TC), a well-known antibiotic of clinical use, is the major disadvantage for its oral administration. With the aim to improve the water solubility of TC, the micelles of formulae SLS@TC and CTAB@TC (SLS = sodium lauryl sulphate and CTAB = cetrimonium bromide) were synthesized. The micelles SLS@TC and CTAB@TC were characterized by melting point (m.p.), thermogravimetric differential thermal analysis (TG-DTA), differential scanning calorimetry (DTG/DSC), attenuated total reflection spectroscopy (FT-IR-ATR), ultra-violet visible (UV/vis) spectroscopy, proton nucleus magnetic resonance (1H-NMR) spectroscopy, and the ultrasonically-induced biregringence technique. The antimicrobial activity of SLS@TC and CTAB@TC was evaluated, by means of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and inhibition zone (IZ), against the Gram negative bacterial strains Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli) and the Gram positive ones of the genus of Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus (S. aureus). Generally, both micelles show better activity than that of TC against the microbial strains tested. Thus, the MIC value of CTAB@TC is 550-fold higher than that of free TC against S. epidermidis. Despite the stronger activity of CTAB@TC than SLS@TC against both Gram negative and Gram positive microbes, SLS@TC is classified as a bactericidal agent (in that it eliminates 99.9% of the microbes), in contrast to CTAB@TC, which is bacteriostatic one . The toxicity of SLS@TC and CTAB@TC was evaluated against human corneal eukaryotic cells (HCECs). Moreover, SLS@TC and CTAB@TC exhibit low in vivo toxicity against Artemia salina, even at concentrations up to threefold higher than those of their MICmax. Therefore, SLS@TC and CTAB@TC can be candidates for the development of new antibiotics.The negligible water solubility of tetracycline ( TC) are considered as ideal for antibiotic drugs /[TC] or [CTAB]/[TC] molar ratios, respectively (w/wTC in SLS@TC and 7.6% w/wTC in CTAB@TC. Both micelles SLS@TC and CTAB@TC are highly soluble in dd water and dimethyl sulfoxide (DMSO).Because of the low solubility of at 36 °C  and the as drawn . Above tectively . These vSLS@TC decomposes with three exothermic steps at 157.0–227.2 °C (mass loss: −0.92%) because of the elimination of water, at 227.2–292.1 °C (mass loss: −62.95%) and 292.1–500 °C (mass loss: −3.75%) °C , 162.4–246.8 °C (mass loss 25.24%), 246.8–339.7 °C (mass loss 63.83%), and 339.7–500.0 °C (mass loss 6.05%). The total mass loss is 95.91% . The strong sharp transitions in DTA diagrams suggest that the melting points of SLS@TC and CTAB@TC are 203–207 and 222–229 °C, respectively  °C , with a s 95.91% . The melectively , in cont191.0 °C , confirmTC interacts with SLS or CTAB in the solid state to give a composite material or a mixture, DSC studies were carried out. DSC diagrams of SLS@TC and CTAB@TC are shown in SLS@TC, while the corresponding ones were observed at 112.3 and 257.6 °C for CTAB@TC. The phase of SLS at 95.8 °C is observed at 110.6 °C in the corresponding one of SLS@TC, suggesting the formation of a composite material  vibrations in the IR spectrum of SLS@TC are observed at 2918 and 2850 cm−1. The vibration band at 1070 cm−1 in the spectrum of SLS@TC is attributed to νasym of the S=O, while the vibration at 1403 cm−1 in the IR spectrum of CTAB@TC is attributed to δ(N+(CH3)3). However, no characteristics bond vibrations such as to νasym (C=O) or C=C  of TC are observed in the IR spectrum of the micelle owing to the low stoichiometry of TC/SLS or TC/CTAB in the micelles.The IR spectra of TC in the micelles was determined by UV/vis spectroscopy. Thus, the UV/vis spectra of five standard solutions in DMSO of TC  were recorded  and 7.9% w/w (CTAB@TC), respectively. These values are in agreement with those found from CMC, namely 8.3% w/w (SLS@TC) and 7.6% w/w (CTAB@TC), which correspond to the 17/1 ([SLS]/[TC]) and 16/1 ([CTAB]/[TC]) molar ratios, respectively. The MIC, MBC, biofilm elimination concentration (BEC), and so on for the micelles SLS@TC and CTAB@TC were determined with respect to TC using its concentrations in micelles, as these were derived by UV/vis spectroscopy. The concentrations of micelles in solutions, however, are far higher to their CMCs, which ensures their stability.Given that both SLS and t 369 nm , any abs1H NMR spectra in DMSO-d6 of TC, SLS, CTAB, SLS@TC, and CTAB@TC (8–6 ppm) are shown in TC are assigned to the amide H[NH2−] group (arom2–4 of TC (SLS@TC (7.52 (br), 7.12–7.10 (d), and 6.91–6.87 (d) ppm) and CTAB@TC (7.56–7.53 (t), 7.14–7.12 (d), and 6.93–6.91 (d) ppm) confirms the encapsulation of the antibiotic in the micelles.The −] group , while t–4 of TC  38]. Th. Th1H NMα clockwise or counterclockwise relative to the λ/4-plate. The transmitted intensity of the laser beam was detected by a fast photodiode and monitored in a digital oscilloscope.For measuring the transient acoustically-induced birefringence, we used an instrument depicted in δ(t)I = δ(t)I − αI is observed. δ(t)Ι is the light intensity attributed to phase retardation δ and αI corresponds to the light intensity at an angle α when the acoustic field is off. The intensity change is related to the induced optical retardation δ(t) through the following equation:When birefringence is induced by ultrasound in the liquid sample, a change in the transmitted intensity Δd is the optical path-length and λ is the laser wavelength. The applied ultrasonic pulse and the acoustically-induced birefringence are presented in maxn corresponds to the steady state birefringence when the acoustic field is on, τ is the characteristic relaxation time for the rise and decay of the birefringence signal, and β is assigned to the width of the relaxation time distribution. A simple exponential function (β = 1) is inadequate to describe the experimental data, implying a distribution of the relaxation times. The smaller the value of β, the larger the distribution of the relaxation times. A representative fitting example of the normalized birefringence decay Δn/Δmaxn is presented in The induced birefringence is associated with the retardation through the following:For all measurements, the liquid sample was kept in a sealed cuvette under isothermal conditions corresponding to 37 °C. The entire volume of the liquid was exposed uniformly to the applied acoustic field. As no flow is permitted in and out of the cell, possible directional flow or liquid sharing is missing.SLS@TC and CTAB@TC dilute solutions, a relatively slower response is observed because of the peculiar nature of the underlying dynamics, where a self-organization into larger supramolecular aggregates takes place, leading to a dynamically complex structure. Thus, both SLS@TC and CTAB@TC solutions cannot be considered as a dispersion of anisotropic particles in a liquid matrix with no polarization and steric effects.The application of the acoustic pulse allows us to observe the molecular motion dynamics directly in the time-domain by monitoring the induced birefringence trace. In general, the acoustic field will induce several types of dynamic processes in isotropic liquids and solutions at equilibrium involving intermolecular interactions. As the acoustic field is switched on, molecules are forced to orient along the propagation of the ultrasound and the whole system becomes un-axial and birefringent. After switching off the acoustic field, the induced orientation will fade away through a diffusion process of the molecules. The rotation is strongly influenced by the polarization of the neighboring molecules through dipole–induced-dipole interactions (DID). The strength of these interactions is subject to the distance and relative position between molecules. After the system is not exposed to the acoustic field, the DID interactions will level off to a minimum free-energy state, while molecules will continue to rotate because of their inertia, but under the restrictions posed by the neighboring molecules. These constraints induce molecular libration relaxation on a femtosecond timescale, which is ultrafast and beyond the limits of the ultrasonically-induced birefringence technique. Molecular liquids and their mixtures exhibit usually fast birefringence response ), exhibiting analogous behavior with other complex liquids ,41. Withequation :(5)τ=ηVhSLS and CTAB micelles and to quantitatively follow the volume change. The relative micelle volume changes are shown in TC in the parental micelles for both surfactants. We also calculated the theoretically predicted volume change between the initial and final complex micelle. We considered the diameter of the initial SLS micelle as approximately two times the size of the SLS (19.5 Å) and the diameter of the initial CTAB micelle as two times the size of the CTAB (24.2 Å), respectively. The diameter of the SLS@TC and CTAB@TC, after the incorporation of TC in the parental micelle, is calculated as the sum of the parental micelle diameter plus the mean diameter of the TC molecule (45.11 Å). The calculation of the mean TC diameter was carried out in vacuum (without any interactions), with the use of the Gaussian 09 series of programs /[TC] and [CTAB]/[CT] were 16/1 and 17/1, respectively, equal to their CMC values. The solution was stirred for 3 h and concentrated to dryness using a rotary evaporator. The oily remains were re-dissolved in 10 mL of diethyl ether and the solution was filtered off. The pure brownish solid micelles were obtained from slow evaporation of the clear ether solution.SLS@TC: yield 30%; TC content in SLS@TC: 5.22 ± 1.68% w/w; melting point: 204–207 °C (change of colour); IR (cm−1): 2918 vs, 2851 s, 1608 m, 1468 m, 1216 s, 1080 s, 823 s, 722 m, 631 w, 586 w, 421 m; 1H-NMR (ppm) in DMSO-d6: 7.53–7.50 (t), 7.12–7.09 (d), 6.90–6.87 (d), 3.69–3.66 (t), 2.51 (s), 1.49–1.46 (t), 1.25 (s), 0.87–0.84 (t) ; UV/vis (DMSO): λmax = 271, 369 nm.CTAB@TC: yield 25%; TC content in CTAB@TC: 4.96 ± 0.16% w/w; melting point 237–243 °C (change of colour); IR (cm−1): 3015 w, 1463 s, 1402 m, 1247 m, 1205 s, 1079 m, 962 s, 911 m, 829 m, 718 s, 626 m, 591 s; 1H-NMR (ppm) in DMSO-d6: 7.51–7.52 (t), 7.13–7.11 (d), 6.92–6.90 (d), 3.05 (s), 2,51 (s), 2.41 (s), 1.66–1.53 (t), 1.25 (s), 0.87–0.84 (t) ; UV/vis (DMSO): λmax = 271, 369 nm.Bacterial Strains: For the antibacterial experiments, the strains of Staphylococcus epidermidis (ATCC® 14990™), S. aureus subsp. aureus (ATCC® 25923™), and P. aeruginosa PAO1 and Escherichia coli were used.SLS@TCEffects of , CTAB@TC, TC, SLS, CTAB on the growth of microbial strainsand : This study was performed according to standard procedure, which is also described elsewhere [8 cfu/mL. The final inoculum size for broth dilution is 5 × 105 cfu/mL. The total volume of the culture solution treated by micelles, TC, and surfactants, as well as the total volume of the positive and negative control, was 2 mL. The range of concentrations of micelles, TC, and surfactants is 0.02–300 μΜ. The growth is assessed after incubation for 20 h. The minimal inhibitory concentration (MIC) was determined as the concentration of the compound that inhibits the visible growth of the bacterium being investigated. The optical density of the solution at 620 nm versus concentrations graph leads to the MIC determination [lsewhere ,27,28,29mination ,27,28,29Minimum bactericidal concentration testing: This study was performed according to standard procedure, which is also described elsewhere [lsewhere ,27,28,29lsewhere ,27,28,29lsewhere ,27,28,29Determination of the inhibition zone (IZ) through the agar disk-diffusion method: This study was performed according to standard procedure, which is also described elsewhere [8 cfu/mL) of the tested micro-organism. Filter paper disks (9 mm in diameter), which were previously soaked by micelles, TC, and surfactants (1 mM), were placed on the agar surface. The Petri dishes were incubated for 20 h and then the diameters of the inhibition zones were measured [lsewhere ,27,28,29measured ,27,28,29Effects on biofilm formation by micelles: Bacteria with a density of 6.7 × 106 cfu mL−1 were inoculated into LB medium for P. aeruginosa or tryptic soy broth for S. aureus  and cultured for 24 h at 37 °C. Afterwards, the content of each tube was carefully removed and the tubes were washed with 1 mL 0.9% saline dilution. The negative control contained broth only. After the bacteria were incubated with 50–600 μΜ of the micelles for 20 h at 37 °C, the content of each tube was aspirated; each tube was washed three times with 1 mL methanol and 2 mL 0.9% saline and left to dry. Then, the tubes were stained for 15 min with crystal violet solution (0.3% w/v). Excess stain was rinsed off with 1 mL methanol and 2 mL 0.9% saline solution and then 3 mL 0.9% saline solution. The tubes were left to dry for 24 h and the bounded crystal violet was released by adding 30% glacial acetic acid. The optical density of the solution yielded was then measured at 550 nm to give the biofilm biomass.Sulforhodamine B assay. These studies were performed in accordance with the previously reported method [SLS@TC, 0.01–0.5 μΜ for CTAB@TC, 15–60 μΜ for TC and SLS, and 1–12 μΜ for SLS. HCECs were exposed with micelles, TC, and surfactants in IC50 values for a period of 48 h.d method ,27,28,29Evaluation of genotoxicity by micronucleus assay. The micronucleus assay was carried out as previously described [4 cells/well cells per well) in glass cover slips, which were afterwards placed in six-well plates, with 3 mL of cell culture medium, and incubated for 24 h. HCECs were exposed with micelles at their IC50 values for a period of 48 h. Afterwards, the coverslips were washed three times with Phosphate Buffer Solution (PBS), and with a hypotonic solution (75 mM KCl) for 10 min at room temperature. The hypotonized cells were fixed by at least three changes of 1/3 acetic acid/ethanol. The coverslips were also washed with cold methanol containing 1% acetic acid. The coverslips were stained with acridine orange (5 μg mL−1) for 15 min at 37 °C. After, the coverslips were rinsed three times with PBS to remove any excess acridine orange stain. The number of micronucleated cells per 1000 cells was determined using a fluoresce microscope. The MN were identified according to the previously established criteria: (1) the area of MN should correspond to 1/256 and 1/9 of the area of the main nuclei; (2) the shape of MN should be round or oval; (3) the MN should be readily distinguished from artifacts; (4) MN are not linked or connected to the main nucleus; (5) MN may touch, but not overlap the main nuclei, and the micronuclear boundary should be distinguishable from the nuclear boundary; and (6) the MN have the same staining intensity as the main nuclei [escribed ,27,28,29Evaluation of toxicity with brine shrimp assay: Brine shrimp assay was performed by a method previously described [min, MICmax, 2 × MICmax, and 3 × MICmax. The final volume of each well is 1 mL with NaCl 0.9%. The brine shrimps were observed after of 24 h using a stereoscope. Larvae are considered dead if they do not exhibit any internal or external movement in 10 s of observation. Each experiment was repeated three times.escribed . Here, 1escribed . The conescribed . An aliq


JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.Familial hypocalciuric hypercalcemia (FHH) is a group of autosomal dominant disorders caused by dysfunction of the calcium sensing receptor (CaSR) and its downstream signaling proteins, leading to generally asymptomatic hypercalcemia. During pregnancy, distinguishing FHH from primary hyperparathyroidism (PHPT) is important, as the latter is associated with adverse outcomes and can be treated surgically during pregnancy, whereas the former is benign. This case report highlights the difficulties in diagnosing FHH during pregnancy. A 32‐year‐old woman was found to have asymptomatic hypercalcemia at 14‐weeks’ gestation. Investigations showed a corrected calcium (cCa) of 2.61 mmol/L (2.10 to 2.60), ionized Ca (iCa) of 1.40 mmol/L (1.15 to 1.28), 25OHD of 33 nmol/L (75 to 250), and PTH of 9.5 pmol/L (1.5 to 7.0). The patient was treated with 2000 IU cholecalciferol daily with normalization of 25OHD. The urine calcium / creatinine clearance ratio (CCCR) was 0.0071, and neck US did not visualize a parathyroid adenoma. Upon a retrospective review of the patient's biochemistry from 2 years prior, hypercalcemia was found that was not investigated. The patient was monitored with serial iCa levels and obstetric US. She delivered a healthy boy at 38‐weeks’ gestation. Postnatal iCa was 1.48 mmol/L and remained elevated. Her son had elevated iCa at birth of 1.46 mmol/L (1.15 to 1.33), which rose to 1.81 mmol/L by 2 weeks. He was otherwise well. Given the familial hypercalcemia, a likely diagnosis of FHH was made. Genetic testing of the son revealed a missense mutation, NM_000388.3(CASR):c.2446A > G, in exon 7 of the CaSR, consistent with FHH type 1. To our knowledge, there are only three existing reports of FHH in pregnancy. When differentiating between FHH and PHPT in pregnancy, interpretation of biochemistry requires an understanding of changes in Ca physiology, and urine CCCR may be unreliable. If the decision is made to observe, clinical symptoms, calcium levels, and fetal US should be monitored, with biochemistry and urine CCCR performed postpartum, once lactation is completed © 2020 The Authors.  A 32‐year‐old woman was referred to our endocrine pregnancy clinic for hypercalcemia, incidentally detected on her first trimester screening, at 14‐weeks’ gestation. She had no past medical history and her medications included a multivitamin and cholecalciferol 1000 IU daily. She was gravida 2, parity 1, with a healthy 2‐year‐old daughter. She had mild nausea and lethargy, but no polyuria, bony pain, constipation, or nephrolithiasis. Her physical examination was unremarkable, with no palpable neck masses. There was no known family history of calcium disorders.Initial investigations showed corrected calcium (cCa) of 2.61 mmol/L (2.10 to 2.60), 25OHD of 33 nmol/L (75 to 250), and elevated PTH of 9.5 pmol/L (1.5 to7.0). Repeat biochemistry early in the second trimester showed ionized calcium (iCa) of 1.40 mmol/L (1.15 to1.28), phosphate of 0.86 mmol/L (0.80 to1.50), and magnesium of 0.73 mmol/L (0.70 to1.10); all other electrolytes were normal ; however, he was otherwise well and examined normally, so this was not treated. Repeat biochemistry of the son at 2 weeks of age found an elevated iCa of 1.81 mmol/L and cCa of 3.29 mmol/L (2.20 to 2.80), with a low PTH of 0.6 pmol/L (1.5 to 7.0). He was feeding well with normal growth. Given the magnitude of the hypercalcemia, he was admitted for i.v. hydration and changed to low calcium formula feeds. His calcium declined over subsequent days, but remained elevated; his PTH increased to 8.2 pmol/L  was made. Subsequent investigations showed mild hypercalcemia (cCa 2.49 mmol/L) in the patient's 2‐year‐old daughter; however, her partner and parents had normal calcium levels, and no other family members were tested. Genetic testing of the son revealed a missense mutation, NM_000388.3(CASR):c.2446A > G:p., in exon 7 of the calcium sensing receptor (CaSR), consistent with FHH type 1.FHH is a group of autosomal dominant disorders caused by dysfunction of the CaSR and its downstream signaling proteins, characterized by chronic, nonprogressive hypercalcemia, which is generally asymptomatic.11, which mediates intracellular signaling from the CaSR. FHH type 3 is caused by mutations of the adaptor‐related protein complex 2 subunit sigma 1 (AP2S1) gene, which encodes the adaptor‐related protein 2 complex that facilitates endocytosis of the CaSR.FHH type 1 is caused by an inactivating mutation in the gene encoding the CaSR and accounts for the majority of cases. FHH type 2 occurs as a result of an inactivating mutation in the G protein subunit alpha 11 (GNA11) gene encoding subunit αThe CaSR is expressed in the parathyroid glands and kidneys, as well as many other organs In pregnancy, PHPT has been associated with maternal pre‐eclampsia, pancreatitis, miscarriage, and hypercalcemic crisis, as well as fetal intrauterine growth retardation, parathyroid hypoplasia, and subsequent neonatal hypocalcemia.2D3. The maternal kidneys are the primary source of 1‐alpha hydroxylase, with a relatively minor contribution from placental 1‐alpha hydroxylase.2D3 and PTHrP act on the skeleton to increase bone turnover, and 1,25(OH)2D3 acts on the intestines to increase the absorption of calcium, which doubles from around the 12th week of gestation.Distinguishing between FHH and PHPT can be difficult, as 20% of those with FHH have elevated PTH levels, and 10% to 20% of those with PHPT have normal PTH levels.The CCCR, based on paired serum and 24‐hour urine levels, is considered the best test to differentiate FHH from PHPT. Findings presented at the Fourth International Workshop on PHPT proposed that a CCCR <0.01 is suggestive of FHH, CCCR >0.02 makes FHH unlikely, and a CCCR of 0.01 to 0.02 is uncertain, with genetic testing indicated in such cases.Unfortunately, the absorptive hypercalciuria of pregnancy makes interpretation of urine CCCR difficult. Of the three existing case reports of FHH in pregnancy, only two had urine CCCR reported, with values of 0.0199 and 0.019, both within the range considered uncertain in nonpregnant individuals Table .15, 16 OIn practice, diagnosing FHH in pregnancy requires a high level of suspicion. In women with newly diagnosed hypercalcemia in pregnancy, we recommend a multidisciplinary approach involving the endocrinology, obstetrics, and pediatrics departments. When differentiating between FHH and PHPT in pregnancy, the interpretation of biochemistry requires an understanding of calcium physiology in pregnancy, and physicians should be aware that the urine CCCR may be unreliable. If the decision is made to observe, clinical symptoms, calcium levels, and fetal US should be monitored closely, with biochemistry and urine CCCR performed postpartum and once lactation is completed. Confirmation of FHH with genetic testing, as in this case, provided additional reassurance in the postpartum period, and should be performed if available during the pregnancy.No authors have any disclosures to declare.


Lymph nodes metastasis (LNM) and distant metastasis (DM) are important prognostic factors in colorectal cancer (CRC) and determine the following treatment approaches. We aimed to find clinicopathological factors associated with LNM and DM, and analyze the prognosis of CRC patients with T1 stage.A total of 17 516 eligible patients with T1 CRC were retrospectively enrolled in the study based on the Surveillance, Epidemiology, and End Results (SEER) database during 2004‐2016. Logistic regression analysis was performed to identify risk factors for LNM and DM. Unadjusted and adjusted Cox proportional hazard models were used to identify prognostic factors for overall survival. We performed the cumulative incidence function (CIF) to further determine the prognostic role of LNM and DM in colorectal cancer‐specific death (CCSD). LNM, DM, and OS nomogram were constructed based on these models and evaluated by the C‐index and calibration plots for discrimination and accuracy, respectively. The clinical utility of the nomograms was measured by decision curve analyses (DCAs) and subgroups with different risk scores.P < .001), whereas N stage, carcinoembryonic antigen (CEA), and tumor size occupied the largest percentage in DM nomogram . OS nomogram was formulated to visually to predict 3‐, 5‐, and 10‐ year overall survivals for patients with T1 CRC. The calibration curves showed an effectively predictive accuracy of prediction nomograms, of which the C‐index were 0.666, 0.874, and 0.760 for good discrimination, respectively. DCAs and risk subgroups revealed the clinical effectiveness of these nomograms.Tumor grade, mucinous adenocarcinoma, and age accounted for the first three largest proportion among the LNM nomogram scores  database. Then, we construct the nomograms to predict the probability of lymph nodes metastasis in colorectal cancer with T1N0‐2M0 stage, and to estimate the probability of distant metastasis in T1 colorectal cancer. Therefore, we aimed to evaluate T1 CRCs by LNM, DM, and OS nomograms developed in the Surveillance, Epidemiology, and End Results (SEER) database.22.1http://seer.cancer.gov/seerstat/). SEER database currently collects and publishes cancer incidence and survival data covering approximately 34.6 percent of the US population (https://seer.cancer.gov/about/overview.html). Within the SEER database, we identified 17 516 adult patients who were diagnosed as suffering from only one primary, T1 colorectal cancer from January 2004 to December 2016. The flowchart of cases selection is shown in Figure The records of patients were downloaded from the SEER 18 registry database using SEER*Stat 8.3.6 software , left colon , and unknown . The histology variable was classified as “Adenocarcinoma,” “Mucinous adenocarcinoma,” or “Other/Not stated.”2.3Univariable and multivariable analyses were performed to identify independent risk factors and prognostic factors in T1 colorectal carcinoma in the SEER cohort. Binary logistic regression models2.4https://www.r‐proje ct.org/). The Chi squared tests or Fisher's exact tests were used to compare categorical variables. Survival variables were compared by Wilcoxon tests. The nomograms, C‐index, calibration curves, Kaplan‐Meier curves, cumulative incidence curves DCAs, and clinical impact curves were conducted using R 3.6.1 with relevant packages and functions, such as rms, rmda, survival, cmprsk, and stdca . A two‐tailed value of P < .05 was statistically significant.All the statistical analyses were calculated in R software  and the study cohort M . The median follow‐up time of the study cohort N and M were 54.0 months (interquartile range 24.0‐89.0 months) and 53.0 months (interquartile range 23.0‐89.0 months), respectively. LNM occurred in 2291 of 17 309 patients (13.24%) in the study cohort N and DM was present in 207 of 17 516 patients (1.18%) in the study cohort M. The characteristics of the patients are shown in Table 3.2P < .001). Patients with mucinous carcinoma had significantly higher risk of LNM than patients with adenocarcinoma . Compared with patients who had well‐differentiated CRC, those with poorly differentiated , and undifferentiated carcinoma  were at higher risk of LNM.Independent risk factors for LNM were determined by univariable and multivariable binary logistic regression analyses. These significant risk factors for LNM included year of diagnosis, age at diagnosis, race, gender, marital status, tumor location, histology, tumor size, number of regional nodes examined, grade, survival status, and CEA Table . In termTo more intuitively display the risk factors for LNM in CRCs with T1N0‐2M0 stage, a nomogram model was established Figure . Additio3.3P = .005). Compared with patients whose tumor size were 1‐9mm, those with over 30 mm of tumor size  were at higher risk of DM. Patients were especially at a higher risk of DM when they suffered from lymph nodes metastasis . The DM often occurred when the tumor grade was moderately differentiated , poorly differentiated , or undifferentiated . CEA‐negative patients were less prone to DM compared with the CEA‐positive .We further evaluated the association between DM and clinicopathological variables. Univariable and multivariable binary logistic regression analyses showed that age at diagnosis, tumor size, N classification, grade, and CEA were significant risk factors for patients with T1 CRC Table . PatientBased on the significant risk factors identified in the multivariable regression analysis, a nomogram was constructed to predict the probability of DM in patients with T1 colorectal carcinoma Figure . Each va3.4P = .001) and distant metastasis , P < .001) were significantly associated with overall survival  and DM , P < .001) were significantly connected with cancer‐specific death using Gray method  = 1.20, 95%CI = 1.08‐1.34), , P = .003.5P < .001), aged 65‐79 , and aged over 80  were at higher death risk. The death probability often increased when lymph nodes metastasis occurred . Patients with distant metastasis had significantly higher death risk than patients without distant metastasis .Using univariable and multivariable COX regression analyses, we found that age at diagnosis, race, gender, marital status, histology, tumor size, number of regional nodes examined, N classification, M classification, grade, and CEA were significant prognostic factors for overall survival in T1 colorectal cancer Table . CompareP < .001) and age over 80 years . Patients of black descent were more prone to CCSD compared with Caucasians . Unmarried status was a significant factor associated with CCSD . Compared with patients whose tumor size were 1‐9mm, those with over 30mm  were at higher risk of CCSD. Patients of higher N stages suffered from a higher risk of CCSD . The CCSD often occurred when the CRC tumor existed distant metastasis . Patients with lower CEA levels were at lower CCSD risk .To study the colorectal cancer‐specific death (CCSD) of T1 colorectal carcinoma, competing risk model was performed. These significant prognostic factors included age at diagnosis, race, marital status, tumor size, N classification, M classification, and CEA Table . In termThe significant factors identified by the COX regression analyses were used to develop the nomogram to predict the probability of overall survival in patients with T1 CRC. The plot of the OS nomogram is shown in Figure 3.6P < .001)   . Incidence of LNM among these subgroups was significantly different  Figure . Similar) Figure . Low‐, m) Figure .4Due to different clinical features, especially for lymph nodes and metastasis status, patients with submucosal invasive(T1) colorectal cancer have a different prognosis. The treatments are distinct in various situations. About 90% patients with T1 CRC are at stage I and only need to remove the lesions under endoscopy to have a similar prognosis of surgery, avoiding the adverse effects of surgery, and improving the quality of life. T1 CRCs with lymph nodes metastasis, accounting for 6.8%‐17.8%,Recently such studies are increasing, but there are still many shortcomings and limitations. First, former studiesTherefore, we divided the included population into N subgroup (T1M0NX CRC for LNM) and M subgroup (T1NXMX CRC for DM and OS). The incidence of LNM and DM, and OS of T1 CRC were analyzed, and the corresponding nomograms were constructed. Three nomograms were established and validated for predicting LNM, DM, and OS in patients with T1 CRC. LNM nomogram includes nine factors: age at diagnosis, race, gender, marital status, tumor location, histology, tumor size, grade, and CEA, whereas DM nomogram incorporates five factors, namely, age at diagnosis, tumor size, N classification, grade, and CEA. OS nomogram for predicting 3‐, 5‐, and 10‐year overall survivals involves 10 factors: age at diagnosis, race, gender, marital status, histology, tumor size, N classification, M classification, grade, and CEA.All the nomograms indicated good agreement between predictions and observations. C‐index of the LNM nomogram, DM nomogram and OS nomogram were calculated with values of 0.666, 0.874, and 0.760, respectively. These nomograms reveal good clinical utility in the proper threshold probability range. Furthermore, based on the interquartile scores from the nomograms, low‐, medium‐, and high‐risk groups were identified to plot stacked bar charts and Kaplan‐Meier survival curves, which intuitively indicated the discrimination ability of the nomograms.P < .001). Consistent with previous findings in T1 CRC,In this population‐based study, we found that tumor grade III‐IV, mucinous adenocarcinoma, and age 18‐ to 49‐year old accounted for the largest proportion among the LNM nomogram scores. As a significant factor, that degree of differentiation has been reported to be closely associated with LNM in T1 CRC.For the DM nomogram, the largest proportion in risk scores were N2 stage, positive CEA, and tumor size over 30mm. Not surprisingly, N classification was a significant predictor for the risk of LNM in T1 CRC. Of note, patients with the worse N stage are more prone to occur distant metastasis. Preoperative CEA has been found to be predictive of distant metastasis in T1 colorectal cancer after radical surgery.In term of the OS nomogram, age over 80 years old, M1 stage and N2 classification take up the largest percentage of the risk score for overall survival. It is not surprising that patients with distant metastasis and N2 classification have poor prognosis. Consistent with our researches, it was reported that older patients with early stage CRC are significantly related to shorter overall survival.In this database‐based study, we screen 17 516 eligible patients with a median follow‐up of 53 months from real‐world data. We analyzed the data by appropriate statistical methods and found these convincing conclusions. However, there were some limitations in our study. This was a population‐based retrospective analysis lacking important treatment information, such as surgical methods and chemoradiotherapy protocols. In addition, these data lacked the description of the distant metastasis site and the detection of key molecules of colorectal cancer, like KRAS and BRAF. Finally, these models were developed from the SEER database and were not verified by external data, being continuously modified based on the application in the future.In conclusion, based on independent risk factors from a large population database, we constructed three nomograms which can accurately predict the LNM, DM, and OS of T1 CRC patients at different stages. Moreover, our nomograms were demonstrated to have high accuracy and reliability by the validation of discrimination and calibration, as well as perform well in clinical utility. Therefore, they can help doctors to make clinical decisions for patients with T1 CRC, including diagnostic investigations, individual treatment, and follow‐up management strategies.KG conceived and designed the study, performed the study, analyzed the data, prepared figures and/or tables, and authored or reviewed drafts of the paper. YF and MS conceived and designed the study, performed the study, analyzed the data. LY, H S. W, and LS performed the study and authored or reviewed drafts of the paper. SR conceived and designed the study, performed the study, authored or reviewed drafts of the paper, and approved the final draft.


Bladder cancer (BCa) is the most common urinary malignancy. The standard surgical treatment for patients with muscle‐invasive BCa is cystectomy plus urinary diversion. Ileal conduit (IC) or orthotopic neobladder (ON), which have different indications, are the most commonly performed urinary diversions.We sampled 5480 BCa patients from the Surveillance, Epidemiology, and End Results (SEER) database from 2004 to 2015. Kaplan‐Meier method with the log‐rank test was used to assess cancer‐specific survival (CSS) and overall survival (OS). Univariate and multivariate Cox's proportional hazard model was conducted to evaluate the hazard ratio of cancer‐specific mortality and all‐cause mortality before and after propensity score matching (PSM).P < .05). After 1:1 PSM, 409 matched pairs were selected. Univariate and multivariate analysis showed that the ON group had better CSS and OS probabilities than the IC group in the overall cohort . However, subgroup analysis revealed that only patients with pathological T2 stage benefited from ON diversion after PSM in the context of CSS (P = .016) and OS (P <.001).We identified 5480 patients who received radical cystectomy (RC) plus IC (n = 5071) or ON (n = 409) with a median follow‐up period of 33 months . Patients in the ON group tended to be male and younger, with a higher percentage of married individuals, early pathological T stage, lymphadenectomy, and non‐radiotherapy (all Young, married, and male patients with early pathological T stage, especially T2 stage, were more suitable to receive RC plus ON surgery, which could improve their probability of survival. Young, married, and male patients with early pathological T stage, especially T2 stage, were more suitable to receive RC plus ON surgery, which might improve the survival probabilities. Patients were included if they met the following criteria (n = 7531): (a) year of diagnosis between 2004 and 2015; (b) using the International Classification of Diseases for Oncology 3/WHO site recode: C67.0‐67.9 (urinary bladder); (c) using the surgery codes C61 and C64 . The following exclusion criteria were also applied: (a) patients with a prior history of other malignancy (n = 2021); (b) unknown death status (n = 30) ” column in the SEER database, lymphadenectomy included 1 to 3 removed regional lymph nodes, 4 or more removed regional lymph nodes, and unknown number of removed regional lymph nodes. Surgical approach was categorized as RC plus ID or ON according to the “RX Summ‐Surg Prim Site (1998+).”2.3U tests were used to determine the significant differences between categorical and continuous variables and surgical approach. The CSS and OS curves were created by the Kaplan‐Meier method with the log‐rank test. To estimate the impact of IC or ON on prognosis, univariate and multivariate cox regression analysis were carried out, and the results were presented as hazards ratios (HR) and 95% confidence intervals (95% CI). A fewer number of patients were treated by ON than by IC diversion (409 vs 5071). Moreover, there were also imbalances in baseline characteristics, such as age, gender, marital status, T stage, lymphadenectomy and radiotherapy; therefore, PSM was used for more objective comparison. For PSM, patients receiving IC or ON were matched 1:1 with a caliper set at 0. The matching algorithm was nearest neighbor matching, and the estimation algorithm was logistic regression. The PSM was described and adjusted for different variables, including age, gender, sex, marital status, TNM stages, lymphadenectomy, radiotherapy, and chemotherapy. All reported P‐values were two‐tailed and a P < .050 was considered significant.SPSS version 23.0  was used for all statistical analyses and propensity score matching (PSM). Chi‐square test and Mann‐Whitney 33.1P > .050). However, patients in the ON group tended to be younger  and male , with a higher percentage of married individuals , early T stage , lymphadenectomy , and nonradiotherapy  before PSM. After 1:1 PSM adjusting for age, gender, sex, marital status, TNM stages, lymphadenectomy, radiotherapy, and chemotherapy, there were no significant differences between the IC and ON groups  or ON (n = 409) were included (Table S1). There were no significant differences in terms of race, primary tumor site, histology, N stage, M stage, grade, and chemotherapy between the two different urinary diversions . When stratified by T stages, patients with each T stage in the ON group also showed longer CSS and OS than those in the IC group , with a total of 3034 (59.8%) and 170 (41.6%) deaths in the IC and ON groups respectively. Among all of the deaths, 2178 (43%) and 127 (31.1%) patients died specifically from BCa in the IC and ON groups respectively. The 5‐year CSS and OS probabilities for the IC group and the ON group were 55%, 72.9%, and 45.1%, 66.0% respectively .Before PSM, univariate analyses revealed that age, marital status, primary tumor site, histology, TNM stages, grade, lymphadenectomy, urinary diversion, and radiotherapy were all associated with cancer‐specific mortality (CSM) and all‐cause mortality (ACM) (Tables S2 and S3). After controlling for other factors, urinary diversion was an independent risk factor for CSM and ACM in the multivariate analyses [ON vs IC, HR: 0.692, 95% CI: 0.576‐0.831, 3.3P = .014; HR: 0.671, 95% CI: 0.548‐0.823, P < .001, respectively) (Table P = .016) and OS (P < .001)  Figure .4RC and urinary diversion are two important steps of the operation for BCa patients. There are different choices of urinary diversions after RC, including uretero‐cutaneostomy, IC, continent cutaneous urinary diversion, ureterocolonic diversion, and ON according to the patients’ preference, performance status, life expectancy, and oncological control. Among these urinary diversions, IC and ON are the most commonly utilized ones in the clinical practice.Consistent with a previous study,P = .04 and P = .001, respectively). However, in the subgroup analysis this statistical significance disappeared when further stratified into organ confined and nonorgan confined diseases.P = .016) and OS (P < .001) benefit from ON diversion.There have been many studies on the impact of ON and IC diversions on the HRQoL of BCa patients, but the influence of ON diversion on cancer control has not been adequately studied. One systemic review, which included 29 studies, found no significant differences in QoL after radical cystectomy between continent and incontinent urinary diversion.This study has some limitations as well as some strengths. Among limitations, first, the study was of a retrospective nature, which means that the significant survival outcomes could be the effect of strict selection criteria for ON diversion. As a consequence, a randomized controlled trial to compare the long‐term oncological outcomes between ON diversion and IC diversion is important and urgent. Second, the documentation on lymphadenectomy was not very precise about the scope of lymph node dissection, which may have had an impact on survival outcomes.5As a continent diversion, ON diversion showed some advantages in terms of survival outcomes compared to IC diversion. Young, married, and male patients with early pathological T stage, especially T2 stage, were more suitable to receive RC plus ON surgery, which could improve their probability of survival.The authors declare that they have no competing interests.Xiaohong Su: Data curation, formal analysis, investigation, methodology, project administration, writing‐original draft. Kaihui Wu, Shuo wang and Wei Su: Data curation, formal analysis, investigation. Bingkun Li and Chuanyin Li: Data curation, formal analysis, investigation. Xiangming Mao: Conceptualization, project administration, supervision, and writing‐review and editing.TableS1‐S3Click here for additional data file.


Objective: To investigate the value of using 18F-FDG PET/CT in combination with serum lactate dehydrogenase (LDH) for prognostic evaluation of newly diagnosed small cell lung cancer (SCLC).Methods: We reviewed 118 patients with pathologically proven SCLC who underwent 18F-FDG PET/CT imaging evaluation in our hospital. Among these patients, 64 patients had extensive disease (ED) and 54 patients had limited disease (LD). The maximum standardized uptake value (SUVmax) of primary tumor was measured. A Cox proportional hazards model was used to evaluate age, sex, performance status, serum LDH, tumor stage and SUVmax on the prediction of overall survival (OS) and median survival time (MST) of patients. Subgroup analysis was performed based on the SUVmax in combination with serum LDH.Results: According to the Receiver Operating Characteristic (ROC) curve, the optimal cut-off value of SUVmax was 10.95. The AUC was 0.535 (95% CI: 0.407–0.663). The patients were divided into four groups according to the SUVmax (higher or lower than 10.95) and LDH (higher or lower than 245 U/L). The univariate and multivariate analyses showed that curative thoracic radiotherapy, Prophylactic Cranial Irradiation (PCI) and the combination of primary tumor SUVmax ≤ 10.95 and LDH ≤ 245 U/L were prognostic factors of OS in patients with all patients (p < 0.05). Smoking status, PCI, the combination of primary tumor SUVmax ≤ 10.95 and LDH ≤ 245 U/L were prognostic factors of OS in patients with LD (p < 0.05). N stage and PCI were significant predictors in both of univariate and multivariate analysis of OS for ED SCLC (p < 0.05). Among all patients, 27 had low SUVmax and normal LDH, and their MST was 36 months (95% CI: 12.98–59.02). Ninety-one patients had high SUVmax and/or high LDH, and their MST was 20 months (95% CI: 15.47–24.53). The difference between these two groups was significant (p = 0.045). In patients with LD, 16 patients had low SUVmax and normal LDH, and their MST was 72 months (95% CI: 26.00–118.0). Thirty-eight patients had high SUVmax and/or high LDH, and their MST was 27 months (95% CI: 20.80–33.21). The difference between these two groups was significant (p = 0.012). In patients with ED SCLC, 10 patients had low SUVmax and normal LDH, with an MST of 18 months . The difference of MST between these two groups was not statistically significant (p = 0.686).Conclusion:18F-FDG PET/CT in combination with serum LDH were prognostic factors of overall survival in patients with SCLC. The prognosis of patients with LD SCLC who had low SUVmax of primary tumor and normal LDH was better than those with high SUVmax and/or high LDH. Small cell lung cancer (SCLC) is the most common neuroendocrine tumor of the lung. It accounts for 10–15% of lung cancers . SCLC haThe main prognostic factors for SCLC include tumor staging, the patient’s physical condition, such as physical status, immune function status, etc. and tumor biological characteristics. The principal prognostic factors of SCLC are staging, physical status, weight loss, tumor burden, and elevated serum biomarkers. Staging is the principal consideration for prognosis and treatment strategies. Patients with SCLC are conventionally staged according to a two-stage system as limited disease (LD) and extensive disease (ED). This system was developed by the Veterans Administration Lung Cancer Study Group (VALG) and has been widely used for a long time. LD is defined as disease confined to one hemithorax that can be encompassed in a tolerable radiation field, whereas ED is defined as disease extending outside the thorax or the existence of malignant pleural effusion. The TNM staging is applied in SCLC . CurrentSerum lactate dehydrogenase (LDH) is used as an important prognostic factor in lymphoma, multiple myeloma, lung cancer and nasopharyngeal carcinoma. Serum LDH is elevated in 50–60% of patients with SCLC at diagnosis and is also critical in the prognosis of SCLC .18F-FDG PET/CT is increasingly used for clinical diagnosis and prognosis  was calculated using the following formula: maximum pixel value with the decay-corrected region-of-interest activity (MBq/kg)/[injected dose (MBq)/body weight (kg)]. The PET images were evaluated by two experienced physicians of nuclear medicine. SUVmax of the primary tumor was automatically measured on the workstation.Images were acquired with two integrated PET/CT devices . PET was performed to cover the identical axial field of view after CT scan. PET images were iteratively reconstructed with CT data for correcting attenuation. For quantitative analysis, irregular regions of interest were placed over the most intense area of The general characteristics of the patients, including KPS score, smoking status, body weight, serum LDH, overall survival time (OS), and median survival time (MST) were recorded.p values of less than 0.05 were considered statistically significant. Survival curves were calculated by the method of Kaplan and Meier. The log-rank method was used to test the statistical significance of the differences between survival curves and to perform comparisons adjusted for other prognostic factors. p values for difference between proportions were calculated with Fisher’s exact test (two-tailed). Correlations between LDH levels  and specific metastatic sites were assessed with the product moment correlation coefficient.All statistical analyses were performed using SPSS . Statistical difference between the groups were analyzed using one-way ANOVA and t tests where appropriate. The clinical characteristics of the 118 patients including age, gender, performance status (KPS), tumor stage, body weight, smoking status and treatment management are presented in max in the primary tumors of all patients was 11.85 (6.0–32.8). The ability of SUVmax in the primary tumors to predict prognosis was depicted by the ROC curve. The AUC of SUVmax was 0.535 (95% CI: 0.407–0.663). The point which showed the maximal sum of sensitivity and specificity was determined to be the cut-off point. As a result, the optimal cut-off value of SUVmax was 10.95. max.The median SUVp = 0.003), with patients with LD had lower LDH level.The median serum LDH in all patients in this study was 226.6  U/L. The median serum LDH in patients with LD was 222.8 (118.5–530.3) U/L, with elevated levels in 19 patients  and normal levels in 35 patients . The median serum LDH in patients with ED SCLC was 241.2  U/L, with elevated levels in 31 patients  and normal levels in 33 patients . Serum LDH was significantly different in patients with LD and ED , PCI  and the combination of primary tumor SUVmax ≤ 10.95 and LDH ≤ 245 U/L   .TABLE 2Umax ≤ 10.95 and LDH ≤ 245 U/L were significant predictors, whereas T stage, N stage, curative thoracic radiotherapy, SUVmax, and serum LDH alone, were not significant prognostic factors (p = 0.013), PCI , the combination of primary tumor SUVmax ≤ 10.95 and LDH ≤ 245 U/L   .TABLE 4Umax, serum LDH alone, and the combination of primary tumor SUVmax ≤ 10.95 and LDH ≤ 245 U/L were not significant prognostic factors (p = 0.004), PCI   .TABLE 6Umax was 10.95. The AUC was 0.535 (95% CI: 0.407–0.663). The patients were divided into four groups according to SUVmax and serum LDH. Group A for primary tumor SUVmax > 10.95 and LDH > 245 U/L, Group B for primary tumor SUVmax > 10.95 and LDH ≤ 245 U/L, Group C for primary tumor SUVmax ≤ 10.95 and LDH > 245 U/L, and Group D for primary tumor SUVmax ≤ 10.95 and LDH ≤ 245 U/L.Base on the ROC curve, the optimal cut-off value of SUVp = 0.057). Among the patients with ED, there were 22 patients in group A, with an MST of 21 months (95% CI: 11.16–30.84); 22 patients in group B, with an MST of 14 months (95% CI: 9.40–18.60); nine patients in group C, with an MST of 14 months (95% CI: 0–33.00); and 11 patients in group D, with an MST of 11 months (95% CI: 8.68–13.32). There was no significant difference of MST among the four groups (p = 0.742).Among the patients with LD, there were 12 patients in group A, with an MST of 28 months (95% CI: 12.92–43.08); 19 patients in group B, with an MST of 28 months (95% CI: 19.33–36.67); seven patients in group C, with an MST of 27 months (95% CI: 11.79–42.21); and 16 patients in group D, with an MST of 72 months (95% CI: 26.00–118.0). There was no significant difference of MST among the different groups (max ≤ 10.95 and LDH ≤ 245 U/L), with an MST of 36 months (95% CI: 12.98–59.02). There were 91 patients with SUVmax > 10.95 and/or LDH > 245 U/L (group A + B + C), and their MST was 20 months (95% CI: 15.47–24.53). The difference of MST between these two groups in the study was statistically significant (p = 0.045)  .FIGURE 3max ≤ 10.95 and LDH ≤ 245 U/L), with an MST of 72 months (95% CI: 26.00–118.0). There were 38 patients with SUVmax > 10.95 and/or LDH > 245 U/L (group A + B + C), and their MST was 27 months (95% CI: 20.80–33.21). The difference of MST between these two groups was statistically significant (p = 0.012)  .FIGURE 4max ≤ 10.95 and LDH ≤ 245 U/L), with an MST of 18 months (95% CI: 13.69–22.32). There were 54 patients with SUVmax > 10.95 and/or LDH > 245 U/L (group A + B + C), and their MST was 12 months (95% CI: 10.61–13.39). The difference of MST between these two groups was not statistically significant (p = 0.686)  .FIGURE 518F-FDG uptake in tumors. SUV can reflect the metabolic information of tumors. Previous studies indicated that the SUVmax of primary tumors is helpful for predicting prognosis of patients with cancers  in SCLC. Compared with stage IV patients, stage I-III patients had lower SUVmax in the primary tumors. In this study, SUVmax alone did not distinguish difference of OS or PFS. However, among patients with stage IV and receiving chemotherapy, patients with higher SUVmax had worse prognosis than those with lower SUVmax. The relationship between SUVmax and prognosis appears to be more complicated in SCLC than in NSCLC.The application of rmation.  analyzedmax was associated with poor survival with significance. 18F-FDG PET could be important for tumor staging and follow-up, because there was a significant negative correlation between SUVmax and survival. The overall survival in PET-negative patients was significantly better than that in PET-positive cases. . After adjusting for age, sex, performance status, tumor stage, and treatment modality, patients with higher SUVmax (>11) were characterized by a significantly shorter median OS (p < 0.001) and PFS (p = 0.002) compared with patients with lower SUVmax. The high SUVmax was an independent prognostic factor for survival in LD-SCLC patients and might be a possible imaging biomarker for risk stratification in LD-SCLC.In a study by Pandit et al. , a high e cases.  also fouh et al.  found thn et al.  found thmax, SUVmean, MTV, and total lesion glycolysis of the primary tumor were not significantly associated with OS, LRF, and DFS in univariate analysis. MTV was significantly associated with DFS (p = 0.024) in univariate but not multivariate analysis in 120 patients with LD who received platinum-based chemotherapy and a median radiation dose of 45 Gy. Kim’s study . The prognosis of all SCLC patients with primary tumor SUVmax ≤ 10.95 and a normal level of serum LDH was better than that of patients with primary tumor SUVmax > 10.95 and/or serum LDH, with longer overall survival time . These results indicate that the prognosis is better in the cases of low SUVmax and LDH, i.e., when both the input and output of glycolysis are low.This retrospective study shows that SUVThere are some limitations in this study. First, it is a retrospective study with a small sample size. Second, two different PET/CT scanners were used in this study. However, our regular quality control ensured scanning consistency. In addition, volume metabolic parameters (such as MTV and TLG) were not included in the study. Because nearly half of the cases in this study were ED SCLC, it is difficult to achieve a Volume Of Interest (VOI) including all lesions of the whole body excluding physiological or benign avid lesions automatically. Moreover, manual drawing is more likely to be influenced by the operator. Therefore, volume metabolic parameters were not used in this study. Further studies with large sample size and multiple centers and prospective studies are needed.18F-FDG PET/CT and serum LDH could be a prognostic factor of overall survival of patients with SCLC. The prognosis of patients with limited disease of SCLC who have low SUVmax of the primary tumor and normal serum LDH was better than those with high SUVmax and/or high serum LDH.Combination of The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by IRB Sun Yat-Sen University Cancer Center. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.XL: Conceptualization, methodology, software, data curation, visualization, investigation, writing-original draft preparation, and writing-editing. ZX, YH, and XZ: Data curation and visualization. WF: Conceptualization, supervision, validation, and writing-reviewing.This work was supported by the Natural Science Foundation of Guangdong Province, China, No. 2018A030310239.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.


Veratrum nigrum L. is a well-known traditional Chinese medicine with a lot of pharmacological activities including antihypertensive, anticancer, and antifungal effects. In the current experiment, a rapid and sensitive UPLC-MS/MS method that takes only 7 min run time has been established and validated for simultaneous determination of eight bioactive compounds including cyclopamine, jervine, veratramine, polydatin, quercetin, apigenin, resveratrol, and veratrosine in rat plasma. The chromatographic separation of analytes and internal standard was performed on a Phenyl-Hexyl column  with the mobile phase consisting of water (0.1% formic acid) and acetonitrile at a flow rate of 0.3 mL/min. An electrospray ionization (ESI) source was used to detect the samples in both positive and negative ion modes. The intra- and interday precisions of the compounds were less than 9.5% and the accuracy ranged from -10.8% to 10.4%. The extraction recoveries of the compounds were in the range of 85.1 ± 1.5% to 102.6 ± 8.0%, and the matrix effect ranged from 91.2 ± 4.5% to 113.8 ± 1.5%. According to the results of the stability test, the eight compounds have good stability under various conditions and the relative standard deviation (RSD) less than 13.2%. The pharmacokinetic parameters of the eight compounds in rat plasma after oral administration of Veratrum nigrum L. extract were successfully determined by the established UPLC-MS/MS method. Veratrum nigrum L., known as “LiLu”, is a famous traditional Chinese medicine that consists of the dried rhizomes and roots of Veratrum nigrum L. [Veratrum nigrum L. have a lot of pharmacological activities such as antithrombotic activity, anticancer, β2-adrenoceptor agonist, antihypertensive, and antifungal effects. It also prevents obesity and alleviates allergy and contact dermatitis etc. [igrum L. . Despiteigrum L. , 3. It higrum L. , while aigrum L. –7. The migrum L. , 9. Pharigrum L. . More antis etc. –18.Veratrum nigrum L. is mainly focused on the components of higher content such as jervine, resveratrol, and veratramine [Veratrum nigrum L. extract.Since traditional Chinese medicine has become more and more popular throughout the world in view of its curative effects and efficacy, it is indispensable to figure out the pharmacokinetic changes of its active ingredients in the body. As we all know, the efficacy of traditional Chinese medicine is generally based on the synergy of various ingredients. Therefore, it is essential to simultaneously determine the main bioactive components in biological samples in regard to multicomponent pharmacokinetic studies. Up to now, the reported pharmacokinetic research of the components in atramine –24. TherVeratrum nigrum L. extract. This study would provide some references for further pharmacology and pharmacodynamics studies of Veratrum nigrum L. extract.UPLC-MS/MS has the characteristics of high sensitivity and accuracy, and it is well known that UPLC-MS/MS plays a significant role in the quantification of the main components of medicinal products for pharmacokinetic studies –27. In tMethanol and acetonitrile (chromatographic purity) were purchased from Merck KGaA . Formic acid (chromatographic purity) was obtained from ROE . A Milli-Q water purification system  was used for preparing the ultrapure water. Cyclopamine, jervine, veratramine, polydatin, quercetin, apigenin, resveratrol, and hupehenine  were purchased from Chengdu Must Bio-Technology Co., Ltd . Veratrosine was purchased from Chengdu Chroma-Biotechnology Co., Ltd. . The chemical structures of the analytes are displayed in ® CSHTM Phenyl-Hexyl  column, and the temperature was maintained at 20°C. The flow rate and injection volume were 0.3 mL/min and 5 μL, respectively. Quantitative parameters are recorded in An Agilent 1290 ultraperformance liquid chromatography coupled to an Agilent 6470 series triple quadrupole mass spectrometer with an Agilent Jet Stream (AJS) ESI source was used for analyzing all samples of this study. 0.1% formic acid in water (A) and acetonitrile (B) were used as mobile phases with the following gradient elution method: 0-2 min, 15%-25% B; 2-4 min, 25%-50% B; 4-6 min, 50%-90% B; and 6-7 min, 90%-95% B. The separation was performed on a Waters ACQUITY UPLCVeratrum nigrum L. extract, 200 g dried rhizome of Veratrum nigrum L. plant was accurately weighed and extracted by refluxing with 70% (v/v) ethanol (ratio of 1 g: 15 mL); repeat three times for two hours each time. After that, the obtained extracts were filtered and mixed, and then the solvent was evaporated under reduced pressure . Finally, the dried Veratrum nigrum L. extracts were pulverized into fine powders and stored in a desiccator until analysis. The contents of veratrosine, jervine, cyclopamine, veratramine, polydatin, quercetin, apigenin, and resveratrol in Veratrum nigrum L. extract powder were 9.4, 7.8, 5.4, 89.7, 74.3, 0.5, 0.6, and 2.7 μg/g, respectively.To prepare the μL of IS into 100 μL blank rat plasma, resulting in concentrations: 2.4, 4.8, 12, 24, 60, 120, 300, and 600 ng/mL for veratrosine; 4, 8, 20, 40, 100, 200, 500, and 1000 ng/mL for jervine; 0.4, 0.8, 2, 4, 10, 20, 50, and 100 ng/mL for cyclopamine, polydatin, quercetin, and apigenin; 1.6, 3.2, 8, 16, 40, 80, 200, and 400 ng/mL for veratramine; and 0.8, 1.6, 4, 8, 20, 40, 100, and 200 ng/mL for resveratrol. Quality control (QC) samples at three levels  were prepared in the same manner. All the samples were kept at 4°C until analysis.Cyclopamine, jervine, veratramine, polydatin, quercetin, apigenin, resveratrol, veratrosine, and hupehenine  were separately weighed and dissolved with methanol as standard stock solutions (1 mg/mL). The calibration solutions were prepared by adding appropriate volumes of mixture working solution and 20 μL plasma sample was incorporated with 20 μL methanol and 20 μL of IS (20 ng/mL), and vortexed for 30 seconds. 1 mL ethyl acetate was then added to the sample and vortex-mixed for 3 min. The samples were centrifuged , and the supernatant was carefully collected to a clean tube and evaporated to dryness under a nitrogen stream. The obtained residue was reconstituted in 100 μL methanol and centrifugation at 12000 × g for 10 min. 5 μL of the supernatant was injected for UPLC-MS/MS measurement.100 Veratrum nigrum L. extract to evaluate any potentially endogenous plasma matrix components that could result in chromatographic interference.The specificity was carried out by comparing chromatograms of blank rat plasma samples, with blank plasma spiked with analytes and postdosing plasma samples gained from the rats given oral administration of x2 was used to describe the regression relationship. The limit of quantification (LOQ) was used to assess sensitivity of the method.We constructed a calibration curve by analyzing the mixed standard solution and IS calibration samples to assess the linearity. The calibration curve equation was calculated using the peak area ratios of analytes against the internal standard as the standard concentration. A linear regression equation with weight coefficient 1/Intraday and interday accuracy and precision were assessed by preparing and analyzing six duplicate quality control (QC) samples with three certain concentration levels  within 1 day or on 3 consecutive days. Accuracy was determined by the relative error (RE) value, while the intra- and interday precision was defined as the relative standard deviation (RSD).The matrix effect and extraction recovery were determined with six replicates of QC samples. The extraction recovery was investigated by comparing the peak areas of the eight compounds using QC samples of three concentrations and the peak areas of the postextraction spiked samples. Matrix effect was investigated by comparing the peak areas of postextraction spiked samples with the peak areas of the corresponding working solutions on three QC levels to assess the matrix effect of the samples.The QC samples of three concentration levels were placed under various conditions including being stored at autosampler for 12 h, under three freeze-thaw cycles, at room temperature for 6 h and stored at -80°C for 7 days to study the stability of the samples.Veratrum nigrum L. extract to a concentration of 0.2 g/mL suspension. The rats were orally administered at a dose of 2 g/kg with the suspension, and approximately 220 μL blood samples was obtained from the ophthalmic venous plexus into centrifuge tubes at predose, and 0.03, 0.08, 0.17, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 36, 48, 60, 72, 84, 96, 108, and 120 h postadministration in rats. After centrifugation at 4000 × g for 10 min at 4°C, the collected plasma was transferred into clean tubes and frozen at -80°C until next analysis. Pharmacokinetic parameters were calculated using “Drug and Statistics 3.0” (DAS 3.0) . The animal protocol was approved by the Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine (TCM-LAEC20190060).Six male Sprague-Dawley rats  were used for this experiment. The rats had free access to water and fasted for 12 h prior to the study. 0.5% CMC-Na aqueous solution was used to dissolve the ® UPLC® C18 column  and ACQUITY UPLC® CSHTM Phenyl-Hexyl column  on chromatographic peaks and elution time. The results showed that the ACQUITY UPLC® CSHTM Phenyl-Hexyl column  had better separation and sensitivity for the eight compounds, and the analysis time was shorter. Meanwhile, the matrix effect and extraction recovery results were more in line with the experimental requirements. The CORTECS® UPLC® C18 column  is prone to compound residues during the analysis, which could affect the accuracy of the analysis. Therefore, the former was chosen as the analytical column in the study. The results showed that 0.1% formic acid/water-acetonitrile was the best choice of mobile phase for this study, when compared to methanol-0.1% formic acid/water or methanol-water as it resulted in satisfactory peak shapes of all eight compounds.To achieve better separation of the eight compounds well, UPLC conditions consisting of the column type as well as the proportion and composition of the mobile phase were optimized since they make a vast difference in achieving the goals. This experiment investigated the influence of chromatographic columns CORTECSVeratrum nigrum L. extract. It has a good response in positive ion mode and is a nonendogenous substance. The extraction recovery of hupehenine is stable and the peak time is suitable. Mass spectrometry parameters are optimized in the mass spectrometer to enable the compounds to have a better response. The optimized MS parameters were set according to the following: drying gas flow rate at 11 L/min, capillary voltage at 4000 V, temperature at 320°C, and nebulizer at 15 psi.The selected internal standard hupehenine is a form of steroidal alkaloids, which is the same type of compound as the main compounds of Preparation of sample with an appropriate method is a critical step in pharmacokinetic analysis. We tested liquid-liquid extraction (LLE) and protein precipitation (PPT) methods and compared the effects of these two methods on sample pretreatment. The results demonstrated that of the two methods, LLE with ethyl acetate has the characteristics of better recovery and efficiency of the eight compounds compared with PPT extraction with acetonitrile and methanol. Considering these factors, ethyl acetate extraction method was selected to be the sample pretreatment method of this study.Typical chromatograms of blank plasma (a), blank plasma incorporated with eight analytes and IS (b), and plasma samples after dosing (c) are illustrated in The accuracy and intraday and interday precision were assessed according to the RE and RSD values. The mean recovery rates of the eight compounds were ranged from 85.1 ± 1.5% to 102.6 ± 8.0%, and the mean matrix effect ratio for all analytes was ranged from 91.2 ± 4.5% to 113.8 ± 1.5% . The resThis experiment investigated different stabilities of the QC samples at three concentration levels through the following four conditions: stored at autosampler for 12 h after preparation (postpreparation stability), at room temperature for 6 h (short-term stability), at three freeze-thaw cycles (free-thaw stability), and at -80°C for 7 days (long-term stability). The RSD value of the replicate QC samples was less than 13.2% as listed in Veratrum nigrum L. extract. The calculated pharmacokinetic parameters are listed in The validated analytical method was applied to study the plasma samples obtained from the rats which were intragastric administrations of Cmax, the maximum plasma concentration and time to reach Cmax of each compound (Tmax). The T1/2 was the elimination half-life and it reflects the elimination rate of the drug in the body. The area under the plasma concentration-time curve AUCt). The mean residence time (MRT) of these ingredients was also investigated.Some important pharmacokinetic parameters were calculated in this study including Veratrum nigrum L. extract, cyclopamine, quercetin, and apigenin in rat plasma were only detected at the first few plasma sampling points, which made it difficult to form a complete pharmacokinetic curve; hence, we excluded the three analytes in the following results.In the study, after oral administration of Cmax of jervine at 515.0 ± 242.4 ng/mL showed that the blood concentration of jervine was higher than the other compounds and its absorption was more complete. The Tmax of veratrosine, polydatin, and resveratrol were 0.08 ± 0.00 h, 0.26 ± 0.29 h, and 0.18 ± 0.17 h, respectively, showing that the absorption of these three compounds was rapid. The Tmax of jervine and veratramine were both longer than 32 h, demonstrating that these two components were slowly absorbed in the rats, which is consistent with the chronic toxicity of Veratrum nigrum L. reported in literatures and is also consistent with the death time of rats in the preliminary experiments [As shown in eriments .t)(0 −  and the AUC(0 − ∞) of all five compounds were close by indicating that the monitoring time of this study was appropriate. The AUCt) after oral administration of


Plant materials were extracted with 95% ethanol or distilled water and then concentrated and dried. Statistical analysis of data was done using one-way ANOVA at p value of 0.05. The ethanolic extracts of Garcinia mangostana L., Glycyrrhiza glabra L., and Nigella sativa L. could inhibit the production of superoxide anion with the IC50 values of 13.97 ± 0.38, 28.62 ± 1.91, and 71.54 ± 3.22 μg/ml and nitric oxide with the IC50 values of 23.97 ± 0.91, 46.35 ± 0.43, and 78.48 ± 4.46 μg/ml, respectively. These extracts could promote cell proliferation and accelerate wound recovery at the rate of 2.02 ± 0.03, 2.12 ± 0.03, and 2.65 ± 0.05% per hour, respectively. Three established markers from these three plants were selected according to the selection criteria. Alpha-mangostin, glycyrrhizin, and thymoquinone were found to be the active markers for wound closure activities. The ethanolic extracts of G. mangostana, G. glabra, and N. sativa could scavenge superoxide anion and inhibit the production of nitric oxide; therefore these extracts could assist in surpassing the inflammatory phase and protected the cells surrounding the wound area. Most importantly, these extracts also increased the proliferation and accelerated wound closure, indicating that these plant extracts could be promoting wound healing processes and support the use of TTM.Skin ensures that a constant internal environment can be maintained in an ever-changing external environment. When a wound occurs on the skin, the inflammatory and proliferative phases are initiated in response to injury. Thai traditional medicine (TTM), using medicinal plants and ancient knowledge, has been used to treat wounds. Eight Thai medicinal plants, most commonly used to treat wounds, were evaluated for their  Additionally, the primary reactive oxygen species (ROS) or the superoxide anion radical (O2−) are formed from molecular oxygen (O2) during cellular metabolism in the mitochondrial electron transport chain (METC) and others [The skin forms a barrier that protects the body from pathogens, maintains moisture, and preserves the temperature of the body. A wound is a type of injury on the skin which causes physical, chemical, electrical, or thermal damage. When a wound occurs, the healing process is immediately initiated in response to the injury to regenerate the tissue. Wound healing consists of 3 phases, that is, inflammation, proliferation, and remodelling, a complex process associated with multiple cells, and molecules . When a d others . MyofibrNigella sativa L.) appeared in most of the recipes in Mukkharoka scripture for the treatment of wound and inflammation in oral cavity. Previous studies found that N. sativa and its major compound thymoquinone have been shown for their therapeutic benefits such as antibacterial, antioxidant, and anti-inflammatory activities [Glycyrrhiza glabra L.) was found as the main ingredients of Am-ma-ruek-ka-va-tee recipe while dry fruits of Indian nightshade (Solanum indicum L.) and dry fruits of Thai nightshade (Solanum trilobatum L.) were found as the main ingredients of Phra-sa-ma-vaeng recipe in the National List of Essential Medicines of Thailand. These medicines were used to treat sore throat, cough with sputum, and ailments in the oral cavity. The major compounds of these extracts had been proved to be glycyrrhizin (Glycyrrhiza glabra) and steroidal glycoside (solanidine and solasodine in Solanum spp.) respectively, with reported activities such as antibacterial, anti-inflammatory, antioxidant, and anticancer activities [Phyllanthus emblica L.) fruits were found as the main ingredient in Tri-pha-la recipe; TTM used this recipe for symptoms in the upper respiratory tract and to balance the internal body. Gallic acid, protocatechuic acid, and tannins were found in its extract with bioactivities such as anti-inflammatory, antioxidant, and analgesic activities [Garcinia mangostana L.) had traditional use such as wound cleansing liquid on the skin and feet. The major compound of G. mangostana, α-mangostin, had various proven bioactivities such as antioxidant, antibacterial, antifungal, anticancer, and anti-inflammatory activities [Mimusops elengi L. was used for toothache; it consisted of many compounds such as steroids, flavonoid, terpenoids, glycoside, saponin, and tannin with bioactivities such as antioxidant and anti-inflammatory activities [Syzygium aromaticum L.) has been used in throat lozenge for pain relief and its major active compound is eugenol; therefore S. aromaticum extract many bioactivities such as antioxidant and antihyperglycemic activities [Thai traditional medicine (TTM) is based on Thai ancient knowledge that has been compiled into various scriptures through experiences and observations by traditional doctors and passed onto the next generations. The list of medicinal plants most commonly used to treat wounds was selected from the Mukkharoka scripture, the National List of Essential Medicines of Thailand, and traditional usages. From the above criteria, the seed of black cumin , bullet wood (M. elengi L.), black cumin (N. sativa L.), Indian gooseberry (P. emblica L.), Indian nightshade (S. indicum L.), Thai nightshade (S. trilobatum L.), clove bud (S. aromaticum L.), and mangosteen (G. mangostana L.) were purchased from herbal shops in Thailand. The voucher specimens were deposited at the Herbarium of the Southern Center of Thai medicinal plants at the Faculty of Pharmaceutical Science, Prince of Songkla University, Songkhla, Thailand. The identities of the herbal materials were done by comparison with the authentic herbarium specimen  and 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) were from Sigma, USA. RPMI medium powder 1640 (RPMI 1640), DMEM medium powder, fetal bovine serum (FBS), penicillin-streptomycin (P/S), trypsin-EDTA, and trypan blue were purchased from Gibco, USA. Phosphate buffer saline (PBS) was from Amresco (USA) and other chemicals were from Sigma and Merck. α-Mangostin, asiaticoside and thymoquinone were purchased from Sigma, USA. Glycyrrhizin was purchased from Tokyo chemical industry (TCI), Singapore.Nitro blue tetrazolium chloride (NBT) and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma, Germany. Hanks' balanced salt solution (HBSS) was purchased from Gibco, USA. Lipopolysaccharide  for 3 days and then filtered through Whatman No. 1 filter paper. The marc was remacerated twice. The filtrates were combined and evaporated to dryness. The ethanolic crude extracts from 8 plants were stored at −20°C until bioactivity testing. Aqueous extracts from 8 plant materials were obtained by decoction with distilled water for 15 min and then filtered through Whatman No. 1 filter paper. The decoction process was repeated twice with the marc. Filtrates were combined, lyophilized, and kept at −20°C until use.μm filter paper. All 16 extracts were stored at −20°C until use.Ethanolic extracts were dissolved in sterile DMSO while aqueous extracts were dissolved in sterile distilled water and filtered through 0.22 2. The cells were harvested with 0.25% trypsin-EDTA by centrifugation at 1,500 rpm for 5 min and then diluted with fresh medium into a suspension at the concentration of 1 × 106 cells/ml. Subsequently, 100 μl of cell suspension was seeded into each of 96-well plates and incubated for 24 hrs to form a monolayer. After incubation, the supernatant was replaced with a fresh complete medium containing 10 ng/ml of LPS together with various concentrations of plant extracts and incubated for 24 hrs subsequently, a 100 μl of supernatant liquid was transferred from the original plate (plate 1) to another plate (plate 2). The nitric oxide reduction from RAW 264.7 cell line was determined by measuring the accumulation of nitrite in the supernatant after the addition of 100 μl of Griess reagent  and the content of developed azo dye was determined at 570 nm. Prednisolone was used as a positive control.Murine macrophage leukemia cell line (RAW 264.7), purchased from American Type Culture Collection , was cultured in RPMI 1640 with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in humidified atmosphere containing 5% CO50 value was calculated.The viability of cells was determined by MTT assay after incubation with LPS and medicinal plant extracts for 24 hrs. MTT solution (5 mg/ml in PBS) was added to 96-well plates (plate 1) and incubated for 2 hrs, and then the supernatant was discarded. A solution of 0.04 M HCl in isopropanol was added to dissolve formazans formed in the cells and the absorbance measured at 570 nm . The pla2. HL-60 cells were adjusted at the concentration of 5 × 105 cells/ml and induced by 1.3% DMSO in RPMI 1640 for 6 days. In this way, HL-60 cells, differentiated into neutrophils, had smaller cell size with increased expression of the NADPH oxidase on the plasma membrane [6 cells/ml. Subsequently, 200 μl of cell suspension was added to the tube with 500 μl of medicinal plant extract in Hanks' balanced salt solution (HBSS) and incubated at 37°C for 15 min. Then 50 μl of phorbol 12-myristate 13-acetate (PMA) solution (2 mg/ml in DMSO) and 250 μl of nitro blue tetrazolium (NBT) solution (1.25 mg/ml in HBSS) were added and incubated for 1 hr. The produced superoxide anion (O2−) was reduced with NBT solution giving the blue formazans, and then 2 ml of 1 M HCl at 4°C was added to the tube and centrifuged at 4,000 rpm for 10 min. The supernatant was discarded and the formazans were dissolved with DMSO and the absorbance was measured at 572 nm. Propyl gallate was used as a positive control.Human promyelocytic leukemia cell line (HL-60), purchased from the American Type Culture Collection , was cultured in RPMI 1640 with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in humidified atmosphere containing 5% COmembrane . After d50 was calculated.The viability of the cells was determined by the MTT assay in parallel with the above NBT assay. The same procedure was executed without the addition of NBT; after 1 hr incubation, it was centrifuged at 1,500 rpm for 5 min and replaced the supernatant with HBSS containing MTT solution (5 mg/ml in PBS) and was further incubated for 2 hrs. The tubes were subjected to centrifugation at 4,000 rpm for 10 min; the supernatant was discarded before dissolving formazans with DMSO and the absorbance was determined at 570 nm . The pla2. Cell proliferation assay, modified from that of Mosmann [5 cells/ml. Then 100 μl of the cell suspension was seeded into each of the 96-well plates and incubated for 24 hrs. After incubation, the supernatant was replaced with medicinal plant extracts in basic growth media . Asiaticoside was used as a positive control. After 24 hrs incubation, MTT solution (5 mg/ml in PBS) was added and further incubated for 2 hrs. The formazan was dissolved in DMSO and determined at 570 nm. The viability was calculated as percentage of proliferation [Murine embryonic fibroblast cell line (3T3-CCL92), purchased from the American Type Culture Collection , was cultured in DMEM with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in humidified atmosphere containing 5% CO Mosmann , was useferation  and plan5 cells/ml. Subsequently, 500 μl of cell suspension was seeded into each of the 24-well plates and incubated for 24 hrs. to allow the cells to adhere into a monolayer. A linear wound was generated on the monolayer with a sterile 200 μl plastic pipette tip with the wound width controlled at 550 ± 50 μm. The cellular debris was removed by washing with phosphate buffer saline (PBS). The medicinal plant extract in basic media  was added, and then the photographs of wound areas were taken at 0 and 24 hours [After the viability assay results were evaluated, the medicinal plant extracts that exhibited equal or higher viability than the control were selected for the study of wound closure and spreading ability of murine embryonic fibroblast cells (3T3-CCL92). The scratch wound closure assay was done to investigate the wound contraction phase. The reduction in wound area was measured by ImageJ software and the percentage of recovery area and the acceleration rate relative to the control, for each dose and time, were calculated. The cells were harvested with 0.25% trypsin-EDTA and centrifugation at 1,500 rpm for 5 min after which the supernatant was replaced with a fresh medium to form a suspension at the concentration of 2 × 10This study chose 4 bioactivities that would represent the wound healing potential of 16 plant extracts. These were production inhibition of nitric oxide, superoxide anion radical reduction, cell proliferation, and wound closure assays. The plant extracts that gave positive results with cell proliferation and wound closure assays were reviewed for their major active markers. These compounds were also tested for the above activities.μl at low rate 1.0 ml/min. The mobile phase consisted of 0.1% v/v phosphoric acid (solvent A) and acetonitrile (solvent B). Total running time was 60 min and the gradient program was as follows: 5% B for 0–5 min, 5% B to 50% B for 35 min, 50% B to 95% B for 15 min, and 95% B to 5% B for 5 min. The compounds were quantified using Agilent lab software.The HPLC  equipped with an UV-detector was set at 253 nm. The chromatographic separation was performed at room temperature on an XDB-C18 analytical column  injection volume 10 p=0.05, while the acceleration rate of recovery was determined by using linear regression to compare slopes of recovery rate at p=0.05.All results are presented as mean ± SEM. Statistical analyses were performed with the standard program using one-way ANOVA with Bonferroni's multiple comparisons test at G. mangostana, N. sativa, S. aromaticum, and P. emblica yielded greater than 10%, that is, 23.81, 22.07, 18.79, and 13.11%, respectively. S. trilobatum, S. indicum, G. glabra, and M. elengi produced medium-to-low yield, that is, 7.67, 6.77, 6.08, and 1.23%, respectively. With decoction, those giving higher yields were P. emblica, S. trilobatum, G. glabra, N. sativa, S. aromaticum, S. indicum, and G. mangostana with 22.11, 19.64, 15.33, 13.31, 13.15, 12.98, and 12.73%, respectively. M. elengi gave the lowest yield of 0.62%.The selected medicinal plants were extracted with two methods, maceration and decoction, producing 8 ethanolic and 8 aqueous extracts as shown in G. mangostana exhibited the highest anti-inflammatory activity with the IC50 value of 23.97 ± 0.91 μg/ml. The second and third active extracts were the ethanolic extracts of G. glabra and M. elengi with IC50 values of 46.35 ± 0.43 and 78.48 ± 4.46 μg/ml, respectively. Other ethanolic extracts and all of the aqueous extracts did not show activity . Later, thymoquinone and α-mangostin, the major compound of each plant which passed the criteria of selection   .P. emblica, the aqueous extract of S. aromaticum, and ethanolic extract of G. mangostana showed the highest activity against superoxide anion with IC50 values of 11.30 ± 0.66, 12.03 ± 0.79, and 13.97 ± 0.38 μg/ml, respectively, which were not significantly different from propyl gallate . Aqueous extracts of P. emblica and G. mangostana and ethanolic extracts of S. indicum and G. glabra exhibited high antioxidant activity with IC50 values of 14.59 ± 1.15, 16.89 ± 1.26, 26.83 ± 1.24, and 28.62 ± 1.91 μg/ml, respectively. The extracts showing low activity were ethanolic extracts of M. elengi and N. sativa with IC50 value of 71.30 ± 3.29 and 71.54 ± 3.22 μg/ml, respectively, while other extracts showed no antioxidant activity . The major compounds, α-mangostin and thymoquinone, which passed the criteria of selection . Four in vitro studies were chosen to demonstrate whether their ethanolic and aqueous extracts could influence wound healing. At the first stage of wound healing, inflammation occurs; this involves RNS and ROS from our immune cells. These free radicals are essential for ridding of bacteria but it could also cause damage to cells surrounding the wound. In this regard, the tests for anti-inflammation and antioxidant were used. At the second stage of wound healing fibroblast cell proliferation commences and the wound reduces its size. To demonstrate these activities of the 16 plant extracts, fibroblast cell proliferation and scratch wound closure assays were chosen.Eight Thai medicinal plants were selected from Mukkharoka scripture, the National List of Essential Medicines of Thailand, and traditional usages. These were G. mangostana and G. glabra could reduce superoxide anion, inhibit nitric oxide production, promote fibroblast proliferation, and accelerate wound closure. These two extracts would be useful for the first and second stages of wound healing as described above. Although the ethanolic extract of N. saliva showed the lowest activity against superoxide anion and no activity against nitric oxide, it showed the highest wound closure activity among all extracts and asiaticoside, the reference compound. N. sativa extract would be beneficial at the second stage of wound healing. For this reason, these three ethanolic plant extracts had passed the selection criteria in testing their markers for these activities. These markers were α-mangostin for G. mangostana, glycyrrhizin for G. glabra, and thymoquinone for N. sativa.The results showed that ethanolic extracts of G. mangostana ethanolic extract, the results showed that IC50 values of antioxidant and anti-inflammation activities were 13.97 ± 0.38 and 23.97 ± 0.91 μg/ml, respectively. Its marker, α-mangostin, also showed stronger activities with the IC50 values of 2.65 ± 0.52 and 15.15 ± 0.14 μg/ml, respectively. Both extract and marker could promote cell proliferation to the maximal values of 52.68 ± 1.99 and 26.72 ± 2.21%, respectively. The acceleration of wound closure was also increased at the rates of 2.02 ± 0.03 and 2.18 ± 0.03% per hour, respectively. Our results were in accordance with previous studies which showed that the ethanolic extract of G. mangostana and its major compound, α-mangostin, exerted strong anti-inflammatory activity through the production inhibition of nitric oxide and PGE2 [in vivo study also showed wound healing activity of G. mangostana extract [Concerning and PGE2  and also extract .G. glabra inhibited superoxide anion and nitric oxide production with the IC50 values of 28.62 ± 1.91 and 46.35 ± 0.43 μg/ml, respectively. Its marker, glycyrrhizin, exhibited antioxidant activities with the IC50 value of 40.85 ± 2.30 μg/ml but has no activity against nitric oxide production inhibition. G. glabra extract also promoted proliferation to the maximal values of 52.13 ± 3.95 and accelerated wound closure at the rate of 2.12 ± 0.03% per hour. Glycyrrhizin, on the contrary, had no activity on promoting fibroblast proliferation but could increase the acceleration of wound closure at the rate of 2.18 ± 0.02% recovery area per hour. These results indicated that the ethanolic extract of G. glabra was active for all activities but not entirely due to glycyrrhizin. Our results were in agreement with a recent study on G. glabra extract against superoxide anion and nitric oxide at IC50 values of 38.4 and 62.1 μg/ml, respectively [G. glabra extract also showed better healing than dexpanthenol cream [The ethanolic extract of ol cream .N. sativa exhibited no activity against production inhibition of nitric oxide  and low activity against superoxide anion with the IC50 value of 71.54 ± 3.22 μg/ml. However, it was able to increase proliferation to the maximum of 15.47 ± 0.87% and gave the highest wound closure activity with the acceleration rates of 2.65 ± 0.05% of recovery area per hour. Its marker, thymoquinone, showed high activities against nitric oxide and superoxide anion with the IC50 value of 1.55 ± 0.14 and 9.56 ± 1.11 μg/ml which were not significantly difference from reference compounds . It also promoted wound closure activity with the acceleration rates of 2.35 ± 0.05% of recovery area per hour without any proliferative promoting activity. The results showed that the ethanolic extract of N. sativa gave good results with proliferation and wound closure activities while thymoquinone exhibited high activities as antioxidant and anti-inflammation through production inhibition of superoxide anion and nitric oxide activities. Therefore, the ethanolic extract of N. sativa could be good choice for the wound at the second phase. Our result was in accordance with a previous study that reported nitric oxide inhibition by N. sativa extract at the concentration higher than 100 μg/ml [The ethanolic extract of 00 μg/ml .G. mangostana, G. glabra, and N. sativa may be applied to treat the wound in various phases. They can be divided into two groups. The first group consists of the ethanolic extracts of G. mangostana and G. glabra which can be applied together with the saline washing solution to remove foreign bodies and bacteria. This is due to the fact that the ethanolic extracts of G. mangostana and G. glabra can reduce inflammation and protect the wound surrounding cells. They have been shown to effectively inhibit Staphylococcus aureus and Staphylococcus aureus MRSA with the Minimal Inhibitory Concentration (MIC) values of 9.8 and 4.9 μg/ml, respectively [N. sativa which can be applied on open wounds or wounds that are not completely close, to accelerate wound recovery. Glycyrrhizin and thymoquinone enhanced wound closure without any cell proliferation activities and may be beneficial in the second phase of wound treatment as well as α-mangostin. Interestingly, G. mangostana and N. sativa yielded high percentage of their ethanolic extracts which made them more attractive for future utilization.Our results suggested that ethanolic extracts of ectively . The secOur findings demonstrated that 3 commonly used Thai medicinal plants are effective for wound treatment and supported their uses in Thai traditional medicine. Their effectiveness can be scientifically explained in terms of the activities of these plant extracts and their active markers that promote wound healing through the inhibition of nitric oxide and superoxide anions and through increasing fibroblast cell proliferation and accelerating wound closure. Further studies are necessary to explain the wound healing property of these herbs and to develop them into a potential wound healing agent.