presented.A new species,  Tylencholaimus de Man, 1876 is common in most soils all over the world and contains more than 50 valid species. It is mainly characterized by having small body, cap-shaped lip region, weak odontostyle and knobbed odontophore. The types of female genital system and tail of the genus are various   . Peña-Saor tail) , b, c red others . HoweverTylencholaimus and is described as Tylencholaimushelanensis sp. n.With detailed examinations based on light microscopy, SEM observations and phylogenetic analysis of 18S rDNA and the D2–D3 region of 28S rDNA, one nematode population from Inner Mongolia, China, was identified to be a new member of Soil samples were collected from the rhizosphere soil of unidentified grasses from Helan Mountain, Alxa Left Banner, Alxa League, Inner Mongolia, China. Nematode populations were extracted from the soil samples by using the modified Baermann funnel method . Then spPageBreakhttp://www.ncbi.nlm.nih.gov/Genbank/).A single nematode was picked into 10 μL mixed solution (distilled water: 2×buffer for KOD FX = 1:1) and cut using a sterilized needle. 1 μL 20 μg/mL proteinase K was added and then reacted at 65 °C for 1 h and 95 °C for 15 min to release the genomic DNA. PCR reactions were performed in a 10 μL reaction mixture containing 5 μL of 2×buffer for KOD FX, 0.3 μL of each primer (10 μM) , 2 μL of dNTPs (200 μM), 1 μL of DNA, 1.2 μL of distilled water and 0.2 μL of KOD FX polymerase (1 U/ μL). Two overlapping fragments of the 18S rDNA were amplified using two primer sets, 988F (5'–CTCAAAGATTAAGCCATGC–3’) and 1912R (5'–TTTACGGTCAGAACTAGGG–3’) for the first fragment, and 1813F (5'–CTGCGTGAGAGGTGAAAT–3’) and 2646R (5'–GCTACCTTGTTACGACTTTT–3’) for the second one . For theTylencholaimus spp. were also included. The sequence alignment was done by using the software MEGA v.6 and the conservative regions were selected by using the online Gblocks server (http://molevol.cmima.csic.es/castresana/GBLOCKS_server.html). Substitution saturation was tested by DAMBE and the model of base substitution was evaluated using MrModeltest v2.3. The best-fit models were selected by AIC (Akaike Information Criterion) in MrModeltest v2.3. Phylogenetic trees were constructed by using MrBayes v3.1.2 running the chain for 5,000,000 generations for the 18S rDNA and 1,000,000 generations for the D2-D3 region of the 28S rDNA, respectively, with a sample frequency of 100 generations and setting the ‘burnin’ at 2500. The topologies were used to generate a 50% majority rule consensus tree with posterior probabilities (PP) for appropriate clades. The software Figtree v.1.3.1 was used to visualize and edit the phylogenetic trees.The sequences of the new species were compared with sequences in GenBank using BLAST. The sequences of other dorylaimid species and the outgroup taxa were chosen according to previous studies , the seqPageBreakTaxon classificationAnimaliaDorylaimidaTylencholaimideahttp://zoobank.org/F0BFD129-C8C7-442C-8A1F-A7C144310D0D38°40.311’N, 105°50.905’E; 22 August 2014; collected by Dong-Wei Wang, Wen-Jia Wu, Lu Yu, and Hui Xie. Female holotype (M51.B.a) and six female paratype specimens  are deposited in the Lab of Plant Nematology/Research Center of Nematodes of Plant Quarantine, South China Agricultural University, Guangzhou, Guangdong 510642, China.Seven females from Qinghai Province; pars dilatata and a slender part, 0.9–1.3 times the uterus long; anterior oviduct 83–107 μm and the posterior one 61.5–92 μm long. Sphincter present at the junction of oviduct and uterus. Uterus simple and with a wide lumen, the anterior one 66–85 μm and the posterior one 58–72 μm long. Vulva transverse. Vagina showing ‘+’ shape in ventral view, extending 44.5–46% inwards the corresponding body width. The walls of pars proximalis vaginae recessed inward in the middle, making pars proximalis vaginae violin-shaped, 12–13 μm long and 13–15 μm wide, with poorly developed musculature surrounding only the part adjacent to pars distalis vaginae. Pars refringens lacking, pars distalis vaginae 7 μm long. No sperm observed in the genital system. Prerectum 2.4–4.2 times and rectum 0.9–1.2 times the body diameter at anus level. Tail hemispheroid with blunt rounded to flat terminus. One caudal papilla opening in tail terminus.Female. Body robust and cylindrical, tapering towards the anterior end. Habitus variable, almost straight or slightly twisted after fixation. Cuticle two layers, 1.0–2.0 μm thick in anterior region, 1.5–2.5 μm at mid-body, and 2.5–3.5 μm on tail; outer layer with fine transverse striations, the inner one loose and often shrunken after fixation. Lateral chord occupying about one-third of the body diameter at mid-body, lateral pores indistinct. Lip region cap-shaped, offset from the body by a constriction, 2.4–2.8 times as wide as high or 25% in average of the body diameter at posterior end of the neck region wide. Lips not amalgamated, the outer part of each lip not distinct from the inner one. Labial and cephalic papillae distinct but not interfering with the contour. Amphidial foveae cup-shaped, opening at the level of the constriction, apertures 0.4 times on average as wide as the lip region. Odontostyle straight with a distinct lumen, 8–9.5 μm long, 0.9–1.0 times as long as the lip region width, its aperture about one-third of its length. Odontophore rod-like with small basal knobs, 9–11 μm long, 1–1.3 times as long as the odontostyle. Guiding ring single. Nerve ring situated at 35–42% of the neck length. Anterior part of pharynx slender and expanded gradually, basal expansion occupying 39–43% of the total neck length. Pharyngeal gland nuclei locations  are as fFore measurements see Table Tylencholaimushelanensis sp. n. were obtained. The inter-individual variabilities of the 18S rDNA sequences and the 28S rDNA sequences are one gap and two base pair differences, respectively. Two sequences for each of the genes were deposited in GenBank (accession numbers: KU992903 (1746 bp long) and KU992904 (1747 bp long) for 18S rDNA, KU992905 and KU992906 (both 840 bp long) for D2-D3 region of 28S rDNA). The BLAST search for the 18S rDNA showed the highest similarity (94% and 95%) to the sequence of an unidentified species of Tylencholaimus (AJ966510). For the D2-D3 region of 28S rDNA, both sequences PageBreakshowed the highest similarity (79%) to the sequences of Xiphinemabrevicollum Lordello & Da Costa, 1961 (AY580057). In the 18S rDNA phylogenetic reconstructions  Peña-Santiago, 2008, T.crassus Loof & Jairajpuri, 1968, T.paracrassus Monteiro, 1970, T.sinensis Li, Baniyamuddin, Ahmad & Wu, 2008 and T.teres Thorne, 1939 in having a body length about 1 mm or less, female genital system didelphic-amphidelphic, odontostyle less than 10 μm and ‘V’ value less than 62 in average, but can be differentiated by having panduriform pars proximalis vaginae. In addition, the new species differs from T.congestus (T.cosmos (T.crassus (T.paracrassus (T.sinensis (PageBreakvs. 53–63 μm and the posterior one 61.5–92 μm vs. 45–50 μm long), prerectum 2.4–4.2 (vs. about 5) times the body diameter at anus long, longer rectum (22–28 μm vs. 18–20 μm). From T.teres (ongestus  by havinT.cosmos , the new.crassus  by longeacrassus , the newsinensis  by lip r T.teres , it diffTylencholaimus, a cylindrical, spindle, convex, or pyriform pars proximalis vaginae has been described or illustrated. The violin-shaped structure in Tylencholaimushelanensis sp. n. is described here for the first time. This enriches the diversity of the pars proximalis vaginae and makes this characteristic more valuable for identification. In fact it is so distinctive that in the 18S rDNA and 28S rDNA Bayesian trees, Tylencholaimushelanensis sp. n. forms a monophyletic clade with 100% support. In the 18S rDNA tree, Tylencholaimushelanensis sp. n. is sister to a clade including T.teres and T.proximus. As mentioned previously, Tylencholaimushelanensis sp. n. is close to T.teres in morphology, but differs from the latter by several morphological characteristics such as a wider amphid aperture, a shorter prerectum, longer odontostyle and tail, and the fragments of their 18S rDNA sequences in common showed ten nucleotide differences. The new species does not otherwise show close relationships to T.teres in the 28S rDNA Bayesian trees, while the other close relative inferred from the 18S rDNA Bayesian tree, T.proximus, has a prodelphic genital system that is different to the didelphic-amphidelphic genital system of Tylencholaimushelanensis sp. n., and thus can be easily differentiated from the new species morphologically.In addition to the above characteristics used to differentiate the new species from its conspecifics, the pars proximalis vaginae of the new species should be noticed. Among the known didelphic species of Tylencholaimus species were not all grouped together in one clade in both the 18S rDNA and 28S rDNA Bayesian trees, suggesting that Tylencholaimus is not monophyletic. The deeper evolutionary relationships among Tylencholaimus currently cannot be further clarified due to because the few molecular data available for Tylencholaimus, especially 28S rDNA sequences, available on GenBank. For example, the relationship of the new species and T.proximus inferred from the 18S rDNA Bayesian tree was close, but this relationship cannot be confirmed because the 28S rDNA sequence of T.proximus is unavailable. Thus, the detailed relationships of Tylencholaimus species cannot be further resolved until more molecular data of Tylencholaimus are obtained.The sequences of PageBreak


The prevalence of functional constipation (FC) is 3–27%, and FC has been reported to cause discomfort in daily life and various complications. The treatment for FC depends on laxatives, and thus, effective and non-toxic alternative treatments are needed.We conducted a randomised, sham-controlled parallel-design, pilot trial. Participants with FC were randomly assigned to either the real acupuncture (RA) or sham acupuncture (SA) group. The RA consisted of eight fixed acupuncture points  and four additional points targeted to the individual based on Traditional Korean medicine (TKM). SA consisted of shallow acupuncture insertion at 12 non-acupuncture points. Twelve sessions were provided over 4 weeks. The outcome measures were weekly defecation frequency (DF), spontaneous complete bowel movement (SCBM), Bristol stool scale (BSS) score and constipation assessment scale (CAS) score. The participants were followed for 4 weeks after the treatment.Thirty participants were enrolled (15:15). The mean DF were 5.86 ± 5.62, 5.43 ± 3.39 and 5.79 ± 3.64 in the RA group and 3.73 ± 1.62, 5.00 ± 1.77 and 5.40 ± 1.96 in the SA group at weeks 1, 5, and 9, respectively. The increases in weekly SCBMs were 2.50 ± 3.86 and 2.71 ± 4.01 with RA and 2.33 ± 2.74 and 1.93 ± 2.25 with SA at weeks 5 and 9, respectively (mean difference [MD] 0.78). The BSS scores were 0.57 ± 1.72 and 1.09 ± 1.30 with RA and 0.15 ± 1.06 and 0.14 ± 0.88 with SA at weeks 5 and 9, respectively (MD 0.95). The CAS score changes were − 3.21 ± 2.91 and − 3.50 ± 3.98 with RA and − 2.67 + ±2.82 and − 2.87 ± 2.95 with SA at weeks 5 and 9, respectively. Greater improvements were observed in subgroup analysis of participants with hard stool. The numbers of participants who developed adverse events (AEs) were equal in both groups (four in each group), and the AEs were not directly related to the intervention.This clinical trial shows feasibility with minor modifications to the primary outcome measure and comparator. Acupuncture showed clinically meaningful improvements in terms of SCBMs occurring more than 3 times per week and in these improvements being maintained for 4 weeks after treatment completion. As this is a pilot trial, future studies are warranted to confirm the efficacy and safety.KCT0000926 (Registered on 14 November 2013).The online version of this article (10.1186/s12906-018-2243-4) contains supplementary material, which is available to authorized users. Functional constipation (FC) refers to persistently difficult, infrequent or seemingly incomplete defecation without other organic diseases and without meeting the Irritable Bowel Syndrome criteria , 2. The Constipation patients experience decreases in health-related quality of life, and both patients and healthcare providers must manage the economic burden of this condition , 10. OneLifestyle changes and pharmacological or surgical treatments are used to manage constipation , 12, 13.Acupuncture is a treatment modality originating from traditional Chinese medicine. Acupuncture has been used to treat various digestive diseases , 20, andThus, we conducted a randomised, sham-controlled, parallel design pilot trial to explore the possible efficacy and safety of acupuncture for treating FC and assess the feasibility of performing a full-sized randomised controlled trial (RCT) in the future.www.CONSORT-statement.org), and the CONSORT flow chart of the study is shown in Fig. This was a randomised, sham-controlled, participant/assessor-blinded pilot trial conducted in a university hospital setting in South Korea from November 2013 to October 2015. Thirty participants with FC were registered and assigned to either the real acupuncture (RA) or sham acupuncture (SA) group; 15 participants were in each group. Twelve RA or SA sessions were performed over 4 weeks, and follow-up assessments were performed every 2 weeks for 4 weeks after intervention completion in each group. This study followed CONSORT guidelines , hypersensitivity reaction, being pregnant, expecting to be pregnant, actively nursing, receiving acupuncture, moxibustion, cupping or herbal medicine treatments for constipation within the past 4 weeks, or taking any medications or probiotics for constipation within the past 2 weeks.Eligible participants who were willing to comply with the study protocol and provided written consent were asked to maintain a defecation diary for a one-week screening period. This diary was used by Korean medical doctors (KMDs) at visit 1 to confirm that the patients satisfied the Rome III criteria.This study was approved by the institutional review board (IRB) of the Daejeon Oriental Hospital, Daejeon University (djomc-112) and performed in compliance with the Declaration of Helsinki. All subjects were provided with verbal and written information on possible benefits, harms and the possible treatments for constipation other than the treatments offered in our clinical trial before signing the consent form. We obtained written informed consent from the subjects who decided to participate in the trial by their voluntary will. The safety assessment was performed at every visit by KMDs.As this was a pilot clinical trial to explore the efficacy and safety of acupuncture for FC and assess trial feasibility, the sample size was not calculated to provide sufficient statistical power to support the hypothesis but was instead based on the number of participants available for enrolment and the minimum number of participants required for the purposes of a pilot study . AccordiThe 30 enrolled participants were randomly allotted into either the RA or SA group at a 1:1 ratio. The randomisation list was generated with a block-randomisation method using SAS® Version 9.1.3  by an independent statistician. The block size was randomly determined.After the randomisation list was created, it was coded, sealed in an envelope, and stored in a locked cabinet. The group allocations were sealed in opaque double envelopes and numbered in order according to the randomisation list. Upon participant enrolment, an envelope was opened to determine their group allocation. The KMDs in charge of the intervention verified the group allocations of the participants.Assessors and participants were blinded. As practitioners cannot be blinded due to the nature of the intervention, they merely conducted acupuncture without unnecessary conversation or sharing information with other researchers. Assessors simply asked essential questions, which were specified in the case report form. Participants were scheduled to visit at different times to minimise both the exchange of information and the risk of bias.Each group received 12 intervention sessions (3 times over 4 weeks). We used same acupuncture needles , and conducted intervention and evaluation with the same number of times in same schedule.The RA group received RA on eight fixed acupuncture points  and four individualised acupuncture points. Sa-am acupuncture was used as the individualised acupuncture. The Sa-am acupuncture is a type of acupuncture based on traditional Korean medical (TKM) principles. The traditional theory about yin and yang, five element, and meridian are used in the application of Sa-am acupuncture. That is, five shu points of each meridian are used according to the creation and control cycles of five-element theory. Acupuncture points are selected based on the relationship of Governor, Mother and Son. For example, in case of deficiency of organ, governor point of its own meridian and of its governor meridian are sedated, while mother point of its own meridian and of its mother meridian are tonified. The set of these four acupuncture points is called ‘Jeong-Gyeok’. In case of excess of organ, the governor points should be tonified while son point of its own meridian and of its son meridian should be sedated. The set of these four acupuncture points is called ‘Seung-Gyeok’. In the present study, we used large intestine (LI) Jeong-Gyeok for LI deficiency, stomach (ST) Jeong-Gyeok for stomach deficiency, and liver (LR) Seung-Gyeok for liver excess , 28.Fig.The SA group received minimal acupuncture consisted of shallow acupuncture insertion (1–2 mm in depth) without manual stimulation or de-qi sensation on 12 non-acupuncture points for 30 min , 30. TheEach participant’s usual intake of dietary fibre drinks, coffee, sweet potatoes, or fermented milk was maintained. In the first screening stage, patients taking regular laxatives or had taken a laxative in the 2 weeks before enrollment were excluded according to our eligibility criteria, and subjects were not asked to discontinue their prescribed medication or change their standard medical care. All patients who participated in the present study were allowed to use laxative (rescue medicine) prescribed by the protocol when needed throughout the entire study period. We provided participants with Magnesium Hydroxide 500 mg as a rescue medication and had them take it if needed. The Magnesium Hydroxide has been recommended as effective treatment for constipation and widely used in clinical practice with agreement of more than 90% of Korean clinicians . Each paThe weekly DFs, weekly spontaneous complete bowel movement (SCBMs) and mean Bristol stool scale (BSS) scores were recorded every week during the treatment period and the two follow-up visits. The constipation assessment scale (CAS) scores were evaluated at weeks 1, 3, 5, 7 and 9. Rescue medication use and AEs were investigated at each visit. The primary outcome was changes in DF at week 5; all others were secondary outcomes.Statistical analyses were performed using the full analysis set (FAS) population, which included a population as similar to intent-to-treat (ITT) as possible. All analyses were performed using SAS® Version 9.4 with a significance level of 5% and two-sided tests.For the purpose of this pilot study, the analyses performed were mainly descriptive to present actual changes and treatment effects using the mean difference (MD) and Hedge’s g. The effect size was interpreted as follows: 0.2, small; 0.5, medium; 0.8, large; 1.2, very large; and 2.0, huge , 33.Inter-group differences were examined using a Mixed-effect Model for Repeated Measures (MMRM). Paired-sample t-tests or Wilcoxon signed-rank tests were conducted to analyse the changes in each group after treatment. All AEs reported during the study period were charted, and the difference in the incidence of AEs between the two groups was assessed using the Chi-square test or Fisher’s exact test. The ‘last observation carried forward’ approach was used to replace the missing values, except in the MMRM. The MMRM method does not require an alternative step because it uses the maximum likelihood, which accounts for missing values.A subgroup analysis was performed for participants with severe symptoms. DF and SCBM were relatively even in participants overall, while the BSS and CAS scores showed heterogeneity. Thus, severity was classified based on these two factors. Subgroup A included participants whose proportion of hard stool (BSS score 1–2) was > 25% of total defecation during one week, and subgroup B included participants whose CAS score was ≥7 at visit 1.Thirty-five participants were assessed for eligibility, but 5 (14.3%) were not enrolled .Twenty-eight (93.3%) participants completed the trial. One participant in the RA group was excluded due to a violation of the inclusion criteria identified at visit 1, before any treatment was performed. One participant in the SA group withdrew consent.The treatment-compliance rate, i.e., the percentage of treatment sessions participants actually received of all 12 planned sessions, was 99.4 ± 2.4% and 94.4 ± 19.3% in the RA and SA groups, with an overall rate of 96.8 ± 14.0%.Both groups included more females than males, with 85.7 and 91.7% females in the RA and SA groups, respectively. The mean age (49.6 and 50.0 years old), height, body weight and body mass index were similar in both groups. Factors that may influence defecation, including dietary, exercise, smoking and drinking habits, were not significantly different between groups , DF decreased from 5.86 ± 5.61 to 5.43 ± 3.39 with RA, while it increased from 3.79 ± 1.62 to 5.00 ± 1.77 with SA. The MD was − 1.70 (− 3.94 to 0.55), Hedge’s g was 0.56, and the changes were not significantly different between the groups (DF decreased by 0.07 ± 4.16 with RA but increased by 1.67 ± 1.72 with SA at week 9. The MD between the groups was − 1.74 (− 4.13 to 0.66) at week 9 (g = 0.53). There was no significant difference between the groups at any time-point (Table n = 3), while that in the SA group was 8, 8 and 7 (n = 1).Four participants had a weekly DF > 7 (3 in the RA group and 1 in the SA group). When these participants were excluded, the weekly DF of the RA group was 3.82, 4.91, and 5.36 at weeks 1, 5 and 9, respectively and that of the SA group was 3.43, 4.79, and 5.29. Of patients with abnormally frequent defecation, the weekly DF in the RA group was 13.3, 7.3 and 7.3 at weeks 1, 5, and 9, respectively, (p < 0.05). Inter-group comparisons did not reveal significant differences .RA showed an increase of 2.50 ± 3.83 at week 5 and 2.71 ± 4.01 at week 9, while SA showed corresponding increases of 2.33 ± 2.74 and 1.93 ± 2.25. Weekly changes within each group revealed a significant increase with RA from week 3 and a significant increase with SA from week 5  (Table p < 0.05), but the inter-group analysis showed no significant difference.The increase in BSS score was 0.57 ± 1.72 and 1.09 ± 1.30 at weeks 5 and 9 with RA compared to 0.15 ± 1.06 and 0.14 ± 0.88 in SA . The change in the RA group was significant from week 7 . At weeks 5 and 9 in subgroup B, RA showed increases of 0.78 ± 2.11 and 1.70 ± 1.10, while SA showed decreases of 0.19 ± 1.35 and 0.21 ± 0.95  (Table p < 0.05), and there was no significant difference between the groups.The CAS score in the RA group changed by − 3.21 ± 2.91 and − 3.50 ± 3.98 at weeks 5 and 9, while that in the SA group changed by − 2.67 ± 2.82 and − 2.87 ± 2.95, showing a very small effect between the groups . Both groups showed significant improvements at each time-point compared to baseline .During the overall study period, 1 (7.1%) and 5 33.3%) participants in the RA and SA groups needed to use the rescue medication. The number of days the rescue medication was used was 0.43 ± 1.60 in the RA group compared to 1.07 ± 2.43 in the SA group (Table 3.3% partp = 0.9087). Most AEs were mild or transient, with the exception of enteritis. The types of AEs were common cold, headache and insomnia in the RA group and common cold, headache, rhinitis, pain and enteritis in the SA group. No AEs were directly related to the interventions, and all AEs were completely cured within the trial period.A total of 11 AE cases were reported over the course of 393 intervention sessions (2.8%), including 4 cases in the RA group (2.1%) and 7 cases in the SA group (3.5%). The number of participants who experienced AEs was the same in both groups  (Table The blinding index scores  were 0.39) Table .Table 4BAs this study was not designed for statistical hypothesis verification, we evaluated effect size and clinical importance. The observed change in SCBMs is clinically meaningful, as an improvement ≥1 is usually considered a treatment success , 36. In Considering the specific effects of RA compared with SA, medium to large effects were observed with RA in terms of the BSS score Table . In the We considered the appropriateness of the comparator in terms of comparability with RA and blinding success. The blinding index revealed that participants in both groups tended to think they received RA. Thus, the attempt to blind participants seemed to be successful such that placebo effects, which reflect the psychological reactions of participants, were equally controlled . HoweverFor the purposes of a pilot study, we assessed trial feasibility. Using DF as the primary outcome might have resulted in false-negative results being overlooked because this study included participants with frequent defecation . SCBM woIn participants who had hard stool (subgroup A), RA showed larger effects on SCBM and the BSS and CAS scores compared with those observed in the overall participant population and subgroup B. Accordingly, participants having hard stools could be grouped as potential responders, meriting further studies.The recruitment rate of screened participants, compliance rate and completion rate were acceptable. However, if future studies target participants with severe symptoms, which seemed to be a potential response group in this study, the recruitment rate may be slower; thus, proper strategies are needed. Future studies would be feasible with some modifications to the primary outcome and comparator.A sample size calculation for a future full-sized RCT was conducted based on SCBM. A recent study revealed that sham EA showed a change in SCBM of 0.87 (0.73 to 0.97) . SupposiRegarding safety, 2.1 and 3.5% AEs were reported in the RA and SA groups, respectively, none of which were deemed to be related to the interventions over the course of all treatment sessions. These results corresponded with those of previous studies reporting acupuncture as safe with a low risk of accidents , 46. TheThe treatment mechanism could not be studied in this clinical trial, but some possible mechanisms have been reported in previous studies. ST25 was reported to improve interstitial Cajal cell expression and to recover colonic smooth muscle atrophy in rats with slow transit constipation . EA on aOur study has strengths and weakness, as discussed below. This clinical trial has significance in that it set a sham-intervention control group to evaluate the efficacy of manual acupuncture in FC. The strengths of this study are as follows: it was previously registered on a clinical trial registry to minimise selective-reporting bias; participant blinding was successfully achieved using SA; and validated assessment tools were used, the lack of which has been a noted limitation of previous acupuncture studies. However, this study also has some limitations. First, we included few participants, which might lead to false-negative results. The results of our study cannot be generalised to support the efficacy and safety of acupuncture for FC because the sample size calculation for hypothesis acceptance was not conducted for this pilot trial. Shallow needle insertion in non-acupuncture points showed clinically meaningful changes, which made it difficult to distinguish the specific effect of RA, and careful interpretation was required. Finally, an FAS analysis was conducted instead of an ITT analysis because no data were obtained from one participant after randomisation. If an ITT analysis was conducted with complete follow-up data, it might have yielded different results.This study demonstrated that a future clinical trial would be feasible with some modifications to the primary outcome measure and comparator. An additionally finding is that twelve acupuncture sessions over 4 weeks have possible effects of increasing stool consistency and SCBM, particularly in participants with severe symptoms. In the future, a full-sized randomised controlled trial with a long-term follow-up period should be conducted to confirm these efficacy and safety findings.Additional file 1:Details of Real Acupuncture. (DOCX 28 kb)Additional file 2:Details of Sham Acupuncture. (DOC 36 kb)Additional file 3:Checklist for items in STRICTA 2010. (DOCX 18 kb)


Haslea ostrearia, is known to display antibacterial activities. Previous studies have demonstrated a prophylactic effect of marennine on bivalve larvae challenged with a pathogenic Vibrio splendidus, suggesting that the blue Haslea is a good candidate for applications in aquaculture as a source of a natural antimicrobial agent. Indeed, the genus Vibrio is ubiquitous in aquaculture ecosystems, and regular events of pathogenic invasion cause some of the biggest losses worldwide. To better characterize the effects of marennine on Vibrios, a panel of 30 Vibrio strains belonging to 10 different species was tested, including bivalve pathogenic species . Vibrio strains were first exposed to 10 and 25 µg mL−1 of Blue Water (BW), a concentrated culture supernatant of H. ostrearia containing marennine. This screening evidenced a great diversity in responses, from growth stimulation to a total inhibition, at both the interspecific or intraspecific level. In a second series of experiments, 10 Vibrio strains were exposed to BW at concentrations ranging from 5 to 80 µg mL−1. The highest concentrations of BW did not systematically result in the highest growth inhibition as hormetic responses—opposite effects regarding the concentration—were occasionally evidenced. The relationships between marennine and Vibrio strains appear more complex than expected and justify further study—in particular, on the mechanisms of action—before considering applications as a natural prophylactic or antibiotic agent in aquaculture.Marennine, the water-soluble blue pigment produced by the marine diatom  Haslea ostrearia is characterized by the production of a specific blue-green pigment, named marennine. This water-soluble pigment accumulates at the apices of the cells before its release into the surrounding environment [H. ostrearia is a ubiquitous diatom and is of special interest in the Atlantic French coast , where blooms in oyster ponds induce the greening of oyster gills that increases the market value of bivalves. Blue diatoms other than H. ostrearia have been identified in the last decade, such as Haslea karadagensis , Hasleaean Sea; ), and moava Sea; ). All thn . Despitnown yet .Haslea species in seawater , combinr ponds; ,8, as wer ponds; ). More pillarium , Vibrio uarianus , or Vibrlendidus . The Vibhellfish . Major parennine . Hence, aculture .Haslea and marennine has been considered for aquaculture applications. Marennine biological activities have thus been investigated in vivo, using blue mussel and giant scallop larvae exposed to a concentrated supernatant of H. ostrearia culture enriched in extracellular marennine [V. splendidus strain [H. ostrearia blue pigment, by assessing the effects of marennine on different species and strains of the genus Vibrio that are threatening aquaculture sustainability.At the sight of such results, the use of blue arennine . Low cons strain . This reVibrio strains belonging to 10 species were tested for their sensitivity toward Blue Water (BW), a concentrated supernatant of H. ostrearia culture containing the extracellular marennine. Vibrio chagasii , Vibrio crassostreae , Vibrio fortis , Vibrio harveyi , Vibrio orientalis , Vibrio splendidus , V. tasmaniensis , Vibrio sp. , and V sp.  strains were provided by the Genomics of Vibrio team (Laboratoire de Biologie Intégrative des Modèles Marins (LBI2M), station biologique de Roscoff, France) and were previously described in [Vibrio aestuarianus strains  were provided by the Laboratoire de Génétique et de Pathologie des Mollusques Marins (LGPMM) of the Institut Français de Recherche pour l’Exploitation de la Mer .Thirty ribed in . Vibrio Vibrio strains to BW was assessed with the method described in the Clinical and Laboratory Standards Institute (CLSI) antimicrobial microdilution guidelines [TMBRANDplatesTMpureGradeTM S 96-well Microplates, Thermo Fisher Scientific, Waltham, MA, USA). Bacterial growth was monitored by Optical Density (OD) measurements with a microplate spectrophotometer . Bacterial growth was then recorded using Microplate Manager 6 Software  with OD measurement  of eachVibrio strains were kept at −80 °C in 25% glycerol. Broth cultures were prepared with an autoclaved, cation-adjusted, Mueller–Hinton broth media  by the addition of 1% NaCl  and agar media, prepared with autoclaved, cation-adjusted Nutrient Agar  by the addition of 2.3% NaCl . Prior to the antibacterial assays, each Vibrio strain was inoculated in CaMHB from the −80 °C sample, incubated overnight at 25 °C under moderate agitation (130 rpm), and isolated on CaNA Petri dishes. After 1 day of incubation at 25 °C, plates containing the isolated colonies were kept at 4 °C for no more than a week. Three different colonies per Petri dish were inoculated in CaMHB  and grown overnight at ambient temperature. The next day, the OD (630 nm) of bacteria in the broth culture was measured  and the absorbance was adjusted at 0.1 by dilution in CaMHB. To obtain the bacterial inoculum, the solution was further diluted by 1/100 in CaMHB, as recommended by the CLSI guidelines [idelines . The bacVibrio strains exposed to Blue Water (BW) solutions was studied over a 24 h period. BW was prepared from a concentrated supernatant of H. ostrearia culture containing the extracellular marennine, and was produced at the Station aquicole de Pointe-au-Père, Institut des Sciences de la Mer à Rimouski-Université du Québec à Rimouski  during the spring of 2017. H. ostrearia was cultured in 100 L circular and flat bottom photobioreactors with filtered sea water  at high irradiance (180 μmol photons m−2 s−1), in a 14/10 h light/dark cycle for 3 weeks, until marennine concentration reached around 6–7 µg mL−1, as described in [−1 , and stored in the dark at 4 °C. The BW concentration was assessed with spectrophotometric measurements (UV/Vis Lambda 25 Perkin Elmer spectrophotometer and UV Winlab Perkin Elmer software (version 6.0.4 2011), Waltham, MA, USA) on a syringe-filtered BW solution  and 1 cm path-length quartz cuvettes, using the Beer–Lambert equation (ε677 = 12.13 L g−1 cm−1) as proposed by [The growth of the 30 ribed in . The supVibrio strains corresponding to 10 different species were screened and exposed to three BW concentrations: 0 µg mL−1 (control), 10 µg mL−1, and 25 µg mL−1. In a second series of experiments, 10 Vibrio strains presenting different patterns of sensitivity to marennine were exposed to a dilution range of BW: 0 µg mL−1, 5 µg mL−1, 10 µg mL−1, 25 µg mL−1, 50 µg mL−1, 70 µg mL−1, and 85 µg mL−1. For the screening experiment, the final volume in each well of the microplates was 100 µL, with a final ratio of 1:1 (v/v) bacterial inoculum: BW. In the dilution range experiment, to reach concentrations as high as 70 and 85 µg mL−1, with a BW stock solution of 117 µg mL−1, the final volume in each well was adjusted to 200 µL, with a final ratio of 1:4 (v/v) bacterial inoculum: BW. Microplates were first filled with the BW solutions, and bacterial inocula were then added to each well using a single channel electronic micropipette . After being completed, microplates were sealed with parafilm and placed in the microplate spectrophotometer for the 24 h run. The experiments were conducted in triplicate, with technical triplicates for each condition. A negative control was also run per microplate for only BW and CaMHB.In a first series of experiments, the 30 Bacterial growth kinetics were analyzed using R 3.5.1 software. For the screening experiment, the OD (600 nm) data obtained over the 24 h run were fitted with a bi-phasic logistic growth equation, defined as below:k1 parameter) and for the second phase of growth (k2 parameter), or the growth rate (r1 and r2 parameters). The k2 parameter was chosen in the screening experiment to study the effects of BW on the Vibrio strains.Interpretable metric parameters were then obtained, such as the maximum possible population size in a particular environment for the first phase of growth .Vibrio strains exposed to BW at 0 µg mL−1, 10 µg mL−1, or 25 µg mL−1 were recorded for 24 h ; and V. harveyi #21 and #22. Although BW had no effect on the maximum population size of V. harveyi #21 and #22, it can be noted that the growth rate of these strains was inhibited with 30.1 ± 1.1% and 37.8 ± 2.3% of inhibition for V. harveyi #21, and 38.1 ± 1.6% up to 57.2 ± 1.2% of inhibition for V. harveyi #22 under 10 and 25 µg mL−1, respectively , and the percentage of relative growth was calculated , up to 64.6 ± 0.7% at 85 µg mL−1, with an IC50 (concentration at which the response is inhibited by 50%) at around 50 µg mL−1  up to 50 µg mL−1, but there was no difference in growth inhibition between 50 and 85 µg mL−1, with a maximum inhibition level of about 37% (V. aestuarianus #02/041 between 0 and 25 µg mL−1 (p = 0.351), and the first inhibitory effect was evidenced at 50 µg mL−1 with 12 ± 0.2% of inhibition (p = 0.015), while the maximum effect was observed at 85 µg mL−1 with 23.4 ± 1.7% of inhibition . The second growth phase presented dose-dependent inhibition, with a total blockage of the growth at the highest concentrations tested  and an important increase of the diauxic lag phase for bacteria exposed to 25 µg mL−1 at 7 h, versus 2 h for the control.Different dose response patterns were observed depending on the strain tested, including linear and “U shape” responses . V. fortexposure . For V.  µg mL−1 a. An incbout 37% . A “no ohibition . For V.  to 24 h b. For thV. sp. 38, V. chagasii #13, V. crassostreae #53, and V. splendidus #90 was also inhibited by the BW concentrations tested, but the dose–response curves expressed in function of the control presented a “U” shape, with the growth inhibitory effect first increasing, then decreasing with increasing BW concentrations . Same observations were made for V. chagasii #13 and V. crassostreae #53, with the inhibitory effect significantly higher at 50 µg mL−1 than at 70 µg mL−1 (p < 0.005). For V. splendidus #90, the percentage of maximum inhibition was 33.3 ± 1.5% at 70 µg mL−1, and the inhibition was significantly lower, at 23.0 ± 0.7% at 85 µg mL−1 , but the results are contrasting. Indeed, BW either inhibited or stimulated the growth of the Vibrio strains.The effects of marennine on several H. ostrearia containing marennine) was used in the present work, and not a purified form of the pigment, it could be questioned if the observed effects were induced by marennine or by other molecules possibly present in the non-axenic culture medium  from Haslea or epibiont bacteria). However, previous studies conducted with purified extracellular marennine (EMn) on marine bacteria, including Vibrio species, confirmed that the pigment itself did exert an antibacterial activity [Haslea species should be cultured at large scale to ensure the production of marennine solutions for applications as antimicrobials in aquaculture, BW represents the easiest and cheapest production process, compared to the purified pigment.As BW solution , and characterized by two distinct exponential phases, with a diauxic lag phase in between [Vibrio diauxic growth is not extensively evidenced in the literature, and was mainly illustrated using V. cholerae [V. alginolyticus [Vibrio species used the disc diffusion method [V. aestuarianus [V. tasmaniensis strain [In addition, diauxic growth was evidenced for all the  between . Vibrio cholerae ,23,24 buolyticus . It is wn method ,27,28, auarianus ,14. The s strain ,12 and nVibrio strains: Type 1, which can divide quickly by using the most suitable carbon source to sustain its growth; and Type 2, which grows more slowly, but which would be able to metabolize a different carbon source. According to Solopova et al. [V. cholerae [V. orientalis, V. chagasii, and V. harveyi strains). It can be hypothesized that the subpopulation Type 1 would be able to metabolize part of the sugars constituting the carbon skeleton of the marennine molecule—or, if any, other EPS present in BW solution to sustain its growth—or that the stimulation would result from overcompensation mechanisms by the bacteria under BW exposure, a mechanism that can be observed when cells undergo a disruption in homeostasis [V. harveyi #23 at the highest concentrations tested (from 50 to 80 µg mL−1).Bacterial diauxic growth has long been considered as a bacterial phase of enzymatic acclimation to metabolize a different type of sugar; however, there is now growing evidence that this phenomenon could actually correspond to the presence of two bacterial subpopulations in the isogenic culture, with different phenotypic adaptation and growth strategies . Based oa et al. , the Typa et al. . This phcholerae , and woucholerae ,31,32. Ieostasis . HoweverVibrio strains did not follow a linear or threshold model, with growth inhibition increasing with concentrations. Indeed, some curves presented a “U-shape” response regarding the antibacterial effect, which decreased with increasing BW exposure . On top of this, opposite effects were observed at low and high concentrations, a phenomenon known as hormesis [−1) highly stimulated the growth of V. fortis #9, up to 50%, while the growth was significantly inhibited at higher concentrations (from 50 to 80 µg mL−1). Hormetic dose–response relationships have raised a growing awareness in toxicological and ecotoxicological studies, and have been extensively documented over the past two decades in different models, as plants, algae, or fungi [Escherichia coli exposed to antibiotics, while the hormetic effect was not observed in Luria–Bertani (LB) culture media [A global observation from the results shows that the dose–response curves of most hormesis . Indeed,or fungi ,36,37. Tor fungi . Moreoveor fungi . In the re media .Haslea provincialis purified blue pigment was demonstrated to interact with the outer membrane of the gram-negative bacteria E. coli, rendering it more rigid [V. cholerae also demonstrate a disruption of the bacterial membrane integrity and a deformation of the cell architecture by antibacterial agents, such as polyphenols [Vibrio interact in vivo, and whether the pigment has a direct effect on Vibrio growth or if it could decrease its pathogenicity by fixing on cell membranes, both of which could explain the better survival of bivalve larvae exposed to marennine and challenged with V. splendidus [The mechanisms of action responsible for the antibacterial activity of marennine remain little explored. So far, the re rigid . Recent yphenols  or nanopyphenols . For nowlendidus .Haslea and marennine as antimicrobials in aquaculture.In conclusion, the present work indicates much more complex interactions between marennine and vibrios than a standard linear correlation between dose and effect. Moreover, the antibacterial activity of marennine is dependent on the species, the strain, and possibly the population of bacteria, suggesting that the blue pigment would act on specific targets. Marennine antibacterial mechanisms and low-dose stimulation phenomenon will have to be better understood before considering any application of