Elatine L. contains ca. 25 small, herbaceous, annual species distributed in ephemeral waters in both hemispheres. All species are amphibious and characterized by a high degree of morphological variability. The importance of seed morphology in Elatine taxonomy has been emphasized by many authors. The degree of seed curvature and seed coat reticulation have been traditionally considered very important in recognizing individual species of this genus. Seed morphometric characteristics of 10 Elatine species, including all European native taxa, are provided on the basis of material from two or three populations of each species. A total of 24–50 seeds were studied from each population, altogether 1,260 images were used for the morphometric study. In total, six parameters were measured from SEM pictures: object surface area, profile specific perimeter (object circuit), rectangle of the object (a) length, rectangle of the object (b) width, angle of the seed curvature, and number of pits in the seed coat counted in the middle row. Our study shows that the range of morphological variation of seeds in European species of Elatine is great, both between the species and the populations. Discrimination analysis showed that all six traits significantly differentiate the populations studied , and the greatest contributions were “number of pits”, “rectangle_a”, and “the angle curvature”. Multidimensional scaling based on a correlation matrix of Mahalanobis distance of the six features studied revealed the greatest similarity between the three populations of E. alsinastrum, E. macropoda, and E. hexandra. Regarding interspecific differences, a Kruskal–Wallis tests showed that, in many cases, lack of statistically significant differences between species relative to the studied seed traits. If distinction of species is only based on seeds, especially if only a few seeds are evaluated, the following species pairs can be easily confused: E. alsinastrum and E. orthosperma, E. hexandra and E. macropoda, E. campylosperma and E. hydropiper, as well and E. gussonei and E. hungarica. We found no diversity in seed coat micromorphology within pits that could have potential taxonomic importance. An identification key and descriptions of species are provided on the basis of seeds traits. Elatine L. is one of the two genera in the Elatinaceae, a family in Malpighiales (Potamopithys (Adanson) Seub, and the other taxa are classified into subgenus Elatine Seub. (=Hydropiper Moesz): E. triandra Schkuhr (sect. Triandra Seub. (=Crypta (Nutt.) Seub.); E. brochonii Clavaud, E. campylosperma Seub., E. gussonei (Sommier) Brullo, Lanfr., Pavone & Ronsisv., E. hexandra (Lapierre) DC., E. hydropiper L., E. hungarica Moesz, E. macropoda Guss., and E. orthosperma Düben (section: Elatinella Seub.). Two more taxa of sect. Elatinella occur in the New World . Other taxa classified to sect. Triandra mainly occur in temperate regions of the Old and New World, with the probable center of diversity in North and South America. E. ambigua is another taxon from Europe  and number of perianth lobes (three or four). The shape of leaves is variable, oblong or roundish, petiolate or almost sessile, and depends on environmental conditions. Flowers are sessile or pedunculated, while tiny seeds are oblong, curved or horseshoe-shaped  and seed coat reticulation have been considered very importan tfor recognizing individual species  find statistical differences between Elatine species relative to seed morphological features, (ii) evaluate intra- and interspeciesseed variability, and then (iii) construct a guide to identifying species based onseed morphological features. Due to the small size of seeds the study was made by using SEM micrographs.Consequently, we studied seed morphometric characteristics of 10 Elatine species and from three populations each, with an exception of very rare E. brochonii and E. campylosperma, two populations, so altogether 28 populations were used for the study. The distance between the populations of each species ranged from approximately 10–2,000 km. For the localities of the original material, and the voucher specimens, see Plants studied were collected across Europe. In total, seeds were collected from all 10 Elatine hungarica, E. hydropiper and E. triandra are protected species in Hungary and were sampled with the permission of the Hortobágy National Park Directorate .To determinate the variability and diagnostic features of seeds, 24–50 seeds obtained from several individuals from each population  were useTo examine micromorphology of seed coat, 48 pictures at ×500, ×2,000, ×4,000, and ×7,000 were taken λ was used to measure the discriminatory power of the model . Interpretation of discriminant functions was performed using canonical analysis.To distinguish the characteristics that have the greatest impact on population and species discrimination, multiple discriminant analysis was used. Wilks For visualization of the relationship between species and populations, Mahalanobis distance-based unweighted pair-group method using arithmetic averages to construct (UPGMA) trees was applied. Canonical values were shown using categorized scatterplots. The most discriminative traits were also independently tested by the non-parametric Kruskal–Wallis tests. All calculations were made in Statistica v. 12.5 software.λ = 0.001, p < 0.001). Of these, the greatest contributions were as follows: number of pits, rectangle a (length), and the angle of curvature  between populations studied of each European Elatine species regarding the following traits: (A) surface , (B) profile , (C) rectangle a , (D) rectangle b , (E) the angle of curvature , (F) number of pits   . AccordiE. orthosperma from Finland, Fin1 (for acronyms see E. hungarica from Slovakia (profile: SD 498.1), E. triandra from Poland, PL1 (rectangle b:SD 39.9 and the angle of curvature: SD 33.7), and E. hydropiper from Hungary (number of pits: SD 5.3) , E. triandra from Hungary (rectangle a: SD 15.3), E. brochonii from Spain (profile: SD 51.7 and rectangle b: SD 13.6), E. hydropiper from Hungary and E. macropoda from Spain, SP2 , and E. brochonii from Morocco (number of pits: SD 0.9)  , Table 4 SD 0.9) , Table 4E. alsinastrum, E. macropoda, E. hexandra . The greatest impact was from the following features: number of pits, the angle of curvature and rectangle a ; the angle of curvature, E. gussonei (SD 44.7); and number of pits, E. campylosperma (SD 7.2). The smallest variation was present in E. triandra  and E. brochonii . The characteristics associated with size  revealed that the following species had the smallest seeds: E. brochonii and E. triandra, while the largest seeds in the studied species belonged to E. gussonei and E. hydropiper , especially within dropiper , Table 7E. alsinastrum, E, brochonii, E. hydropiper, and E. orthosperma . The lowest values were found for E. campylosperma   to 84% . The highest values of classification were found for 7%, 55%) .E. hexandra and E. macropoda, and E. campylosperma and E. hydropiper. The spatial distribution of observed characteristics of the analyzed species is depicted as a categorized scatterplot based on canonical analysis values . These findings confirm previous knowledge about the usefulness of these features in Elatine taxonomy  are very difficult to confuse with E. macropoda’s 21 ± 3 narrow pits/row. Our study yielded different results: seeds of E. macropoda populations had similar number of pits [(13–)19–23–(29)] compared to E. gussonei [(17–)23(–32)]. However, because E. hungarica are much more curved than those of E. gussonei, and especially of E. macropoda and E. orthosperma, but somewhat less curved than those of E. hydropiper. Our current study revealed that the range of variation for the feature the angle of curvature of E. hungarica seeds is similar to that of E. gussonei, and more curved seeds are found in E. hydropiper and E. campylosperma. These results are basically consistent with observations of E. gussonei, E. hydropiper, E. hungarica, and E. campylosperma. Regarding the seed testa, a very distinctive network-shape ornamentation pattern of E. triandra as visible 776–854(–971) µm, width (242–)297–334(–389) µm, angle of curvature (55–)61–99(–156)° number of pits in the middle row (23–)32–38(–47), prevailing pit shape rectangular, semilunar membrane absent on the concave side of seeds.Seed length (365–)533–645(–813) µm, width (217–)252–276(–312) µm, angle of curvature: 26–47(–79)°, number of pits in the middle row (12–)14–15(–17), prevailing pit shape hexangular, semilunar membrane absent on the concave side of seeds.Seed length (328)467–560(700) µm, width: (158)201–231(274) µm; angle of curvature: 58–89 (136)°, number of pits in the middle row (16)20–23(28), prevailing network-shape of pits in the seed coat, semilunar membrane absent on the concave side of seeds.Seed length (708–)799–859(–950) µm, width: (230)290–330(391) µm, angle of curvature: 33–56(91)°, number of pits in the middle row: (18)21–23(26), prevailing rectangular shape of pits in the seed coat, semilunar membrane absent on the concave side of seeds.Seed length: (568–)666–732(–830) µm, width: (282–)329–360(–407) µm, angle of curvature (78–)111–134(–167)°, number of pits in the middle row: (13–)19–23(–29); prevailing rectangular shape of pits in the seed coat, usually semilunar membrane absent on the concave side of seeds.Seed length: (593–)656–697(–760) µm, width: (223–)283–322(–381) µm; angle of curvation (15–) 77–118(–180)°, number of pits in the middle row: (16–)19–21(–24), the shape of pits in the seed coat hexagonal and rectangular, semilunar membrane absent on the concave side of seeds.Seed length: (439–)505–549(–615) µm, width: (274–)419–517(–663) µm, angle of curvation: (222–)265–294(–337)°, number of pits in the middle row: (15–)31–42(–59), narrow rectangular or round shape of pits in the seed coat, semilunar membrane present on the concave side of the seeds.Seed length: (548–)602–638(–693) µm, width: (367–)454–512(–599) µm, angle of curvation: (246–)273–291(–318)°, number of pits in the middle row: (22–)37–48(–62), prevailing rectangular shape of pits in the seed coat, semilunar membrane present on the concave side of the seeds.Seed length: (296–)459–567(–730) µm, width: (284–)357–405(–477) µm; angle of curvation: (161–)213–247(–299)°, number of pits in the middle row: (11–)20–26(–35), prevailing hexagonal shape of pits in the seed coat, semilunar membrane present on the concave side of the seeds.Seed length: (539–)627–685(–774) µm, width: (325–)436–509(–620) µm; angle of curvation: (80–)180–247(–347)°, number of pits in the middle row: 17–23(–32), prevailing hexagonal shape of pits in the seed coat, semilunar membrane present on the concave side of the seeds.10.7717/peerj.3399/supp-1Supplemental Information 1Click here for additional data file.


Ardisia japonica is a well-known traditional Chinese medicinal herb used as a diuretic, for treating cough and for stopping uterine bleeding. A simple, sensitive, and reliable LC-MS/MS method was developed to determine six active compounds in rat plasma and this method was further applied to the pharmacokinetic study of these compounds after oral administration of Ardisia japonica extract. Acetonitrile was used to precipitate the protein in the plasma samples. Using acetonitrile and formic acid aqueous solution (0.05%) as the mobile phase, the separation of the six compounds and internal standards was achieved at a flow rate of 300 μL min−1 on an Eclipse plus C18 column at an elution time of 16 min. A tandem mass spectrometer having an electrospray ionization (ESI) source was used in the detection of the analytes and internal standards using multiple reactions monitoring (MRM) in the negative ionization mode. The LLOQ was 2, 2, 4, 2, 1, and 0.4 ng mL−1 for gallic acid, bergenin, epicatechin, epicatechin gallate, isoquercitrin, and quercetin-3-rhamnoside, respectively. The validated method was applied to the pharmacokinetic study of gallic acid, bergenin, and quercetin-3-rhamnoside in rat plasma after oral administration of A. japonica extract to rats. Ardisia japonica (Thunb.) Bl, a low growing shrub belonging to the family Myrsinaceae, is one of the well-known traditional medicinal traditional herbs being probably one of the most studied among over 500 other species. It is referred to as “Ai di cha” in Chinese and is very famous for its use in treating various respiratory tract infections such as pneumonia, bronchitis, and tuberculosis. It has also been widely used in treating asthma and conjunctivitis and possesses bacteriostatic and hemostatic effects Various mobile phases consisting of different solutions of acetonitrile, methanol, and formic acid were optimized in order to obtain short run times, well-resolved peaks, and peaks with symmetrical shape. In the end, better response, good separation, and increased sensitivity of the compound peaks were obtained using a mobile phase consisting of acetonitrile and 0.05% aqueous formic acid solution. Also, no interference was observed between the tested compounds and internal standards. Typical chromatograms of blank plasma, plasma spiked with the compounds and IS, and plasma with the IS are shown in  A. japonica extract orally to rats as shown in The method's specificity was evaluated by looking at the MRM chromatograms of blank plasma sample, blank plasma spiked with reference standards, and IS and then plasma sample after givingx. Representative mean calibration curves are as follows: Y = 0.0157X − 0.0112, Y = 0.00125X + 0.000782, Y = 0.00223X + 0.00102, Y = 0.00579X − 0.00933, Y = 0.00582X + 0.00202, and Y = 0.0156X − 0.000007 for gallic acid, bergenin, epicatechin, epicatechin gallate, isoquercitrin, and quercetin-3-rhamnoside, respectively, where y represented the peak area ratio of analytes to IS and x denoted the concentration of analytes present in the plasma samples. Also, the LLOQs of the analytes are 2, 2, 4, 2, 1, and 0.4 ng mL−1 for gallic acid, bergenin, epicatechin, epicatechin gallate, isoquercitrin, and quercetin-3-rhamnoside, respectively. LLOQs of these analytes were sufficiently applied for the pharmacokinetic study in rats.The calibration curves for the 6 compounds were linear within the stipulated ranges as shown in The intraday and interday variabilities of the LLOQ and QC samples are shown in As shown in The stabilities of all analytes were tested through the analysis of the three levels of QC samples as shown in  A. japonica extract. The content of gallic acid, bergenin, epicatechin, epicatechin gallate, isoquercitrin, and quercetin-3-rhamnoside are 2.1467 μg/mg, 24.9 μg/mg, 0.696 μg mg−1, 0.198 μg mg−1, 0.0916 μg mg−1, and 2.13 μg mg−1, respectively. Therefore, the administration dosages of gallic acid, bergenin, epicatechin, epicatechin gallate, isoquercitrin, and quercetin-3-rhamnoside after administration of A. japonica extract were 2.00 mg kg−1, 23.16 mg kg−1, 0.65 mg kg−1, 0.18 mg kg−1, 0.09 mg kg−1, and 1.98 mg kg−1, respectively.The six compounds were determined in A. japonica extract at a dose of 0.93 g kg−1 to rats. The 3 compounds including gallic acid, bergenin, and quercetin-3-rhamnoside were determined and their plasma concentration-time profiles are illustrated in −1 and the Tmax was 1.40 ± 1.13 h after oral administration of the A. japonica extract. The area under the curve (AUC0–24 h) and the half-life, T1/2, were 194 ± 44 ng (mL h)−1 and 3.21 ± 4.57 h, respectively. The Cmax and Tmax for bergenin and quercetin-3-rhamnoside were 288 ± 107 ng mL−1, 0.86 ± 0.52 h and 7.08 ± 8.28 ng mL−1 and 0.50 ± 0.60 h with the area under the curve for each compound being 1743 ± 666 ng (mL h)−1 and 6.44 ± 11.99 ng (mL h)−1, respectively. Both compounds exhibited a Tmax less than one hour implying that they had a fast rate of absorption into plasma from the gastrointestinal tract after oral administration, compared to gallic acid which achieved maximum plasma concentrations after one hour.The method having been validated was successfully applied to the determination of plasma concentration of 3 of the compounds in rats, after oral administration of A. japonica extracts to rats.A sensitive and simple LC-MS/MS method for simultaneous measurement of 6 compounds in rat plasma was developed and subsequently validated. The method proved to be accurate, precise, and specific and was successfully applied to investigate the pharmacokinetics of gallic acid, bergenin, and quercetin-3-rhamnoside after an oral administration of


Trimester-specific reference ranges for T3, T4, and TSH need to be established in different communities. Neither Sudan nor other African countries have established trimester-specific reference ranges for TSH, free T3 (FT3), and free T4 (FT4) in healthy pregnant women. This study aimed to establish trimester-specific reference ranges for TSH, FT3, and FT4 in healthy pregnant Sudanese women.P < 0.001).We performed a longitudinal study, which included 63 women with singleton pregnancies who were followed since early pregnancy until the third trimester. The study was performed in Saad Abu-Alela Hospital, Khartoum, Sudan, during January to October 2014. An equal number of age- and parity-matched non-pregnant women were enrolled as a control group. Basic clinical and obstetrics data were gathered using questionnaires. TSH, FT3, and FT4 levels were measured. Median (5th–95th centile) values of TSH, FT3, and FT4 were 1.164 IU/ml (0.079–2.177 IU/ml), 4.639 nmol/l (3.843–6.562 nmol/l), and 16.86 pmol/l (13.02–31.48 pmol/l) in the first trimester. Median values of TSH, FT3, and FT4 were 1.364 IU/ml (0.540–2.521 IU/ml), 4.347 nmol/l (3.425–5.447 nmol/l), and 13.51 pmol/l (11.04–31.07 pmol/l) in the second trimester. These values were 1.445 IU/ml (0.588–2.460 IU/ml), 4.132 nmol/l (3.176–5.164 nmol/l), and 12.87 pmol/l (9.807–23.78 pmol/l) in the third trimester, respectively. TSH levels increased throughout the trimesters. FT3 and FT4 levels were significantly higher in the first trimester compared with the second and third trimesters. TSH, FT3, and FT4 levels were significantly lower in pregnant women compared with non-pregnant women (The present study is the first to establish trimester-specific reference ranges of TSH, FT3, and FT4 in Sudanese women with normal pregnancies. Our results suggest that pregnancy is likely to suppress TSH, T3, and T4 levels in healthy women. Pregnancy may be associated with alterations in iodine homeostasis and other physiological changes that ultimately result in alteration of thyroid function , 2. SeveMany studies have been conducted worldwide to establish TSRRs in different countries –10. HoweTo the best of our knowledge, no other studies have been conducted to establish TSRRs for healthy pregnant women either in Sudan or in other African countries. The current study was conducted to establish TSRRs for healthy pregnant Sudanese women. Our findings are expected to assist in accurate interpretation of laboratory results, proper diagnosis, and management of thyroid dysfunction among Sudanese women during pregnancy.We conducted a longitudinal study in Saad Abu-Alela Hospital  during January to October 2014. Women with a singleton pregnancy were enrolled early in pregnancy (once their pregnancy was confirmed) and followed until the third trimester. After patients signed an informed consent, the sociodemographic, medical, and obstetrics history  was recorded from each woman using a questionnaire. Pregnancy and its duration were confirmed by ultrasound, which was conducted by the practicing obstetricians (MAA and IA) as part of antenatal investigations. During the current longitudinal study, an equal number of non-pregnant women (pregnancy was excluded by ultrasound) matched for age and parity were included as controls. Pregnant and non-pregnant women who had a previous history of thyroid disease, hypertension, renal disease, diabetes, liver disease, and those on medication that might affect thyroid function were excluded from the study.2). BMI during the first trimester is likely comparable with pre-conception BMI, which was difficult to get in our study population. Hemoglobin was measured for each patient at every visit.Blood pressure was measured using a sphygmomanometer for the case and control groups. During the first visit (in early pregnancy), maternal weight and height were measured. BMI was calculated and expressed as weight (kg)/height  using the immunoassay analyzer AIA 360 TOSOH (Japan), guided by the manufacturer’s instructions as described previously . NoteworA total required sample size of 63 women was calculated using a formula for a longitudinal study and the difference in the mean of the proposed variables  that woP value < 0.05 was considered significant.Data were entered into a computer using SPSS for Windows (version 16.0). The 5th–95th centile and median were calculated for levels of TSH, FT3, and FT4. Levels of TSH, FT3, and FT4 were not normally distributed , and differences were assessed using the Kruskal–Wallis H test (Mann–Whitney U test between two groups). A Among 75 pregnant women who were initially enrolled, 63 (84.0 %) completed the follow-up until the third trimester. The rest (16.0 %) of the patients were lost during follow-up because of a change in address.n = 34.0, 54.0 %), the majority were housewives , and few of them had a rural residence . Hemoglobin level and the other basic characteristics of the women are shown in Table Approximately half of these women were primiparous  , 23 of TThe present study is the first to establish TSRRs of TSH, FT3, and FT4 in Sudan. Pregnancy is likely to suppress TSH, T3, and T4 levels in Sudanese women. TSRRs of TSH in Sudan are comparable with some Western countries, but are relatively lower compared with some Arab, Persian, and Eastern countries. However, TSRRs of thyroid hormones are higher in Sudan compared with other countries. Further research that also considers iodine homeostasis in pregnant women may provide further support for the conclusions of the present study.


Halorubrum lacusprofundi, a cold-adapted halophilic Antarctic archaeon, was able to grow anaerobically on 0.04 M concentration of perchlorate. With increasing concentrations of perchlorate, growth was inhibited, with half-maximal growth rate in ca. 0.3 M NaClO4 and 0.1 M Mg(ClO4)2 under aerobic conditions. Magnesium ions were also inhibitory for growth, but at considerably higher concentrations, with half-maximal growth rate above 1 M. For a purified halophilic β-galactosidase enzyme of H. lacusprofundi expressed in Halobacterium sp. NRC-1, 50% inhibition of catalytic activity was observed at 0.88 M NaClO4 and 0.13 M Mg(ClO4)2. Magnesium ions were a more potent inhibitor of the enzyme than of cell growth. Steady-state kinetic analysis showed that Mg(ClO4)2 acts as a mixed inhibitor (KI = 0.04 M), with magnesium alone being a competitive inhibitor (KI = 0.3 M) and perchlorate alone acting as a very weak noncompetitive inhibitor (KI = 2 M). Based on the estimated concentrations of perchlorate salts on the surface of Mars, our results show that neither sodium nor magnesium perchlorates would significantly inhibit growth and enzyme activity of halophiles. This is the first study of perchlorate effects on a purified enzyme. Key Words: Halophilic archaea—Perchlorate—Enzyme inhibition—Magnesium. Astrobiology 18, 412–418.Effects of perchlorate salts prevalent on the surface of Mars are of significant interest to astrobiology from the perspective of potential life on the Red Planet.  For anaerobic growth, cultures were grown in the dark without shaking, essentially as described previously  test tube reader. Growth rates were calculated by using the slope of a line of regression calculated from early to mid-log phase.For aerobic growth, cultures of halophiles were shaken in the light in test tubes at 42°C in CMHalobacterium sp. NRC-1 (pRK42) , harvested, resuspended in binding buffer  glycerol, 30 mM imidazole, pH 7.4), and lysed by sonication . Cell debris was removed by centrifugation  in an Eppendorf 5417C centrifuge, and resulting crude extract was filtered through a 0.2 μm polyethersulfone filter . The proteins were purified by using Nickel HisTrap HP Columns , equilibrated, and washed with binding buffer, and eluted sequentially with 2 M NaCl, 100 mM phosphate buffer (pH 7.4) supplemented with 40, 80, 100, 200, and 500 mM imidazole.To purify β-galactosidase, cultures of o-nitrophenyl-β-D-galactopyranoside . Active fractions were combined and protein concentration estimated by absorption at 280 nm with a Shimadzu UV-1601 spectrophotometer . To confirm purity, the protein was analyzed by 10% SDS-PAGE followed by staining with Coomassie Blue .Column fractions were tested for activity with 2+ and ClO4− ions on the β-galactosidase enzyme were tested through steady-state kinetics with a Shimadzu UV-1601 spectrophotometer. The assay solution containing 10 μg/mL enzyme was preincubated at 50°C followed by addition of ONPG at three different concentrations , and absorption was recorded at 420 nm. V0, the Michaelis constant (Km), as well as Vmax, were determined with UV Probe ver. 4.23 software . kcat was calculated from given values of Km and Vmax. After the kinetic experiments were recorded, Lineweaver-Burk plots for each concentration of the inhibitory ion were used to determine whether the inhibition was competitive or noncompetitive. KI was determined by using Dixon plots, which use the reciprocal velocity of multiple steady-state kinetic experiments at multiple substrate and inhibitor concentrations to extrapolate the concentration of inhibitor that results in half-maximal inhibition 2 for H. lacusprofundi and 0.07 M for Halobacterium sp. NRC-1 (M Mg(ClO4)2 for H. lacusprofundi and 0.2 M for Halobacterium sp. NRC-1. These results indicate a slightly greater inhibition for the divalent cation compared to the monovalent cation at equivalent perchlorate concentration, for example, 50% inhibition with 0.12 M magnesium perchlorate  versus 0.3 M sodium perchlorate for H. lacusprofundi.Next, we determined the effects of magnesium perchlorate on growth rate of the two halophiles, by replacing an equivalent concentration of sodium chloride in their media, with the total anion concentration unchanged. Growth reduction by 50% was observed with 0.12 p. NRC-1 . Cessati2+ concentration on growth by addition of MgCl2 to the growth media. Halorubrum lacusprofundi retains a substantial growth rate up to 0.9 M Mg2+ and shows a 40% decrease in the growth rate with 1.2 M Mg2+ 2, catalytic efficiency was decreased 70% at 0.18 M, and no measurable activity was observed at 0.23 M and higher 2. Dixon plots for magnesium chloride showed behavior as a competitive inhibitor with a KI of 0.3 M for Mg2+ (KI of 2 M NaClO4 (4)2 to the reaction buffer, mixed inhibition characteristics were observed from the Dixon plot (KI of 0.04 M for Mg(ClO4)2, a considerably more potent effect than when added separately.To characterize the nature of inhibition, kinetic analysis was carried out for β-galactosidase reactions with added MgClfor Mg2+ . The DixM NaClO4 . When thxon plot , with a Halorubrum lacusprofundi was found to grow anaerobically in low concentrations of perchlorate and was inhibited at considerably higher concentrations, with more sensitivity to magnesium perchlorate compared to sodium perchlorate (50% inhibition at 0.3 M sodium perchlorate versus 0.1 M for magnesium perchlorate). Similar results were obtained for inhibition of the purified H. lacusprofundi β-galactosidase enzyme, with greater enzyme activity reduction for magnesium perchlorate compared to sodium perchlorate (50% inhibition with 0.88 M sodium perchlorate and 0.13 M magnesium perchlorate). Interestingly, steady-state kinetic analysis showed that magnesium ions act as a competitive inhibitor for the enzyme, while perchlorate ions act as a noncompetitive inhibitor, with magnesium perchlorate acting as a mixed inhibitor.We investigated the effects of sodium and magnesium perchlorate salts prevalent on Mars on growth and enzyme function of a cold-adapted extreme halophile from Antarctica. et al.,M for the sodium salt and 0.0532 M for the magnesium salt, based on weight/volume  2 when grown on Mars soil simulant .A recent report indicated that UV radiation enhances the toxicity of perchlorates in certain bacteria. When 2+ ions were also inhibitory for growth of both H. lacusprofundi and Halobacterium sp. NRC-1, albeit less potent than perchlorate. Growth media for H. lacusprofundi contains 0.4 M magnesium compared to 0.08 M for Halobacterium sp. NRC-1, reflecting the relatively higher magnesium concentration in Deep Lake where the cold-adapted species was isolated, compared to Great Salt Lake and similar thalassic environments where the mesophile has been found. Addition of Mg2+ up to nearly 1 M concentration did not have any significantly deleterious effect. However, at even higher concentrations, above 1 M Mg2+, growth of both halophiles could be completely inhibited, with the growth rate for the cold-adapted species declining more steeply. Additional studies are needed to understand high magnesium ion tolerance and potential involvement of heavy metal efflux systems 2 suggest synergistic action of magnesium and perchlorate ions to inhibit the growth of both H. lacusprofundi and Halobacterium sp. NRC-1, as well as β-galactosidase enzyme activity. Both H. lacusprofundi and Halobacterium sp. NRC-1 exhibited 50% growth inhibition at 0.1 M Mg(ClO4)2, which is a lower concentration than either magnesium or perchlorate ions alone. However, perchlorate has more detrimental effects on growth than magnesium ions, suggesting that halophilic cells are able to keep excess Mg2+ outside of cells, but the ClO4− ions are able to enter and potentially cause damage to DNA 2 with a KI of 0.04 M.The observed effects of Mg2 and NaClO4 on Mars, while inhibitory for most microorganisms, likely would not preclude the growth and function of haloarchaea, such as H. lacusprofundi, tested in this study. Perchlorate ions do inhibit the growth of haloarchaeal cells, and magnesium ions can cause inhibition of a halophilic enzyme, but with complete inhibition occurring at concentrations much higher than those reported on Mars.In summary, the presence of Mg(ClO


These preclinical findings clearly indicate that MUS can improve the therapeutic index of carboplatin and ensures more effective therapeutic strategy against cancer for clinical development.A newly designed organoselenium compound, methyl substituted umbelliferone selenocyanate (MUS), was synthesized as a primary hit against the myelotoxic activity of carboplatin. MUS was administered at 6 mg/kg b.wt, p.o. in concomitant and pretreatment schedules with carboplatin  in female Swiss albino mouse. MUS treatment reduced (P < 0.001) the percentage of chromosomal aberrations, micronuclei formation, DNA damage and apoptosis in murine bone marrow cells and also enhanced (P < 0.001) the bone marrow cell proliferation of the carboplatin-treated mice. These activities cumulatively restored the viable bone marrow cell count towards normalcy. Myeloprotection by MUS was achieved, in part, due to a significant reduction in the ROS/RNS formation and restoration of glutathione redox pool. Additionally, MUS synergistically enhanced the cytotoxicity of carboplatin against two human cancer cell lines (MCF-7 and Colo-205). Furthermore, MUS can effectively potentiate the antitumour activity of carboplatin against two murine cancers   Like other chemotherapeutic platinum drugs, CBDCA interacts with cellular DNA to form DNA-platinum adduct which generally crosslinks DNA2, resulting in cytotoxicity to cancer as well as normal cells. Hence, the substantial therapeutic benefit of CBDCA treatment is compensated by various clinical complications like cardiotoxicity3, myelotoxicity5, ototoxicity6 and nephrotoxicity7. Myelotoxicity induced by CBDCA limits its therapeutic dose5 because of its unwanted toxicity in highly proliferative organs like bone marrow. CBDCA induces substantial DNA crosslinking. This leads to increases in replication error and eventually generates mutation. In this scenario, protection from CBDCA-induced myelotoxicity without compromising its chemotherapeutic efficacy is a clinical necessity.Despite much advancement in targeted and customized cancer chemotherapy, platinum-based drugs are in routine clinical use in low and middle-income countries. The second generation platinum salt, carboplatin  or CBDCA is generally used for the treatment of head and neck, lung and ovarian cancer8. In a recently developed approach, selenium is considered as a modulator of chemotherapeutic outcome as it can attenuate the chemotherapy-induced toxicity and stimulates the antineoplastic efficacy9. Some biochemical properties of selenium like its (a) incorporation into proteins as selenocysteine and selenomethionine during translation, (b) presence of the functional moiety of 25 selenoenzyme in human11 and (c) dual behavior to act as antioxidant and pro-oxidant depending upon cellular redox environment make it unique among other possible candidates. In addition, this micronutrient is essential for bone homeostasis12 and it protects bone from various diseases like rheumatoid arthritis, osteoarthritis, and osteoporosis13. Thus the efficacy of this trace element in the protection of normal cells, tissues and organs from mutagens can be capitalized to achieve a better therapeutic outcome through the possible use of chemotherapy in higher doses, longer duration or both. In post-SELECT  era, attention is now shifted towards various organic selenium compounds due to their better bioavailability and safety profile16 than inorganic selenium compounds.The concurrent use of nontoxic chemoprotective agent offers effective management of chemotherapy-associated toxicity17, anticancer18 and many others21. Moreover, the nontoxic character of the coumarin-based compounds also motivated us to use this bioactive scaffold22 to synthesize a new coumarin-based organoselenium compound. The oral LD50 and effective dose of MUS were determined in female Swiss albino mice according to OECD guidelines. MUS at its most effective dose were evaluated for its chemoprotective potential against CBDCA-induced myelotoxicity in bone marrow cells of Swiss albino mouse. Therapeutic effects of MUS alone or in combination with CBDCA were also investigated in vitro and in vivo as well. In vitro therapeutic efficacy was evaluated against MCF-7 and Colo-205 whereas in vivo study was carried out in Swiss albino mice bearing Dalton’s Lymphoma or Sarcoma-180 in solid as well as ascites tumour forms.In the present work, a coumarin-based organoselenium compound, methyl substituted umbelliferone selenocyanate or MUS, was designed and synthesized as a potential hit against carboplatin-induced myelotoxicity. Coumarin and its analogues are well known for their various biological activities including anti-steroid50 of the MUS was found to be >2000 mg/kg b.wt. (based on an assumed sigma of 0.5). No sign of toxicity or moribund state were observed among the live animals during the entire duration of the study.The oral LDOptimized oral dose of MUS was selected through the evaluation of some clinical safety  × 106/femur, respectively, whereas the BrdU LI was found to be 54.17% and 55.01%, respectively. In Group 3 animals, CBDCA administration resulted in significant (P < 0.001) reduction of the BrdU LI to 23.82% which, in turn, lowered the viable bone marrow cell count by 43.54% in comparison to Group 1. MUS administration in concomitant treatment schedule (Group 5) increased the BrdU LI to 34.62% (P < 0.001) and the viable cell count by 48.45% (P < 0.01) with respect to Group 3. Pretreatment by MUS (Group 6) increased the BrdU LI to 41.39% (P < 0.001) and the viable cell count by 69.14% (P < 0.001) [(9.32 ± 1.29) × 106/femur] with respect to Group 3. 4MU treatment increased the BrdU LI to 29.74% (P > 0.05) and the viable cell count by 3.99% (P > 0.05) [(5.73 ± 1.04) × 106/femur] in comparison with Group 3. On the other hand, mice treated with CBDCA showed significant (P < 0.001) a high proportion of chromosomal aberrations (CA)  high micronuclei (MN) frequency  increase in the levels of ROS Fig.  and NO  in the bin vitro therapeutic efficacy of CBDCA in combination with MUS  values from which mean CIs were estimated. The combination of 4MU and CBDCA was also tested in a similar way. We were delighted to found that, MUS and CBDCA interacted synergistically in both the cell lines with CIs being significantly lower than 1 (P < 0.05)  and Sarcoma-180 (S-180) to determine the in vivo antitumour potential of MUS in combination treatment. Moreover, ascites and solid tumour, both the forms were evaluated in the course of this study. The results revealed that MUS augmented the cytotoxicity of CBDCA in both the ascites tumour models of DAL and S-180. In the combination treatment group (TCS group) we observed reduction in the ascites tumour volume, packed cell volume as well as tumour cell count compared to only CBDCA treated animals  of MUS (>2000 mg/Kg b.wt.) indicates the non-toxic nature25 of the compound. Dosage regimen was determined as per OECD guideline 407 as the best combination of administration route, dose amount and dose schedule can ensure the highest efficacy and lowest risk. As observed in this study, MUS at 6 mg/kg b.wt. p.o. helped to achieve overall good health, optimum antioxidative capacity without any clinically detectable toxicities. Hence this dose was identified as the most suitable dose to explore the protective capacity of MUS against CBDCA-induced myelotoxicity.Acute oral toxicity threshold  can prevent the induction of DNA damage and (iii) increase the activity of DNA repair enzymes27. Although not conclusive, but in most of the cases DNA repairing system was strengthened rather than increased removal of DNA adduct to cope up with these assaults. It is also documented that, the incidence of DNA damage from oxidative injury are reduced due to the activity of glutathione peroxidase, a well-known selenoprotein that maintains redox balance28. In the present study, the severity of CBDCA induced DNA damage was observed to be reduced after MUS administration. We also observed that reduction in DNA damage led to the lowering of chromosomal aberrations and subsequent micronuclei formation in bone marrow cells.Platinum is a heavy metal transition element and generally damages cellular DNA. In this present work, we have also observed such kind of DNA damage in bone marrow cells after treatment with CBDCA and MUS treatment able to reduce the laddering of DNA and comet formation. As shown, MUS was three times more effective in reducing the comet formation than 4 MU proves the role of selenium in preventing DNA damage. Selenium in various forms (i) is reported to enhance biochemical response against DNA damage 29 and nephrotoxicity3, (ii) heavy metals are known to generate free radicals which can interact with DNA and produce lesions30 and (iii) CBDCA can intercalate within DNA that resulted in the formation of the free radicals generated by platinum can damage DNA in spite of their short lifespan. We examined this hypothesis by measuring cellular oxidative and nitrosative stress. As expected, the oxidative burst was observed in CBDCA-treated murine bone marrow cells  CBDCA induced free radicals are reported to be responsible for neurotoxicity, ototoxicitylls Fig. . The inclls Fig. . Adminisin vitro. But MUS in combination with CBDCA resulted in better synergistic cytotoxicity in vitro. This may be attributed to the fact that MUS regulates other antiproliferative pathways due to the presence of selenium moieties. In vivo studies revealed that administration of MUS in combination with CBDCA significantly increased the cytotoxicity of CBDCA against two murine cancer cell lines i.e. DLA and S-180. As the anti-cancer activity of CBDCA is ROS-dependent, so MUS must behave like a prooxidant wherein oxidative environment of cancer cells acted as catalyst resulting in synergistic cytotoxicity. But in a homeostatic environment of normal bone marrow cells, MUS was found to be antioxidant while interacted with CBDCA. This dual behaviour of MUS may be due to the presence of selenium moiety in MUS since selenium compounds are well known to act as antioxidants and pro-oxidants depending upon cellular redox state31. Tumour cells generally possess a high level of ROS. Selenium compounds due to its characteristic pro-oxidant property in tumour microenvironment can act as selective cytotoxic agents against cancer cells by achieving toxic levels of ROS. However, the chemical form of selenium and the dose employed often dictate the interplay between the pro-oxidant and antioxidant behaviour of selenium compounds. Higher concentration of selenium was proved to act as pro-oxidant due to the generation of ROS33. On the other hand, the higher bioavailability of organic selenium than inorganic selenium made the former one a potential candidate for preclinical and clinical development. Moreover, the metabolism of different forms of selenium also plays a key factor in delivering the pro-oxidant and antioxidant efficacy of selenium36.MUS alone were found to be less cytotoxic towards transformed cells than 4 MU In summary, we have synthesized a new hit, MUS which showed myeloprotective efficacy in combination with CBDCA and also synergizes the therapeutic efficacy of the standard chemotherapeutic drug. The underlying molecular mechanism of MUS is underway for further modification of its structure to produce the potential lead molecule.MCF -7 and Colo-205 cell lines were brought from NCCS, Pune, India. CBDCA was purchased from Fresenius Kabi India Pvt. Ltd., Pune, India. All reagents were purchased from commercial suppliers and used directly unless otherwise stated.2 atmosphere. After cooling to room temperature, 1,5- dibromopentane  was added to the mixture and refluxed for another 9 h. The resulting bromo compound (2a) was extracted with ethyl acetate and purified by column chromatography using petroleum ether/ethyl acetate. The desired selenocyanate (MUS) was then obtained by dropwise addition of a solution of anhydrous potassium selenocyanate  in acetone to a stirred suspension of the precursor bromo-compound  over a period of 1 h at 25 °C. The reaction mixture was stirred for 48 h at room temperature (reaction was monitored by TLC).The product (MUS) was extracted with CHCl3 and purified by column chromatography using petroleum ether/chloroform.The Methyl-substituted umbelliferone selenocyanate (MUS) was synthesized following a two-step procedure as shown in Fig. 2a: Yield: 57%. m.p: 63.1 °C.Compound 1H NMR  δ 7.45 , 6.85 , 6.74 , 6.09 , 3.99 , 3.40 , 2.36 , 1.92 , 1.82  and 1.64–1.60 .  δ 162.08, 161.31, 155.29, 152.55, 125.52, 113.54, 112.61, 111.92, 101.37, 68.18, 33.45, 32.39, 28.19, 24.75 and 18.66.  m/z calcd CMUS: Yield: 62%; m.p.71 oC.Compound max cm−1: 2943.31, 2860.96, 2147.24, 1708.57, 1692.7, 1608.33.IR (KBr) υ1H NMR  δ 7.48 , 6.88–6.80 , 6.80–6.73 , 6.11 , 4.02 , 3.08 , 2.38 , 1.99 , 1.86 , 1.65 .  δ 161.93, 161.34, 155.22, 152.66, 125.60, 113.58, 112.55, 111.90, 101.51, 101.35, 67.99, 30.62, 29.26, 28.29, 25.73, 18.71. m/z calcd Cad libitum. Animals were acclimatized for 7 days before all the starting of an experiment. Experiments like blood sample collection and sacrifice of the animals were performed by strictly following the standard guidelines of Institutional Animal Ethics Committee . All the experimental methods and protocols were approved and in accordance with the Institutional Human Ethics Committee, Chittaranjan National Cancer Institute, Kolkata.Adult (6–8 weeks) Swiss albino female mice (25 ± 2 g) used in this study were bred and maintained in the animal colony of Chittaranjan National Cancer Institute . The mice were maintained at a controlled temperature (22 ± 2 °C) and humidity (60 ± 5%) under alternating light and dark conditions (12 h/12 h) in standard cages. Standard food pellets and drinking water were provided 50 of MUS was determined by Up-and-Down-Procedure as per the OECD guidelines 425. After administration of each dose, animals were continuously observed for the first 4 h and then periodically up to 48 h for any adverse symptoms. The animals were kept under observation for next 14 days for any delayed mortality. The oral LD50 of MUS at 95% confidence interval was analyzed by AOT425 software according to OECD guideline.Oral LDth day, 24 h after last treatment.MUS was orally administered as a suspension of 5.5% propylene glycol in water. Animals were divided into four groups, containing six animals (n = 6) in each group. Vehicle control group: Each animal was given 5.5% propylene glycol in water for 28 days. MUS (3 mg/kg b.wt.)- treated group: The compound was orally administered at the dose of 3 mg/kg b.wt. for 28 days. MUS (6 mg/kg b.wt.)- treated group: The compound was orally administered at the dose of 6 mg/kg b.wt. for 28 days. MUS (12 mg/kg b.wt.)- treated group: The compound was orally administered at the dose of 12 mg/kg b.wt. for 28 days. The animals were treated for 28 days and sacrificed on the 29Group 1 (vehicle-treated group): 5.5% propylene glycol in water was administered orally for 17 days along with the intraperitoneal injection of normal saline from day 8 to 17. Group 2 (MUS only treated group): MUS in 5.5% propylene glycol was orally administered at 6 mg/kg b.wt. for 17 days. Group 3 (CBDCA treated group): CBDCA was intraperitoneally administered at 12 mg/kg b.wt. from day 8 to 17. Group 4 (4 MU pretreatment group): 4MU in 5.5% propylene glycol was orally administered at 3 mg/kg b.wt. for 17 days along with the intraperitoneal injection of CBDCA at 12 mg/kg b.wt. from day 8 to 17. Group 5 (MUS concomitant treatment group): MUS in 5.5% propylene glycol was orally administered at 6 mg/kg b.wt. from day 8 to 17 along with the intraperitoneal injection of CBDCA at 12 mg/kg b.wt. from day 8 to 17. Group 6 (MUS pretreatment group): MUS in 5.5% propylene glycol was orally administered at 6 mg/kg b.w. for 17 days along with the intraperitoneal injection of CBDCA at 12 mg/kg b.wt. from day 8 to 17. All experimental animals were sacrificed on the 18th day, 24 h after last treatment.The animals were divided into six groups Fig. , containBefore sacrifice, all animals were fasted for 4 h and anaesthetized with i.p. injection of ketamine-xylazine solution (87.5 mg/kg b.wt. ketamine and 12.5 mg/kg b.wt. xylazine). Blood samples from retro-orbital venous plexus were collected in a heparinized vial and subjected to haematological analysis. Cells from spleen, thymus and femoral bone marrow were collected by aspirating with 1X PBS through 26 gauge needle.6 cells/ml were incubated with BrdU in RPMI at 37 °C in a humidified incubator with 5% CO2 for 1 h. Then the cells were smeared and fixed on slides followed by incubation with anti-BrdU monoclonal antibody at 37 °C for 30 min. Alkaline phosphatase conjugated anti-mouse immunoglobulin was then added to it followed by 30 min incubation at 37 °C. The slides were then incubated with BCIP/NBT substrate solution at 25 °C for 30 min and visualized under a light microscope37. Cells (≥100) were counted from the randomly selected area of each slide and percentages of labelled cells were expressed as BrdU labelling index.1 × 1038, slides were scored under a light microscope at ×1000 magnification. Percentage of aberrated metaphase plate was noted as a chromosomal aberration (CA).0.03% colchicine  was administered 90 min before sacrifice. Bone marrow cells were collected in warmed (37 °C) 1% sodium citrate solution and fixed in acetic acid/ methanol (1:3) on slides. After flame drying and Giemsa staining38, the slides were visualized under a light microscope at ×1000 magnification. Nuclei with a diameter between 1/16 and 1/3 of the original nucleus which was on the same plane, stained uniformly and non-overlapping with an original nucleus are considered as micronuclei. The percentage of MN in each group was reported after a counting of ≥1000 cells from randomly selected zones on the slide.The cells from the femoral bone marrow were flushed out by 0.075 M KCl solution and incubated at 37 °C for 10 min. After which the cells were harvested by centrifugation, smeared on slides, air-dried and fixed in absolute methanol. Following Giemsa staining4) was layered in 1.0% low melting agarose onto half-frosted slides pre-coated with a thin layer of 1.0% normal melting agarose39. The third layer of 0.5% low melting agarose was added to the top. The slides were kept for 2 h at 4 °C in lysis solution  after which electrophoresis was performed for 30 min in buffer . Then the slides were neutralized , dried at room temperature and stained with ethidium bromide. Various parameters40 like damaged cell (%), average tail length [migration of the DNA from the nucleus (μm)] and Olive tail moment [product of tail length and the fraction of total DNA in the tail (arbitrary units)] were measured using KOMET 5.5 software, Andor Technology, USA.The femoral bone marrow cells (2 × 106) was isolated using HiPurATM Multi-sample DNA Purification Kit as per manufacturer instruction. Electrophoresis was performed in 1.5% agarose gel containing ethidium bromide (0.5 μg/ml) along with 1 kb DNA ladder. Photograph of banding patterns was taken by a gel documentation system.Genomic DNA from femoral bone marrow cells  from 5–6 zones/slide were counted to determine the number of apoptotic cells. Apoptotic index (AI) was determined as the percentage of the labelled nuclei with respect to the total number of nuclei counted.Femoral bone marrow cells were smeared on slides, permeabilized using 0.1% Triton X-100 and then incubated with TUNEL reaction mixture containing the TdT  and fluorescein-dUTP (2′-deoxyuridine 5′-triphosphate) at 37 °C for 1 h in a humidified chamber6) and incubated in dark for 30 min at 25 °C to allow the formation of DCF42 which was analyzed  using spectrofluorometer to measure the levels of ROS. The level of nitrate (NO3−) and nitrite (NO2−) in 1 × 106 bone marrow cells were measured by adding Griess reagent  to it43. Absorbance at 545 nm was measured using NaNO2 as standard.10 μM DCFH-DA was added in femoral bone marrow cells (2 × 106) were assessed by a kinetic assay44 in which the catalytic amounts of GSH caused a continuous reduction of DTNB to TNB at 412 nm. Quantification was done by parallel estimation of standard curves of known GSH and GSSG concentrations.The levels of GSH and GSSG in femoral bone marrow cells (2 × 104 cells) into 96-well plates and incubated at 37 °C in a humidified incubator with 5% CO2 for 24 h. Different concentrations of 4MU, MUS, and CBDCA in media were added to each well and incubated for 48 h. Then MTT (0.4 mg/ml) solution was added to each well followed by 4 h incubation. After removal of the supernatant, the Formosan crystals formed in the cell were dissolved in DMSO and the absorbance was measured by multiwell spectrophotometer at 570 nm. Cell viabilities were expressed as percentages relative to untreated controls.The Anti-proliferative potential of 4MU, MUS, and CBDCA alone or in combination was determined by MTT assay. MCF-7 and Colo-205 cells were seeded  values45 from which mean CIs were estimated. The effect of combined cytotoxicity was categorized by combination index method (CIs) using the following equation: CI = (DMUS/DMUSx) + (DCP /DCPx) + (DMUS × DCP/DMUSx × DCPx), with DMUS, DCP being each drug  concentration in the mixture, required to induce x% cytotoxicity and DMUSx, DCPx is the concentration of each drug required to induce the same cytotoxicity when used alone. The interaction between 4MU and MUS is considered synergistic if CI < 1, additive if CI = 1 and antagonistic if CI > 1.MUS and CBDCA each were added at their corresponding IC4 cells (MCF-7 and Colo-205) were seeded in 6-well plates. After 80% confluent growth, CBDCA, MUS and 4 MU were added in their corresponding IC25 values alone and in combinations, and incubated for 48 h, after which the cells were harvested. In the case of in vivo experiment, femoral bone marrow cells were isolated and harvested by centrifugation. Thereafter apoptotic or necrotic cell death (in vitro and in vivo) was determined by using Annexin-V/PI kit . Briefly, 1 × 106 cells were suspended in 100 μl of binding buffer. After that Annexin V-FITC and propidium iodide (PI) was added in 1:1 ratio and incubated at room temperature for 15 min in dark. 400 μl of binding buffer was then added and percentages of apoptotic or necrotic cells were analyzed with a flow cytometer.5 × 10in vivo (ascites and solid tumour) against two murine cancer cell lines viz Dalton’s Lymphoma (DAL) and Sarcoma-180 (S-180). These cell lines were maintained in Swiss albino mouse by intraperitoneal transplantation of 1 × 106 viable tumour cells once in a week. In each cancer model (two ascites and two solid), animals were divided into four groups . The day of tumour cell inoculation was counted as “day 0”. In the ascites tumour model, treatment was started from day 1 and in solid tumour model treatment was started after 7 days . The experimental groups were divided as below-Anti-tumour activity was evaluated ups Fig. , containT (tumour control): 5.5% propylene glycol in water (p.o.) and normal saline (i.p.) for 10 days.TC (CBDCA treated group): 12 mg/Kg b.wt. CBDCA (i.p.) for 10 days.TCU (CBDCA + 4MU treated group): 4MU was given ate dose of 3 mg/kg b.wt. in 5.5% propylene glycol (p.o.) and CBDCA at the dose of 12 mg/Kg b.wt. (i.p.) for 10 days.TCS (CBDCA + MUS treated group): MUS was given ate dose of 6 mg/kg b.wt. in 5.5% propylene glycol (p.o.) and CBDCA at the dose of 12 mg/Kg b.wt. (i.p.) for 10 days.th day for the ascites tumour and 17th day for the solid tumour). After sacrifice, ascitic tumour cells and solid tumour tissues were collected and washed in isotonic PBS solution.All experimental animals were sacrificed 24 h after the last treatment (113) = 4/3πr12 × r2; where r1 is the width and r2 is the length of the solid tumour measured by Vernier calliper. Tumour-growth inhibition rate (% TIR) was calculated according to the formula (P − Q)/P × 100, where ‘P’ represents the mean tumour volume of the respective control group and ‘Q’ represents the mean tumour volume of the treated group.In ascites models, the percentage of tumour cell inhibition (%) was calculated as (A − B)/A × 100, where ‘A’ represents the mean tumour cell count of the respective control group and ‘B’ represents the mean tumour cell count of the treated group. The solid tumour volume was calculated as tumour volume .Supplementary Information


Mechanistic studies reveal that a structural reconstruction of Rh single atoms in Rh/CoO occurs during the catalytic process, facilitating the adsorption and activation of reactants. In kinetic view, linear products are determined as the dominating products by analysing reaction paths deriving from the two most stable co-adsorbed configurations. As a bridge of homogeneous and heterogeneous catalysis, single-atom catalysts can be potentially applied in other industrial reactions.Rh-based heterogeneous catalysts generally have limited selectivity relative to their homogeneous counterparts in hydroformylation reactions despite of the convenience of catalyst separation in heterogeneous catalysis. Here, we develop CoO-supported Rh single-atom catalysts (Rh/CoO) with remarkable activity and selectivity towards propene hydroformylation. By increasing Rh mass loading, isolated Rh atoms switch to aggregated clusters of different atomicity. During the hydroformylation, Rh/CoO achieves the optimal selectivity of 94.4% for butyraldehyde and the highest turnover frequency number of 2,065 h Despite the advantages of using heterogeneous catalysts, most successful rhodium hydrogenations are carried out with homogeneous catalysts. Here the authors report a supported single atom rhodium catalyst providing high activities and selectivities for propene hydroformylation. Hydroformylation of olefins is one of the most important industrial processes because its corresponding product, aldehydes, can be widely applied as intermediates for synthetic esters, alcohols, carboxylic acids, aliphatic amines and other fine chemicals11345789Single-atom catalysts are a class of heterogeneous catalysts recently developed, in which isolated metal atoms are singly dispersed on supports1111121314151617181920212223242526272827282728−1 and selectivity of 94.4% for butyraldehyde among the obtained atomic-scale Rh-based nanocatalysts. In addition, 95.8% of the original reaction activity for Rh/CoO was preserved with the selectivity for butyraldehyde maintaining at 94.0% after five rounds of reaction, indicating their noticeable stability. Further mechanistic studies revealed that a structural reconstruction of Rh single atoms in Rh/CoO occurred during the catalytic process, facilitating the adsorption and activation of reactants. The kinetic analysis indicates the dominating products of linear products based on the analysis of reaction paths deriving from the two most stable co-adsorbed configurations.Herein, we report the preparation of CoO-supported Rh single-atom catalysts (Rh/CoO) with remarkable catalytic activity and chemoselectivity towards hydroformylation reactions. By increasing Rh mass loading, isolated Rh atoms switched to aggregated nanoclusters of different atomicity. During the hydroformylation of propene, Rh/CoO achieved the highest turnover frequency (TOF) number of 2,065 hfcc) crystalline nature in high purity and uniformity  was injected into the aqueous solution containing CoO nanosheets (100 mg), followed by stirring at room temperature for 4 h. In this process, Rh atoms replaced Co atoms on the surface of CoO nanosheets via the galvanic replacement reaction between the precursor and substrate. X–Y (3RhCl6 solution (10 mM) increased to 0.97 and 4.85 ml, 1.0%Rh/CoO and 4.8%Rh/CoO were obtained, where the mass loadings of Rh were 1.0 and 4.8%, respectively. fcc CoO without the signal of Rh species, because of single-atom structures and extremely low mass loading. In 1.0%Rh/CoO and 4.8%Rh/CoO, the peaks of fcc Rh were observed due to the existence of Rh clusters. In addition, Rh single atoms supported on bulk CoO, denoted as Rh/bulk-CoO, were also synthesized based on similar procedures except for using different CoO supports  of 5.8  compared with that of Rh foil (CN=12.0)  and extended X-ray fine structure (EXAFS) were measured to determine the electronic and local structures of chemical nature of the samples. The Rh K-edge XANES profiles in R space , 0.2%Rh/) of 5.8 . No othe) of 5.8 . Moreove) of 5.8 . Their RCN=12.0) , indicatCN=12.0) , where tenerated .−1, 1.4 and 5.2 times as high as that of 1.0%Rh/CoO and 4.8%Rh/CoO, respectively. Moreover, the selectivity for butyraldehyde over 0.2%Rh/CoO reached 94.4%, while only 68.7 and 53.9% of butyraldehyde were generated in the products over 1.0%Rh/CoO and 4.8%Rh/CoO, respectively. This decreasing trend of selectivity against mass loadings was further reviewed by reaction profiles. Nowadays, more than 75% of all hydroformylation reactions are based on Rh triarylphosphine catalysts−1, and the selectivity for linear products reaches >90%. Thus, 0.2%Rh/CoO exhibited comparable catalytic activity and selectivity to Rh-based homogeneous catalysts. As shown in We applied the established atomic-scale Rh-based catalysts in the hydroformylation of propene to evaluate their catalytic performance. A blank test was performed with only CoO nanosheets added, in which no product was observed. in situ diffuse reflectance infrared Fourier transform (DRIFT) and in situ XPS measurements to investigate the adsorption properties of reactants on the surface of 0.2%Rh/CoO. In order to simulate the reaction conditions, 0.2%Rh/CoO was respectively exposed to H2, CO, propene, and the mixed gas (H2:CO:propene=1:1:1) at 100 °C, followed by measurements of in situ DRIFT  calculations. The model was established by replacing two surface Co atoms with two Rh atoms on a CoO thin film and involving a Co vacancy . This mo−1.41 eV . Althoug2 , adsorption energies of configuration II, III, and IV were calculated to be 0.024, 0.210, and 0.089 eV, respectively. The hydroformylation reaction occurs when a H atom attacks one end of C=C bond in olefin, followed by the addition of CO in another end of C=C bond and at last the other hydrogen atom combines with the C atom in C=O as shown in 1/CoO, only several stable configurations existed in the single-atom catalyst, so the variety of addition pathways was limited. On the basis of the relative location between H and the nearest unsaturated C in propene, linear products  were favoured in configuration I and III, while configuration II and IV were expected to generate branched products . Due to the small energy difference (0.024 eV) between configuration I and II, the reaction paths deriving from these two configurations were investigated. As revealed in 1/CoO.As a result, co-adsorbed configurations of reactants and catalysts were established. On the basis of DFT calculations, four stable configurations, respectively donated as configuration I, II, III, and IV were proposed as shown in −1, 1.4 and 5.2 times as high as that of 1.0%Rh/CoO and 4.8%Rh/CoO, respectively. In addition, 0.2%Rh/CoO gave the highest selectivity of 94.4% for butyraldehyde. Mechanistic studies revealed that a structural reconstruction of Rh single atoms in Rh/CoO occurred during the catalytic process, facilitating the adsorption and activation of reactants. In terms of a kinetic view, linear products were determined as the dominating products by analysing reaction paths deriving from the two most stable co-adsorbed configurations. This work not only develops an efficient catalyst for hydroformylation reactions with significantly high selectivity, but also advances the understanding of discrepancy in selectivity between single-atom catalysts and traditional metal nanocrystals. As a bridge of homogeneous and heterogeneous catalysis, single-atom catalysts can be potentially applied in other industrial reactions.In conclusion, we reported a facile synthesis of Rh/CoO which achieved remarkable catalytic performance towards hydroformylation reactions. The configuration of obtained products changed from single atoms to subnanometer clusters and nanoclusters with the increase of Rh mass loading from 0.2% to 1.0% and 4.8%, respectively. In hydroformylation reactions, TOF number of 0.2%Rh/CoO reached 2065, h3 was added into a mixed solution, containing 20 ml of ethylene glycol and 4 ml of distilled water, followed by vigorous stirring for 30 min. Then the mixture was transferred into a 50 ml Teflon-lined autoclave, followed by being sealed and heated at 190 °C for 48 h. After the solution was cooled down to room temperature, the product was collected by centrifugation, washed three times with ethanol and then dried in vacuum overnight.CoO nanosheets were synthesized based on the previously reported method3RhCl6 aqueous solution (10 mM) were dissolved in 10 ml of distilled water, followed by stirring at room temperature for 4 h. The product was collected by centrifugation, washed three times with water, and then dried in a vacuum at 50 °C. Further ICP result determined that the mass loading of Rh was 0.2%, well corresponding to the amount of precursors. When the amount of Na3RhCl6 solution (10 mM) increased to 0.97 and 4.85 ml, 1.0%Rh/CoO and 4.8%Rh/CoO were obtained, where the mass loadings of Rh were 1.0 and 4.8%, respectively. The synthetic procedure of Rh/bulk-CoO was similar to that of 0.2%Rh/CoO, except for using commercial CoO to replace CoO nanosheets.In a typical synthesis of 0.2%Rh/CoO, 100 mg of CoO nanosheets and 0.19 ml of NaE0=23.22 keV) were performed at BL14W1 beam line of Shanghai Synchrotron Radiation Facility (SSRF) operated at 3.5 GeV under ‘top-up' mode with a constant current of 240 mA. The XAFS data on Rh/CoO sample were recorded under fluorescence mode with a Lytle ion chamber or a 32-element Ge solid state detector for different Rh loading samples. The energy was calibrated accordingly to the absorption edge of pure Rh foil. Athena and Artemis codes were used to extract the data and fit the profiles. For the XANES part, the experimental absorption coefficients as function of energies μ(E) were processed by background subtraction and normalization procedures, which were reported as ‘normalized absorption'. For the EXAFS part, the Fourier transformed (FT) data in R space were analysed by applying the 1st shell approximation or metallic Rh model for the Rh–O or Rh–Rh shell, respectively. The passive electron factors, S02, were determined by fitting the experimental Rh foil data and fixing the Rh–Rh coordination number (CN) to be 12, and then fixed for further analysis of the measured samples. The parameters describing the electronic properties  and local structure environment including CN, bond distance (R) and Debye Waller (D.W.) factor around the absorbed atoms were allowed to vary during the fit process.XAFS spectra at Rh K-edge (2 (15.0 bar). The amount of catalysts for 0.2%Rh/CoO, 1.0%Rh/CoO and 4.8%Rh/CoO was 50, 10 and 2.1 mg, respectively, to ensure the same amount of Rh species in catalytic tests. The reaction was performed at 100 °C with stirring at 300 r.p.m. for 2 h. After the completion of the reaction, the gas phase was determined by GC-FID. No signals for propane were detected. The liquid phase of the reaction mixture was collected by centrifugation at 10,000 r.p.m. for 2 min, and analysed by GC–MS by using N,N-dimethylformamide as internal standard. When different olefins other then propene were used as substrate molecules, 5 mmol of olefins, 20 ml of isopropanol and catalysts into a Teflon inlet, the autoclave was pressurized with CO (15.0 bar) and H2 (15.0 bar). The reaction was performed at 100 °C with stirring at 300 r.p.m. for 5 h.The hydroformylation of propene was performed in a 100 ml stainless-steel autoclave (Parr Instrument Company). After the addition of 20 ml of isopropanol and catalysts into a Teflon inlet, the autoclave was pressurized with propene (1.6 bar), CO (15.0 bar) and Hk-point sampling. The transition states are determined by climbing-image nudged elastic band methodAll the calculations were performed within spin-polarized DFT implemented in VASPSl=Rl/(Rb+Rl)=(Rl/Rb)/[1+(Rl/Rb)], where Sl, Rl and Rb represent the selectivity for linear products, the reaction rate for linear products, and the reaction rate for branched products, respectively. The ratio of the two products is described as Rl/Rb=exp θl3−r/[exp θb3−r], where Ea,l−3 and Ea,b−3 are the respective activation energies of step iii for linear and branched products, θl3−r and θb3−r are the respective coverages of the reactant in step iii for linear and branched product, kB is the Boltzmann constant, and T is the reaction temperature. Given the quasi-equilibrium approximation, the ratio of θl3−r to θb3−r can be described as the ratio of the equilibrium constants of the first two elementary steps, and that is θl3−r/θb3−r=exp(−ΔHl−1/kBT) exp(−ΔHl−2/kBT)/[exp(−ΔHb−1/kBT) exp(−ΔHb−2/kBT)], where ΔHl−1 and ΔHl−2 are the respective enthalpies of step i and ii for linear products, while ΔHb−1 and ΔHb−2 are the respective enthalpies of step i and ii for branched products. At 100 °C, Sl is calculated as 96%.The reaction paths deriving from configuration I and II were calculated. The whole reaction involves three elementary steps: the addition of one hydrogen atom (step i), the addition of one CO molecule (step ii), and the formation of the product (step iii). In terms of kinetics, step i and ii were regarded as quasi-equilibrium states, whereas the step iii was the rate-limiting step. Thus, the selectivity for linear products deriving from configuration I and II is depicted as In situ DRIFT experiments were conducted in an elevated-pressure cell (DiffusIR Accessory PN 041-10XX) with a Fourier transform infrared spectrometer (TENSOR II Sample Compartment RT-DLaTGS) at 100 °C. After flowing 1 atm of N2 at the rate of 30 sccm for 0.5 h, the background spectrum of the sample was acquired. Then, 1 atm of H2, CO, propene, or the mixed gas (H2:CO:propene=1:1:1) were allowed to flow into the cell at 100 °C for 1.0 h. Afterwards, 1 atm of N2 was allowed to flow into the cell at 100 °C for 0.5 h. In situ DRIFT spectra of the sample after the treatment with different gases were obtained.In situ XPS measurements were performed at the photoemission end-station at beam line BL10B in the National Synchrotron Radiation Laboratory (NSRL) in Hefei, China. Briefly, the beam line is connected to a bending magnet and covers photon energies from 60 to 1000, eV with a resolving power (E/ΔE) better than 1,000. The end-station is composed of four chambers, that is, analysis chamber, preparation chamber, quick sample load-lock chamber and high pressure reactor. The analysis chamber, with a base pressure of <2 × 10−10 torr, is connected to the beamline and equipped with a VG Scienta R3000 electron energy analyser and a twin anode X-ray source. The high pressure reactor houses a reaction cell where the samples can be treated with different gases up to 20 bar and simultaneously heated up to 650 °C. After the sample treatment, the reactor can be pumped down to high vacuum (<10−8 torr) for sample transfer. In the current work, the sample was treated with different gases (1 bar) at 100 °C for 0.5 h in the high pressure reactor, after which it was transferred to analysis chamber for XPS measurement without exposing to air.λ=1.54178 Å).HAADF-STEM images were collected on a JEOL ARM-200 F field-emission transmission electron microscope operating at 200 kV accelerating voltage. ICP-AES  was used to determine the concentration of metal species. X-ray diffraction patterns were recorded by using a Philips X'Pert Pro Super diffractometer with Cu-Kα radiation .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary figures and supplementary tables.


Zingiber , Hedychium , Curcuma (Ao), Etlingera (Torch ginger), Amomum (Chi Kuk), and Alpinia , which are eaten fresh or cooked as ingredients in the preparation of many Thai dishes. The proximate compositions  varied among the different genera. The plants sampled were generally low in fat content (<1%), which contributed as little as 30% of the total caloric energy. Edible plant parts contained substantially high amounts of potassium (max. 737.21 mg/100 g), calcium (max. 140.15 mg/100 g), and iron (~0.32 mg/100 g). Among the tested samples, torch ginger had the highest vitamin C content (1.05 mg/100 g), total phenolic and total flavonoid contents, as well as 2,2-diphenyl-1-picrylhydrazyl activity. On the other hand, the 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) assay suggested that Hedychium species possessed the highest antioxidant activity (~5.38 mg TEAC/g extract). Our results prove that edible plants of the Zingiberaceae family found in Thailand are rich sources of potentially important nutrients.The nutritional compositions and phytochemical properties of eight edible flowers of the ginger family (Zingiberaceae) commonly found in Thailand are reported herein. The plant genera investigated were  Zingiber (49 spp.), Kaempferia (17 spp.), Hedychium (22 spp.), Curcuma (34 spp.), Globba (42 spp.), Alpinia (17 spp.), Amomum (16 spp.), Etlingera (12 spp.), and Caulokaempferia (14 spp.).Plant species of the Zingiberaceae family are widely distributed in the tropical and subtropical regions of the world , 2. In ACurcuma longa, Boesenbergia rotunda, Alpinia galanga, and Zingiber officinale are typically used to treat diarrhea, stomachache, and flatulence , in traditional medicine and to produce natural dyes . Their ratulence –8. The lMalaysia , and thoin Japan . The flo species . In addit dishes , 12, whili paste . Moreovemachache .Phytochemicals are biological compounds produced by plants throughout primary and secondary metabolisms. Many phytochemicals convey known nutritional, biological, and pharmacological benefits , 16. RecA. zerumbet contain flavonoids and phenolic acids that possess a higher inhibition level of beta-carotene oxidation and a greater radical scavenging activity than its rhizomes , H. forrestii (variety with an orange filament), and Etlingera elatior were used for our experiments. Flowers or inflorescences were either sampled from the living collections of the Queen Sirikit Botanic Garden (QBG), Chiang Mai, Thailand or purchased from local markets in Northern Thailand. Their voucher specimens were taxonimically identified according to Zingiberaceae experts’ comments  and literature , potassium (K), and calcium (Ca) and one microelement, iron (Fe), were estimated according to Xiao et al. , by whicThe total energy of one serving of sample (100 g fresh weight) was calculated according to the following equation :Total e3P) in ultra turrax  during 4 min. The extract was centrifuged at 16,096 × g for 15 min at 4°C, and the supernatant was filtered using filter paper (Whatman no. 1). Ascorbic acid content was then quantified by Shimadzu’s HPLC system on reversed-phase C18 column  under the following conditions: injected volume 20 µL; oven temperature 40°C; solvent potassium phosphate (10 mM); flow rate 1.5 mL/min, and the detection was performed with an ultraviolet wavelength of 242 nm. Total ascorbic content was calculated from calibration curves by comparison with external standard   .g for 10 min at 4°C. The supernatant was then collected and used for further phytochemical analyses (A dried powder (0.5 g) was extracted with 1.0 mL of 95% (v/v) methanol. The mixture was vortexed for 1 min, and the resulting homogenate was centrifuged at 14,000 × analyses .2CO3) solution (w/v) was added to the mixture, which was then incubated for 30 min in the dark at room temperature (25°C). Finally, the absorbance of the solution was measured at 765 nm by using a microplate spectrophotometer , and the TPC was reported as milligrams of gallic acid equivalents per gram of extracted sample (mg GAE/g extract). Gallic acid was obtained from Sigma-Aldrich .Total phenolic content of the samples was calculated following a modified procedure of Ao et al. . The met2) solution in a 96-well plate. After 5 min incubation in the dark, 10 µL of 10% of aluminum nitrate [Al(NO3)3] solution was added, and the mixture was further incubated for 5 min in the dark at room temperature. Finally, 100 µL of 1 M sodium hydroxide (NaOH) and 30 µL of distilled water were added to the mixture and shaken. The absorbance was then measured at 510 nm by using a microplate spectrophotometer . The total flavonoid contents were expressed as milligrams of rutin equivalents per gram of extracted sample (mg RE/g extract). Rutin was obtained from Sigma-Aldrich .Total flavonoid content was assessed according to the method of Hajiaghaalipour et al.  with sli50) value (mg/mL).2,2-Diphenyl-1-picrylhydrazyl radical scavenging activity was determined according to Yen and Hsieh  with sli2S2O8) was added up to the concentration of 2.45 mM. The mixture was incubated at room temperature overnight (16–18 h) and in the dark before use. The stock solution of ABTS was diluted with absolute ethanol to obtain an absorbance value of 0.70–0.90 at 734 nm with a microplate spectrophotometer , this solution was the working ABTS solution. The methanol extract of the sample (1.9 µL) was then pipetted in to 96-well plate, and 7.5 µL of absolute ethanol and 190.6 µL of working ABTS solution were added. The mixture was finally shaken and incubated in the dark at room temperature for 5 min, with an absorbance at 734 nm with a microplate spectrophotometer . The ABTS results were expressed as milligrams of Trolox equivalent antioxidant capacity per gram of extracted sample (mg TEAC/g extract). Trolox was obtained from Sigma-Aldrich .The radical scavenging measurement of ABTS was performed according to Re et al.  and ChanAll experiments were performed to the least in triplicate and reported as means ± SD. Differences between samples were determined by Duncan’s multiple range tests in SPSS statistical program ver. 17 . A probability level of 99% was used in testing the statistical significance of all experimental data.–Zingiber , Hedychium (yellow filament and orange filament), Curcuma (Ao), Etlingera (Torch ginger), Amomum (Chi Kuk), and Alpinia  genera. The main traditional uses of the surveyed Zingiberaceae plants were for medicinal purposes, such as the use of rhizomes to treat stomach ailment and fever, and as an anti-inflammatory agent. The flowers and rhizome of A. galanga, fruits and seeds of A. maximum, the rhizome of H. forrestii, the inflorescence of Z. zerumbet, and all parts of Z. officinale were commonly used to treat stomach pains. This ethnobotanical knowledge confirms previous findings, by which the rhizomes of A. galanga and Z. officinale are used to treat diarrhea, stomachache, and flatulence , followed by A. maximum 94.38 g/100 g, C. plicata 93.60 g/100 g, and Z. zerumbet 93.48 g/100 g. A. galanga had the highest fat content (0.85 g/100 g), followed by H. forrestii (yellow filament), 0.69 g/100 g, H. forrestii (orange filament), and Z. ottensii, 0.63 g/100 g. The higher the moisture content, the more likely plant products become perishable . High fiber contents are beneficial to human health as they minimize the risks of diseases and illnesses by decreasing cholesterol levels. They also reduce the risks of heart diseases and of constipation . These values are comparable to the amount of protein found in African dark green leaves consumed as vegetables (~4%)  inflorescences, a plant of the same family, reached 315 kcal/100 g from a dried basis  followed by galangal  and Phlai Dam . Calcium was also the highest in Phlai Dam (Z. ottensii 140 mg/100 g), followed by torch ginger  and Z. zerumbet (83 mg/100 g). Macroelements are important nutrients that play essential roles in the biochemical pathways of the human body , in minor yet non-negligible amounts (0.32 mg/100 g).The mineral compositions  of the studied edible flowers are presented in Table man body . Potassiman body , 67. Calman body . Iron haman body . In our E. elatior) showed the highest content (1.05 mg/100 g), followed by Phlai Dam (Z. ottensii 0.09 mg/100 g), and Ao . These values are relatively low when compared with Sesbania flowers   . Vitaming/100 g) , 72. In E. elatior) contained the highest levels of TPC at 2.29 mg GAE/g extract, while ginger flower had the lowest content at 0.14 mg GAE/g extract. Chan et al.  while Hedychium sp. had approximately 8.20 mg GAE/g of leaf extract. When comparing these results to our own, there seems to be a 10- to 15-fold increase in TPC between flower and leaf parts in some edible Zingiberaceae species of our study.The TPC ranged from 0.14 to 2.29 mg GAE/g extract. Torch ginger flower  flowers at 42.50 mg RE/g extract, followed by ginger flowers Z. officinale, at 37.50 mg RE/g extract and H. forrestii (orange filament) flowers, with 27.50 mg RE/g extract (Table 50) value. Overall, the percentage of inhibition against DPPH ranged from 9.99 to 68.70%, and the IC50 value ranged from 2.86 to 24.72 mg/mL. Here again, torch ginger (E. elatior) flowers had the highest inhibition (%) and lowest IC50 values, which were 68.70% and 2.86 mg/mL, respectively, while Krathue (Z. zerumbet) showed the lowest inhibition percentage against DPPH, i.e., 9.99%, and its IC50 value was the highest, at 24.72 mg/mL most likely because the Krathue extract had low total contents of phenolic and flavonoid compounds, which primarily act as antioxidants. Our DPPH radical scavenging activity results however differ from those found in previous studies. Yan and Asmah  than those of an ethyl acetate extract (IC50 = 68.24 µg/mL) against DPPH free radicals.The amount of total flavonoid contents ranged from 16.53 to 42.50 mg RE/g extract. From our results, the highest total flavonoid content was found in torch ginger  had the highest ABTS value (~5.38 mg TEAC/g extract), and C. plicata showed the lowest ABTS value (~2.90 mg TEAC/g extract). A previous study by Butsat and Siriamornpun , the high macroelement  content of galangal, ginger , and torch ginger (E. elatior) flowers, and the flowers of torch ginger as a source of strong antioxidants. Our findings thus provide useful information for the development of flower-based plant products, particularly in the food and nutraceutical industries, as well as ethnobotanical purposes to support their future ecological conservation.Edible flowers are historically part of traditional Thai cuisine and culture. They are often used as ingredients in local food and beverages, for medicinal or pharmaceutical purposes and also in religious rituals. This study acknowledges the importance of some flowers of the Zingiberaceae family that are commonly used in local food dishes by Northern Thai people. Our results illustrate their potential as sources of essential nutrients, for instance, the high dietary fiber content of galangal  was removed during the preparation of the database, to protect subject anonymity. The data collections were, however, not submitted to an ethics review process, as none of the data collected were of medical or clinical purpose, would have a direct impact on the participants’ health, or affect the participants’ life and welfare.The project idea was developed by SS; the experimental design was developed by SS and RS; sample collections were performed by AR, KK, RS, and RP; the laboratory experiments were run by AR, KK, and RP; the data were analyzed by AR, KK, and PP; and the manuscript was written by AR, KK, RS, RP, PP, and SS.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


At low moisture contents (≤25.4% w.b.) or low temperatures (≤45 °C), ε″ increased with increasing frequency. However, ε″ changed from decrease to increase with increasing frequency at high moisture contents or temperatures. At low temperatures (25–35 °C), both ε′ and ε″ increased with increasing moisture content. At low moisture contents (15.1–19.5% w.b.), they increased with increasing temperature. The change trends of ε′ and ε″ were different and dependent on temperature and moisture content at their high levels. The penetration depth (dp) decreased with increasing frequency. RF treatments may provide potential large-scale industrial drying application for potato starch. This research offers useful information on dielectric properties of potato starch related to drying with electromagnetic energy.To develop advanced drying methods using radio-frequency (RF) or microwave (MW) energy, dielectric properties of potato starch were determined using an open-ended coaxial-line probe and network analyzer at frequencies between 20 and 4,500 MHz, moisture contents between 15.1% and 43.1% wet basis (w.b.), and temperatures between 25 and 75 °C. The results showed that both dielectric constant ( Potato starch, a representative of tuber starches, is the third-place material with about 6% global starch production3. Drying is the last process in the production of potato starch. During this period, the wet starch cake with about 40% moisture content in wet basis (w.b.) is dried to about 20% w.b. using hot air at 150 °C4. Although high temperature improves heating or drying efficiency, it affects rheological properties of starch granules and final quality of starch products6. Kugimiya et al.7 studied the unrecoverable endothermic gelatinization of potato starch at high moisture contents, and found that gelatinization temperature was about 65 °C. Donovan8 found that when potato starch granules with moisture content lower than 60% w.b. were heated, there were two endothermic transitions. The high endothermic temperature increased with decreasing moisture content, and the low endothermic temperature was found at 66 °C. It indicates that drying below 65 °C is helpful to obtain potato starch with high quality.Starch is a major agricultural product that is consumed and/or used on a daily basis in the whole world. Its application is not only confined to making foods, but also used as raw materials in industry, such as in oil drilling, mining, and making bio-plastics, paper, and textiles13. However, Jiang et al.14 found that drying starch using dielectric heating resulted in poor heat distribution uniformity compared to using conventional thermal energy.Dielectric heating, which incorporates radio-frequency (RF) and microwave (MW) treatments, has been regarded as an advanced and promising technology in drying agricultural products. Some studies have indicated that RF/MW drying has advantages in improving thermal efficiency and product quality, and in shortening drying time when contrasted with conventional drying15 and to improve dielectric heating uniformity. Frequency, moisture content and temperature are main factors that influence the dielectric properties of food materials16. The effects of temperature and moisture content on dielectric properties of several kinds of starches were studied by Ndife et al. at 2450 MHz17. Motwani et al.18 found that the dielectric properties of starch slurries were influenced by starch concentration and gelatinization. However, few reports have been found on studying the dielectric properties of potato starch as related to dielectric drying with RF/MW energy. Moreover, little information was reported on penetration depth which influences drying uniformity. Therefore, the objectives of this study were: (1) to obtain the dielectric properties of potato starch at moisture contents between 15.1% and 43.1% w.b. and at temperatures between 25 and 75 °C over a frequency range from 20 to 4,500 MHz, (2) to analyze the relationship between dielectric properties and penetration depth with above-mentioned factors, and (3) to provide information on designing drying process and on deciding treatment sample thickness for drying potato starch with RF/MW energy.Dielectric properties are main parameters that describe the interaction between electric energy and materials during dielectric heating. Study on dielectric properties of agriculture products is necessary and crucial to develop effective RF or MW drying processε′ and ε″ of potato starch samples with different moisture contents at 35 °C and over the frequency range of 20–4,500 MHz are shown in Fig. ε′ and ε″ were influenced by frequency. ε′ decreased with increasing frequency at all moisture contents  of Equation  and ε″ (b) of potato starch sample with moisture content of 15.1% w.b. at investigated temperatures and over the frequency range of 20–4,500 MHz are shown in Fig. ε′ decreased almost linearly with increasing frequency , then changed slightly with moisture content above 25.4% w.b. Analysis of variance (ANOVA) results showed that there was no significant difference (p > 0.05) between the values of dp at high moisture contents (≥25.4% w.b.). At 65 and 75 °C, when the moisture content was lower than 34.1%, dp increased slightly with increasing moisture content, and then decreased a little. There was no significant difference (p > 0.05) between the values of dp at all investigated moisture contents at 65 and 75 °C.Figure dp at investigated moisture contents and 915 MHz. When the moisture content was between 15.1% and 29.5%, dp decreased with increasing temperature firstly, then changed small or even kept constant. Similar results have been reported for mango puree49, chestnut50, and rapeseed51. When the moisture content was higher than and equal to 34.1% w.b., dp increased slightly and linearly with increasing temperature. ANOVA results showed that there was no significant difference (p > 0.05) between the values of dp at two adjacent temperatures. The increased dp with increasing temperature was also noted on some cereals, such as corn, sorghum and wheat at 915 MHz52, and garlic at 2450 MHz53.Figure dp of potato starch was influenced by frequency, moisture content and temperature greatly, choosing a appropriate thickness during drying using RF/MW energy is very important. Schiffmann54 suggested that for uniform treatment with RF/MW energy, the largest thickness of materials should be two or three times the penetration depth. In this study, the smallest dp at 27.12, 40.68, 915, and 2450 MHz at all investigated moisture contents and temperatures were 77.10, 68.02, 5.26, and 1.41 cm, respectively. If the largest thickness of potato starch was calculated as two times penetration depth, the thickness of potato starch should be smaller than 154.20, 136.04, 10.52, and 2.82 cm when the frequency of electric field was 27.12, 40.68, 915, and 2450 MHz, respectively. Large penetration depths at RF frequencies mean better heating uniformity than at MW frequencies in potato starch. Therefore, RF frequency may provide potential large-scale industrial drying applications for potato starch. It should be mentioned that even if the thickness of treated materials was smaller than the largest thickness, non-uniformity still exists during heating. Some parts might absorb more energy, and other parts might absorb less energy, which cause different temperature and different moisture content. In industry applications, agitating, shaking, moving and other methods can be used to improve heating uniformity and to maintain the temperature of potato starch below 66 °C.Since Edible potato starch, produced by Beijing Dezhongjiaxin Economic and Trade Co., Ltd. , was purchased from a local supermarket in Yangling, Shaanxi, China. Its main compositions in mass were 76.1% carbohydrate, 7.2% protein, 0.5% fat, and 15.1% moisture content in wet basis (w.b.). The carbohydrate, protein, and fat contents were measured according to the methods of CFR 101.9, AOAC 955.04, and AOAC 992.06, respectively. The moisture content was measured according to the standard of AOAC 925.10 with some modification.To obtain samples at different moisture levels from original moisture content (15.1% w.b.) to about 45% w.b. with about 4% interval, 200 g original potato starch of each lot was sprayed with predetermined mass of distilled water. The samples were conditioned about 3–6 days at 4 °C in sealed polyethylene bags before they were warmed up to ambient temperature for moisture content determination and dielectric properties measurement. To ensure the moisture was distributed uniformly throughout the samples, the bags were shaken 3–4 times each day. Finally, eight moisture contents, 14.8 ± 0.1, 19.5 ± 0.1, 22.3 ± 0.2, 25.4 ± 0.1, 29.5 ± 0.2, 34.1 ± 0.2, 37.6 ± 0.3, and 43.1 ± 0.4% w.b. were obtained in this study.4 before reweighing to determine water loss. Moisture content was calculated from the initial and final weights of the samples. The mean value of moisture content of each sample was obtained from three replicates.According to the standard of AOAC 925.1, moisture content was determined by drying triplicate 2–3 g potato starch samples on aluminum moisture dishes at 130 °C in a forced-air oven  until the weight kept constant. The samples were cooled in a desiccator with CaSO56. Therefore, the water activity of each sample was measured at room temperature (25 °C) using an AquaLab water activity meter  with an accuracy of ±0.003 aw. Before water activity measurement, the meter was calibrated using a standard salt solution. Then about 5 g of potato starch was placed in the measurement chamber, and the water activity was measured. Triplicate was conducted for each moisture level, and the mean value was presented.Several studies have shown that there is relationship between dielectric properties and water activityet al.57. In this study, the frequency range of the network analyzer used to obtain the dielectric properties was set from 20 to 4,500 MHz. The dielectric properties were measured at 201 frequencies on a logarithmic scale. The frequency range includes the specific frequencies of 27.12, 40.68, 915 and 2450 MHz, which are generally applied in ISM  applications.The dielectric properties measurement system consisted of a vector network analyzer , an open-ended coaxial probe (85070E), dielectric probe kit software (85070), a constant temperature water bath , a temperature-controlled stainless steel cylindrical sample holder (23 mm in diameter and 25 mm in height), a type-T thermocouple temperature meter, a hydraulic platform, and a computer. The detailed information on the system and the calibration procedure was described earlier by Guo 3 and 0.67 g/cm3. Since the sample density change was small, the influence of density on dielectric properties was not considered in this study.At each moisture level, 20 g potato starch was placed in the temperature-controlled stainless steel cylindrical sample holder, which was placed in the constant temperature water bath. The temperature meter was inserted into the center of potato starch to monitor sample temperature. The water bath was put on a hydraulic platform, which was lifted to ensure the potato starch sample contact tightly with the coaxial probe. The temperature of samples was set from 25 to 75 °C with an interval of 10 °C in sequence. After the temperature of samples reached the set value, dielectric properties measurements over the frequency of 20–4,500 MHz were conducted for three times in 2 min. During dielectric properties measurements, a rubber slice with a hole in the center was used to cover the potato starch to prevent moisture loss. After the dielectric properties data were obtained, the sample height was measured, followed by measuring the sample weight. All measurements were carried out in triplicate. Mean values of nine measurements in triplicate were calculated and used for analysis. The results showed that the weight loss during dielectric properties measurement was less than 5%, and the sample density was between 0.63 g/cmdp, m) is defined as the depth to which the power density reduces to 1/e  of its value at the surface. The depth is a key factor that influences heating uniformity and in designing the thickness of sample during RF/MW heating. When the nonmagnetic materials are under the condition of plane wave incidence, the penetration depth can be calculated as23c is the speed of light in free space, 3 × 108 m/s, f is the frequency of electromagnetic wave in Hz. After obtaining the dielectric properties, the penetration depths of potato starch samples were calculated at eight moisture contents, six temperatures, and 201 frequencies.Penetration depth  and low moisture contents (15.1–19.5% w.b.). At high moisture contents and temperatures, the effects of moisture content and temperature on dielectric properties were complex. The penetration depth decreased with increasing frequency over the whole frequency range. The moisture content and temperature had little effect on penetration depth at their high levels, but had significant influence at their low levels.Permittivities of potato starch were measured between 20 and 4,500 MHz at moisture contents from 15.1% to 43.1% w.b. and temperatures between 25 and 75 °C. The study indicates that the permittivities of potato starch were influenced by frequency, moisture content, and temperature. The new frequency, temperature and moisture dependence permittivity data provide crucial information that can be useful in understanding the behavior of potato starch exposed to radio frequency and microwave dielectric heating. They also provide background information that is helpful to design drying process and apparatus using electromagnetic energy.