Polygonum multiflorum Thunb (PM) are commonly used in clinical practice to treat diverse diseases; however, reports of hepatotoxicity induced by Polygoni Multiflori Radix (PMR) and Polygoni Multiflori Radix Praeparata (PMRP) have emerged worldwide. Thus, it is necessary for researchers to explore methods to improve quality standards to ensure their quality and treatment effects.The raw and processed roots of 18 column . The triple quadrupole mass spectrometer (TQMS) was operated in negative ionization mode with multiple reaction monitoring for the quantitative analysis of six dianthrones. Moreover, compounds 5 and 6 were further evaluated for their cytotoxicity in HepaRG cells by CCK-8 assay.In the present study, an ultra-high performance liquid chromatography triple quadrupole mass spectrometry (UHPLC-QQQ-MS/MS) method was optimized and validated for the determination of dianthrones in PMR and PMRP using bianthronyl as the internal standard. Chromatographic separation with a gradient mobile phase [A: acetonitrile and B: water containing 0.1% formic acid (v/v)] at a flow rate of 0.25 mL/min was achieved on an Agilent ZORBAX SB-C–C4 (1–4), trans-emodin dianthrones (5), and cis-emodin dianthrones (6). The contents of 1–6 in 90 batches of PMR were in the ranges of 0.027–19.04, 0.022–13.86, 0.073–15.53, 0.034–23.35, 0.38–83.67 and 0.29–67.00 µg/g, respectively. The contents of 1–6 in 86 batches of commercial PMRP were in the ranges of 0.020–13.03, 0.051–8.94, 0.022–7.23, 0.030–12.75, 0.098–28.54 and 0.14–27.79 µg/g, respectively. Compounds 1–4 were almost completely eliminated after reasonable processing for 24 h and the contents of compounds 5 and 6 significantly decreased. Additionally, compounds 5 and 6 showed inhibitory activity in HepaRG cells with IC50 values of 10.98 and 15.45 μM, respectively. Furthermore, a systematic five-step strategy to standardize TCMs with endogenous toxicity was proposed for the first time, which involved the establishment of determination methods, the identification of potentially toxic markers, the standardization of processing methods, the development of limit standards and a risk–benefit assessment.The UHPLC-QQQ-MS/MS method was first developed to simultaneously determine six dianthrones in PMR and PMRP, namely, polygonumnolides C1trans-emodin dianthrones (5) and cis-emodin dianthrones (6) could be selected as toxic markers of PMRP. Taking PMR and PMRP as examples, we hope this study provides insight into the standardization and internationalization of endogenous toxic TCMs, with the main purpose of improving public health by scientifically using TCMs to treat diverse complex diseases in the future.The results of the cytotoxicity evaluation of the dianthrones indicated that  Polygonum multiflorum Thunb, including Polygoni Multiflori Radix (PMR) and PMR Praeparata (PMRP), is a commonly used TCMs used to treat various diseases in China and is also popular in many other countries [O-β-d-glucopyranoside (THSG), emodin-8-O-β-d-glucoside, catechin, epicatechin, and physcion-8-O-β-d-glucopyranoside; however, these compounds did not disappear [ountries , 2. PMR ountries –5. Howevountries –8. The cisappear , 10. The3+ ions and degrade easily when heated [trans-emodin dianthrones (5) and cis-emodin dianthrones (6) could be chosen as potential toxicity markers of PMRP. Furthermore, a systematic five-step strategy to standardize TCMs with endogenous toxicity was proposed for the first time, which involved the establishment of determination methods, the identification of toxic markers, the standardization of the processing method, the development of limit standards and a risk–benefit assessment. Taking PMR as an example, it is hoped that these findings will improve the standardization and internationalization of endogenous toxic TCMs and provide indispensable evidence for ensuring safe and effective clinical treatment in the future.Our previous work on PMR toxicity showed that the dianthrones that were first isolated from PMR by our team could have potential hepatotoxicity, and there are many minor dianthrones in PMR –17. Moren heated , 19. Then heated , 21. Hown heated . In additrans-emodin dianthrones (5), and cis-emodin dianthrones (6) to hepatocytes in vitro.In the past several decades, many human liver cell lines have been used for in vitro screening tests to evaluate hepatotoxic drugs and other compounds. The HepaRG cell line has been proven to be suitable human hepatocytes for the assessment of hepatotoxicity in vitro . HepaRG HPLC-grade acetonitrile was purchased from Fisher Scientific . Formic acid was purchased from Merck Inc. . Ethanol was of analytical grade and purchased from Shanghai Chemical Reagent Co. . Water was purified with a Milli-Q water purification apparatus . The immortalized hepatic cell line HepaRG was obtained from the Type Culture Collection of the Chinese Academy of Sciences . The following reagents were also used in this study: RPMI 1640 culture medium , foetal bovine serum , penicillin , staurosporine , 0.25% trypsin–EDTA , CCK-8 reagent , DMSO , and a Victor Nivo multi-mode plate reader .Polygonum multiflorum samples were authenticated by Associate Professors Ji Zhang and Jian-Bo Yang  in accordance with the Chinese Pharmacopoeia  [olume 1) . A vouch1), polygonumnolide C3 (2), polygonumnolide C1 (3), polygonumnolide C2 (4), trans-emodin dianthrones (5), and cis-emodin dianthrones (6) were isolated and purified. The structures of the six dianthrones (1–6) were confirmed by UV, MS, 1H NMR and 13C NMR analyses, which have been reported in the literature [The chemical compounds polygonumnolide C4 . Separation was achieved with a gradient of mobile phases consisting of acetonitrile (A) and water containing 0.1% formic acid (v/v) (B) at a flow rate of 0.25 mL/min. The gradient was programmed as follows: 0–8 min, maintenance at 37% A; 8–10 min, linear change to 60% A; 10–12 min, linear change to 78% A; 12–20 min, linear change to 90% A; 20–22 min, linear change to 37% A; and 22–30 min, maintenance at 37% A. The column temperature was maintained at 30 °C. The injection volume was 2.0 μL.The UHPLC-MS/MS instrument consisted of an Agilent 1200 series UHPLC system equipped with an Agilent 6410B TQMS/MS system . Chromatographic analyses were performed using an Agilent 1200 series UHPLC system  consisting of a quaternary pump, an online degasser, an auto plate-sampler, and a thermostatically controlled column compartment. Chromatographic separation was carried out at 30 °C on an Agilent ZORBAX SB-CAll MS experiments were conducted using an ESI source in negative ion electrospray mode with a 6410B TQMS . The optimal MS conditions were as follows: drying gas temperature, 300 °C; drying gas flow rate, 10 L/min; nebulizer gas pressure, 30 psi; sheath gas temperature, 300 °C; sheath gas flow, 11 L/min and capillary voltage, 4.0 kV. Detection was carried out in MRM mode. All data were processed using MassHunter Workstation software .1), polygonumnolide C3 (2), polygonumnolide C1 (3), polygonumnolide C2 (4), trans-emodin dianthrones (5) and cis-emodin dianthrones (6), were prepared in 70% ethanol. Accordingly, a standard mixture solution was obtained by precisely mixing the six stock solutions with 70% ethanol so that the concentrations were 0.210 (1), 0.214 (2), 0.283 (3), 0.280 (4), 0.318 (5) and 0.280 (6) μg/mL. The mixture solutions were further diluted to generate standard solutions in different concentration ranges. The calibration curves were generated with at least six appropriate concentrations. Bianthronyl (IS) was prepared in DMSO/methanol  at a concentration of 100.44 μg/mL. All standards solutions were stored at 4 °C.Standard stock solutions for the six dianthrones, namely, polygonumnolide C4 (Ninety batches of PMR PMR-01–PMR-90) were collected from different provinces of China, as shown in Table –PMR-90 wPMRP can improve the efficacy and reduce the hepatotoxicity of PMRP after processing. PMRP could be extracted from PMR using the method from the Chinese Pharmacopoeia (2020 edition)  and trad0h, S2h, S4h, S6h, S8h, S10h, S12h, S16h, S20h, or S24h. In addition, ten samples of PMRP-(S0h–S24h) were successfully obtained. Moreover, 15 batches of crude PMR (300 g) were infiltrated by distilled water and steamed at 100 °C for 0, 12, or 24 h. These processed products were then dried in the sunlight. Finally, 45 samples of PMRP- were successfully obtained.The water-steaming method was as follows: A sample (PMR-49) was collected for examination at different points and labelled PMRP-SAn aliquot of 1.0 g of PMR or PMRP (filtered through a no. 3 sieve) was weighed into a stoppered conical flask, 50 mL of accurately measured ethanol–water  was added followed by weighing and ultrasonication  for 30 min. The solution was cooled and weighed again, the loss of weight was replenished with ethanol–water  and the solution was mixed well. This extract was then filtered through a 0.22 μm syringe filter. The filtrate was used as the test solution and analysed with UHPLC-QQQ-MS/MS according to the above procedure.2. The effects of the toxic dianthrone markers on HepaRG cell viability were determined using a CCK-8 assay. According to the experimental operation requirements, the day before detection, HepaRG cells were inoculated in 384-well cell plates at a density of 1000 cells/well with 40 μL of cell suspension inoculated in each well. The cell plates were placed in an incubator at 37 °C with 5% CO2 for overnight incubation. On the day of the experiment, 10 μL of compound working solution  was added to each well, and the plate was incubated at 37 °C in a 5% CO2 incubator in the dark for 72 h. After incubation, 5 μL of CCK-8 reagent was added to each cell well followed by incubation for 4 h. The absorbance at 450 nm was measured on the NIVO instrument, and the inhibition rate was calculated by the following formula:NC is the absorbance of the negative control (cell + medium + DMSO), and ODSTSP is the absorbance of the positive control (cell + medium + 10 μM STSP). According to the inhibition ratios of the compounds, the IC50 values (the concentration corresponding to 50% of the maximum inhibition response) were calculated from the dose–response curves using GraphPad. All tests were conducted in triplicate, and the mean values were finally obtained.HepaRG cells were maintained in RPMI 1640 medium containing 10% FBS, 100 U/mL penicillin and streptomycin at 37 °C with 5% COOptimization of the extraction solvent system: the first step in the preparation of the sample solution was to select a suitable extraction solvent because of its paramount role in achieving good recovery. Five solutions, H2O and 30%, 50%, 70%, and 95% ethanol (v/v in water), were systematically compared by virtue of the peak areas of the six dianthrones in PMR. As a result, 70% ethanol exhibited the highest extraction efficiency among the tested solvents, as shown in Fig. Optimization of solvent volume: extractant volume may be another factor that could affect extraction efficiency. This study aimed to obtain the minimum volume of extractant required to achieve the highest extraction efficiency. Four different volumes of 70% ethanol  were systematically studied. From Fig. Optimization of ultrasonication time: in this study, an ultrasonic process was used to extract the six dianthrones from PMR. From Fig. PMR No. 20191001) was used to optimize the extraction process. Optimization was successfully completed using a three-step approach, described as follows. Step 1. 0191001 wIn conclusion, the optimal sample preparation method was found to be the extraction of a 1.0 g sample with 50 mL of 70% ethanol in an ultrasonic water bath for 30 min.1–6 and the IS are also shown in Fig. 1–6 and IS) identify cleavage of the C10–C10′ bond to yield anthrone-free radicals in the MS/MS product ion spectra. The MS/MS product ion spectra of 1–6 were reported in the article [The chromatographic conditions, especially the composition of the mobile phase, were optimized to achieve the best possible resolution and symmetric peaks of the seven compounds within a suitable run time. Over the course of the tests, four mobile phases were examined, i.e., methanol-, acetonitrile-, and methanol–water containing 0.1% formic acid (v/v), and acetonitrile–water containing 0.1% formic acid (v/v), in different ratios. The acetonitrile–water containing 0.1% formic acid (v/v) combination had the lowest pressure, best baseline stability, and highest ionization efficiency among those tested and was eventually selected as the mobile phase. Both positive and negative ion modes were also tested for MS analysis. The seven compounds showed cleaner mass spectral backgrounds and higher sensitivities in negative mode than in positive mode. The parameters of fragmented voltage and collision energy were optimized to obtain the richest relative abundance of parent ions and outputs for the optimization of MRM conditions. In addition, the MRM transitions and parameters of these seven dianthrone compounds are shown in Table  article .Table 2Pmixed standard solution and a sample of P. multiflorum are shown in Fig. The peaks of the six dianthrones and the IS presented good separation without interference peaks based on the chromatographic and MS conditions mentioned above. The typical MRM chromatograms for a blank test sample, a 2, linearity range, LOD, and LOQ values. All calibration curves showed good linear regression (r2 ≥ 0.9965) within the tested ranges; the LOD (S/N = 3) and the LOQ (S/N = 10) for the six dianthrones were in the ranges of 0.3–0.4 ng/mL and 0.7–1.1 ng/mL, respectively, showing high sensitivity.The developed UHPLC-QQQ-MS/MS method was further validated in accordance with the guidelines of the Validation of the Quality Standard of TCMs  . Table 3The precision of the method was evaluated based on intra- and inter-day precision. The intra-day precision was tested with mixed standard solutions in 1 day. The standard solutions are examined in triplicate on three consecutive days for inter-day precision. The corresponding % RSD values were calculated. The RSDs for the intra-day (n = 6) and inter-day (n = 3) assays were less than 2.73% and 4.63%, respectively  = /amount spiked × 100%. Table m/z values of the six dianthrones with those of the reference compounds, the identification of the target peaks was successful by the UHPLC-QQQ-MS/MS method. The content of each analyte was determined using the respective calibration curves with the IS method. The developed method was successfully applied to analyse the contents of the six dianthrones in PMR and PMRP.Comparing the UHPLC retention times and 1, 2, 3, 4, 5 and 6 were in the ranges of 0.027–19.04, 0.022–13.86, 0.073–15.53, 0.034–23.35, 0.38–83.67 and 0.29–67.00 µg/g, respectively. The total contents of 1–6 ranged from 1.39 to 171.45 µg/g. There were distinct differences in the contents of 1–6 in the 90 batches of PMR. Interestingly, the contents of 5 and 6 in PMR extracted with 70% ethanol were remarkably higher than those of 1–4. The average content order in the 90 batches of PMR was 5 > 6 > 1 > 4 > 3 > 2. According to previous studies [5 and 6 are more toxic than 1–4. Therefore, 5 and 6 could be used as potential toxicity markers of PMRP.The developed and validated UHPLC-QQQ-MS/MS method was subsequently applied to evaluate the six dianthrones from 90 batches of PMR, and the quantification results are summarized in Table 0h, PMRP-S2h, PMRP-S4h, PMRP-S6h, PMRP-S8h, PMRP-S10h, PMRP-S12h, PMRP-S16h, PMRP-S20h, and PMRP-S24h) using the water-steaming method, and the quantification results are summarized in Table 1, 2, 3, 4, 5 and 6 were in the ranges of 0.18–2.09, 0.58–3.67, 0.26–2.04, 0.71–4.07, 0.25–7.20 and 0.22–6.11 µg/g, respectively. The total contents of 1–6 ranged from 2.20 to 20.74 µg/g.The developed and validated UHPLC-QQQ-MS/MS method was subsequently applied to identify the six dianthrones in 10 samples of PMRP  samples consist of standard solutions of different concentrations and they were injected every 24 h. According to the literature , the bes50 values of each compound in the HepaRG cell model were determined. The IC50 values of compounds 5 and 6 were 5.60 μg/mL and 7.88 μg/mL, respectively. These values corresponded to 10.98 μM and 15.45 μM, respectively. The results suggested that compounds 5 and 6 had strong hepatocellular toxicity and could be used as potential toxicity markers.The two potentially toxic compounds, 5 and 6, were evaluated for their cytotoxicity in HepaRG cells by CCK-8 assay. According to the concentration-HepaRG cell inhibition rate curves drawn at different concentrations of the compounds, the ICTCMs with endogenous toxicity have a relatively narrow treatment window. If they are used improperly in the clinic, severe adverse reactions may occur. Attention has been directed towards TCMs with endogenous toxicity because of the negative effects and serious risks they cause to humans. Therefore, it is essential to develop a system to standardize TCMs with endogenous toxicity to guide the clinical use of TCMs. For the first time, in the present study, a systematic five-step strategy standardize TCMs with endogenous toxicity was proposed and involved the establishment of determination methods, the determination of toxic markers, the standardization of the processing method, the development of limit standards and a risk–benefit assessment Fig. .Fig. 5A First, determination methods are expected to be developed to isolate and identify endogenous toxic chemicals in TCMs. The present study innovatively established a UHPLC-QQQ-MS/MS technique to simultaneously detect six dianthrones in PMR and PMRP. UHPLC-QQQ-MS/MS techniques are widely used for applications in chromatography–MS analysis. The method developed herein could not only provide rapid and improved chromatographic separation and a shorter chromatographic run time but can also provide higher sensitivity and selectivity, which are ultimately helpful for determining the contents of dianthrones in PMR and PMRP.trans-emodin dianthrones (5) and cis-emodin dianthrones (6), could be selected as potential toxic markers of PMRP [1–6) in PMRP are as follows. Free dianthrones (5 and 6) may undergo glycosidation and be further converted into the combined dianthrones 1–4. On the other hand, the C10–O-glucopyranoside, which may undergo methylation. Finally, the combined anthraquinones could be converted into free anthraquinones originating from the loss of the glucoside unit. Accordingly, the postulated degradation process of dianthrones in PMRP was speculated as Scheme Second, determining toxic markers and clarifying the mechanism could decrease the toxicity of TCMs. Interestingly, this study showed that dianthrones are widely distributed in PMR and demonstrated that these compounds, especially  of PMRP , 17. TheP. multiflorum preparations (PMPs) as an example, this study illustrated the relationship between different solvent extracts and the contents of dianthrones in PMRs and PMRPs for the first time. Different extracts using ethanol at different concentrations as an extracting agent could significantly influenced the hepatotoxicity of PMR, as reported in the references [2O extract and 30% ethanol extract [Third, standardization of the processing method is of great significance. Taking ferences –30. Ther extract . FurtherP. multiflorum preparations (PMPs) as an example, there are more than 300 Chinese patent medicines (CPMs) containing PMRs and PMRPs in the Chinese Pharmacopoeia and Drug Standard of the Ministry of Public Health of the People’s Republic of China [L = AWδ/M (1) where L is the maximum theoretical limit, W is the body weight (70 kg), and M is the daily ingestion rate of the TCM or TCM preparation (g/day), which could be based on the consumption rate in the Pharmacopoeia of the People’s Republic of China (PPRC), and δ is a safety factor, accounting for the contribution of dietary supplements as a component of daily food intake. According to the judgement of the National Science Foundation (NSF), δ could be 10. A is the acceptable daily intake (ADI), which is defined as the estimated amount of a chemical to which a person can be exposed on a daily basis over a lifetime without suffering a detectable deleterious effect. For some endogenous toxic chemicals, such as pyrrolizidine alkaloids, ADI values have been set by organizations involving the World Health Organization (WHO) and European Food Safety Authority (EFSA), as references. However, for other endogenous toxic chemicals, such as the dianthrones in PMR or PMP, the ADI should be determined under the guidance of Good Laboratory Practice (GLP). In future studies, we will make great efforts to determine the crucial parameter ADI, especially the ADI for trans-emodin dianthrones (5) and cis-emodin dianthrones (6), based on which the maximum theoretical limit could be acquired. A practical maximum theoretical limit is the basis of a practical limit standard, and other factors involved in economic development, human cognition, and even history and culture, are recommended to be considered to maintain a balance between public safety and economic progress.Fourth, considering public confidence in the safe use of TCMs and TCM preparations, the development of a scientific and practical limit standard for TCMs with endogenous toxicity is beneficial and urgently needed. Taking of China . It has of China –33. HoweFinally, it is necessary to establish a benefit and risk assessment model of TCMs with endogenous toxicity to comprehensively evaluate the benefits and risks of TCMs and ensure both their safety and effectiveness. The evaluation of the risk–benefit ratio is determined by many factors and involves the establishment of a value tree of the risk–benefit ratio. The value tree is composed of the characteristics of the disease, clinical efficacy of the TCM, adverse reactions caused by the TCM, etc. On the basis of the severity, duration and incidence of the adverse reactions caused by TCMs with endogenous toxicity, these indexes should be weighed to obtain the estimated risk–benefit ratio. Moreover, it is paramount to build a very large mass spectral database to identify endogenous toxic chemicals, including dianthrones, as well as accumulate a wider range of extensive health risk assessment data on these endogenous toxic chemicals.trans-emodin dianthrones (5) and cis-emodin dianthrones (6) could be considered as potentially toxic markers of PMRP. Furthermore, taking PMR as an example, a systematic five-step strategy to promote the standardization of TCMs with endogenous toxicity was proposed for the first time, covering the research gap in this field. The systematic strategy consisted of the following steps: the establishment of determination methods, the identification of toxic markers, the standardization of the processing method, the development of limit standards and a risk–benefit assessment. Taking PMR and PMRP as examples, we hope this study provides insight into the standardization and internationalization of endogenous toxic TCMs and is conducive to improving the quality standard of these endogenous toxic TCMs and ensuring safe and effective clinical treatment.In the present study, a rapid, sensitive, precise, and reliable UHPLC-QQQ-MS/MS method was developed for the simultaneous determination of six dianthrones in PMR and PMRP for the first time. The results indicated that


Tetrastigma hemsleyanum Diels et Gilg and identify the antioxidant components in vitro. α, α-Diphenyl-β-picrylhydrazyl (DPPH) radical assay, Trolox equivalent antioxidant capacity (TEAC) assay, ferric reducing antioxidant power (FRAP), and hydroxyl radical scavenging method were used to screen the extract with high antioxidant activity. The antioxidant capacity of the extracts was evaluated by the free radical scavenging ability of DPPH. The ability of extracts to scavenge 2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radical was evaluated by TEAC assay. The FRAP method was used to evaluate the ability of extracts to reduce Fe3+. The ability to scavenge hydroxyl radicals produced by the interaction of hydrogen peroxide and Fe2+ was measured by monitoring the change in the absorbance of the reaction mixture at 536 nm. Then, high-performance liquid chromatography-DPPH (HPLC-DPPH) and HPLC-hydroxyl radical scavenging methods were used to screen the antioxidant components in the extract. The molecular weight of the above antioxidant components was investigated using the qualitative analytical method of high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (HPLC-Q-TOF LC/MS). Based on the concentrations of the samples (0.2–4 mg/mL), the DPPH free radical scavenging ability, ABTS+ free radical scavenging ability, hydroxyl free radical scavenging ability, and Fe3+ reducing ability of the ethyl acetate extract (EAE) were stronger than that of the crude extract (CE), petroleum ether extract (PEE), and n-butanol extract (BE). The EAE has higher antioxidant activity than CE, PEE, and BE. Six antioxidant components, rutin, quercetin, isoquercetin, astragalin, kaempferol, and kaempferol-3-o-rutoside, were identified in the EAE.This study aimed to investigate the extract with high antioxidant activity of  Tetrastigma hemsleyanum Diels et Gilg, a Chinese folk herbal medicine, is used for the treatment of children with high fever, pneumonia, hepatitis, viral meningitis, scrofula, carbuncle, and furuncle [Tetrastigma hemsleyanum Diels et Gilg has anti-inflammatory, analgesic [The rhizome of furuncle . Modern nalgesic , antivirnalgesic , antilivnalgesic , 5, antinalgesic , 7, and nalgesic , 9.Tetrastigma hemsleyanum Diels et Gilg. The aboveground part of Tetrastigma hemsleyanum Diels et Gilg contains β-sitosterol, palmitic acid, pentacosane, gallic acid, ethyl gallate, catechin, 7-O-galloylcatechin, and 3,3'- dimethoxy ellagic acid-4-O-β-D-glucopyranoside [α-linolenic acid, kaempferol, quercetin, and kaempferol-3-o-neohesperidin were found in liposolubility extract of Tetrastigma hemsleyanum Diels et Gilg [Tetrastigma hemsleyanum by HPLC-Q-TOF-MS and found that it contained kaempferol-7-o-rhamnose-3-o-glucoside, rutin, isoquercetin, kaempferol rutoside, astragaloside, and quercetin [Tetrastigma hemsleyanum leaves by rapid resolution liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (RRLC-Q-TOF-MS) [A large number of studies are focused on the chemical constituents of ranoside . Taraxer et Gilg –13. In auercetin . Fan ide-TOF-MS) . These r-TOF-MS) . Flavono-TOF-MS) , 17, ant-TOF-MS) –20, anti-TOF-MS) –23, anti-TOF-MS) , and are-TOF-MS) ; their aFree radicals that are produced in the process of life metabolism regulate the signal transmission between cells and cell growth and inhibit the viruses and bacteria . ExcessiTetrastigma hemsleyanum Diels et Gilg, we found that some of the isolated parts were oxidized [Tetrastigma hemsleyanum Diels et Gilg. However, active components responsible for antioxidants in Tetrastigma hemsleyanum Diels et Gilg are yet to be identified. Therefore, by utilizing the antioxidant test, we screened out the extract that harbors high antioxidant activity, and the molecular weight of the potential active components was determined by HPLC-Q-TOF LC/MS.While screening the antitumor extract and active components of oxidized , 47. ThiDPPH, ABTS, TPTZ , and Trolox  were purchased from Sigma , and methanol, ethanol, petroleum ether, ethyl acetate, n-butanol, and other reagents for the antioxidant test were analytically pure and purchased from China National Pharmaceutical Group Co. . Rutin , astragaloside , isoquercetin , quercetin , kaempferol , and kaempferol-3-o-rutoside  standard reference materials were obtained from National Institutes for Food and Drug Control . Chromatography grade and LC-MS grade acetonitrile were obtained from Tedia  and Fisher , respectively.Tetrastigma hemsleyanum Diels et Gilg from Guangxi Province, China, was purchased from Guangxi, China (lot no. 180615), and identified by Professor Xia Miao-fen at Zhejiang Pharmaceutical College.The rhizome of Tetrastigma hemsleyanum Diels et Gilg was soaked in three volumes of 60% methanol at room temperature. Subsequently, the soaking solution was filtered, combined, and concentrated under reduced pressure; the crude extract (CE) (364 g) was prepared by vacuum drying. Then, it was suspended in water and extracted successively with petroleum ether, ethyl acetate, and n-butanol to obtain petroleum ether extract (PEE) (28 g), ethyl acetate extract (EAE) (25 g), and n-butanol extract (BE) (68 g), respectively. The antioxidant activities of each extract were measured using the DPPH assay, TEAC assay, ferric reducing antioxidant power (FRAP), and hydroxyl radical scavenging method to screen the extract with high antioxidant activity. The antioxidant components in this extract were detected by HPLC-DPPH and HPLC-hydroxyl radical scavenging methods. The molecular weight of the antioxidant components was deduced by HPLC-Q-TOF LC/MS. The extracts were solubilized in ethanol solution (80% v/v), except for PEE, which was dissolved in dimethyl sulfoxide (DMSO).The extracts were prepared according to the method of Bao et al.  and Yong−4 M) was mixed with 0.1 mL of the sample solution. After incubation for 30 min in the dark at room temperature, the absorbance was measured at 517 nm using a UV-vis spectrophotometer . The radical scavenging activity (%) was calculated as follows:A1 is the absorbance of the sample, and A0 is the absorbance of the blank group.The DPPH free radical scavenging activity was assessed according to Leong et al.  with a sμL sample solution (0.2–4 mg/mL) with 3.0 mL ABTS+ at 30°C for 10 min, the absorbance was measured at 734 nm by the UV-vis spectrophotometer. The antioxidant capacity was expressed as mg of Trolox/g of samples (TEAC), and the TEAC was calculated as follows:A1 is the absorbance of the sample, A0 is the absorbance of the blank group, b is the slope of the standard curve, a is the intercept of the standard curve, V is the volume of Trolox (μL), and W is the weight of extracts (g).The antioxidant activity against ABTS+ was measured as described by Strail et al.  with som3+-TPTZ was performed according to the method established by Benzie et al. [3·6H2O solution at the ratio of 10 : 1:1. A volume of 3.0 mL FRAP reagent was added to 20 L sample solution (0.2–4 mg/mL) and incubated at 37°C for 30 min. The absorbance of the reaction mixture was measured at 593 nm. FRAP was expressed as the number of moles of FeSO4 required to achieve the same absorbance per g of samples and calculated as follows:A1 is the absorbance of the sample, A0 is the absorbance of the blank group, b is the slope of the standard curve, a is the intercept of the standard curve, and W is the weight of extracts (g).The antioxidant activity against Fee et al.  with som2O2) solution was added and incubated at 37°C for 60 min. The absorbance was measured at 536 nm. The hydroxyl radical scavenging capacity was calculated as follows:A1 is the absorbance of the sample, A0 is the absorbance of the blank control group, and A2 is the absorbance of the sample and reagent blank group.The hydroxyl radical scavenging capacity was evaluated according to the method described by Jin et al.  with somμm) with solvent A (0.1% formic acid) and solvent B (acetonitrile). The gradient condition was as follows: 0 min, 18% B; 0–5 min, 25% B; 5–10 min, 35% B; 10–15 min, 60% B; 15–20 min, 90% B; and 20–30 min, 18% B. The flow rate was 1.0 mL/min, the injection volume was 20 μL, the column temperature was set at 30°C, and the detection wavelength was 254 nm.Herein, we used traditional methods to select the extract with high antioxidant activity, and the antioxidant components were screened using the HPLC-DPPH scavenging method and HPLC-hydroxyl radical scavenging method . The chrμL sample solution (4 mg/L) and 250 μL DPPH solution (500 μM) were mixed and incubated at 37°C in the dark for 30 min. The mixture was then filtered through a 0.45 μm membrane and determined according to the chromatographic conditions in 2.2.5.The majority of the phenolic antioxidants can quench DPPH radicals, and hence, DPPH radicals are often used to screen the antioxidant activities of phenolic compounds . In thisμL H2O2 solution (25 mM) and 200 μL of FeSO4 solution (250 μM) were added to 250 μL sample solution (4 mg/L). The mixture was incubated at room temperature in the dark for 7 min, filtered with 0.45 μm membrane, and measured according to the chromatographic conditions in 2.2.5.Briefly, 200 μm) with solvent A (0.1% formic acid) and solvent B (acetonitrile). The gradient condition was as follows: 0 min, 18% B; 0–5 min, 25% B; 5–10 min, 35% B; 10–15 min, 60% B; and 15–20 min, 90% B. The flow rate was 0.2 mL/min, injection volume was 1 µL, the column temperature was room temperature, the scanning range was 50–1500, and the capillary voltage was 4000 V. Nitrogen was used as dry gas at a flow rate of 10 mL/min, the temperature was 350°C, and the atomizer pressure was 40 psi. The mass spectrogram and chromatogram were recorded under the negative ion source. After acquiring the molecular weight, the antioxidant components in the sample were further identified based on their standard products by HPLC, according to the chromatographic conditions in 2.2.5.The identification experiment was carried out using an Agilent HPLC-Q-TOF LC/MS  and an Agilent HPLC system with a photodiode array detector (1260 series). The chromatographic separation was conducted using a Extend Agilent C18  of three independent experiments in triplicate and analyzed by SPSS (version 17.0). One-way analysis of variance (ANOVA) and Tukey multiple comparisons were carried out to test any significant differences between the means. P > 0.05) and 4 mg/mL (P > 0.05), respectively, while DPPH scavenging activity of EAE and all the other extracts is the largest difference at the sample concentration of 1 mg/mL (P < 0.05). The DPPH scavenging activity of EAE was dose-dependent in the concentration range of 0.2–4 mg/mL and occurred in the following order: EAE > CE > BE > PEE . On the other hand, in the sample concentration range of 0.2–4 mg/mL, the TEAC values of EAE were 150.78 ± 20.94, 241.72 ± 30.34, 281.36 ± 3.17, 265.54 ± 7.79, and 207.59 ± 14.45 mg Trolox/g extract, respectively. The TEAC value was not dose-dependent because the weight of the extract (W) varied in the five different concentrations of each extract. If calculated according to formula . At different concentrations, the reduced ability of EAE was significantly higher than that of other extracts, indicating a high reduction ability of Fe3+. In the concentration range of 0.2–4 mg/mL, the FRAP values of EAE were 0.74 ± 0.06, 1.12 ± 0.04, 1.02 ± 0.04, 0.71 ± 0.01, and 0.68 ± 0.00 mM FeSO4/g extract, respectively . At 0.2, 0.5, and 0.4 mg/mL, the clearance rate of EAE was significantly different from that of other extracts (P < 0.05). In the concentration range of 0.2–4 mg/mL, the scavenging activity of hydroxyl radicals in EAE was 32.69 ± 3.66%, 59.73 ± 2.76%, 56.80 ± 1.69%, 51.28 ± 3.47%, and 47.60 ± 1.63%, respectively.The present study demonstrated that the EAE had the strongest antioxidant activity among the four extracts. Then, we used HPLC to analyze and screen the antioxidant components by HPLC-DPPH and HPLC-hydroxyl radical scavenging methods.When no reagent was added to the sample solution, nine chromatographic peaks were obtained . After tThe response of each component in the EAE extract to the negative ion source is better than that of the positive ion source, which is the same as reported previously , 58. BasTetrastigma hemsleyanum had better scavenging ability to DPPH and ABTS+ free radicals than ethyl acetate extract and hexane extract [3+ was consistent with that of EAE  combined with HPLC-DPPH and HPLC-hydroxyl radical scavenging methods to screen out EAE with high antioxidant activity from


Cellulosimicrobium cellulans strain. We investigated the characteristics of strain D in relation to QNC degradation. Under optimal culture conditions, the QNC degradation rate was 45.9% after 21 days of culture. QNC degradation by strain D in the field was modeled and quantified by a pot experiment. The results show that strain D promotes rice growth and degrades QNC. This research has identified a new bacterial species that degrades QNC, providing a foundation for further research into QNC remediation.Quinclorac (QNC) is a persistent, highly selective, hormonal herbicide of low toxicity. QNC accumulates in soil and affects the growth and development of crops planted subsequent to its application. In this study, we isolated and screened a QNC-degrading bacterial strain, strain D, from rice paddy soil. Morphological analysis, physiological and biochemical tests, and 16S rRNA gene sequencing led us to identify strain D as a IMPORTANCE QNC-degrading bacteria have been isolated from different environments, but there are no reports of Cellulosimicrobium cellulans strains that degrade QNC. In this study, a previously unidentified bacterial strain that degrades QNC, strain D, was screened from paddy soil. The characteristics of strain D that relate to QNC degradation were investigated in detail. The results showed that strain D effectively degraded QNC. Two degradation products of QNC formed by strain D that have not been reported previously, i.e., 3-pyridylacetic acid (m/z 138.0548) and 3-ethylpyridine (m/z 108.0805), were identified using high-performance liquid chromatography–quadrupole time of flight mass spectrometry. Strain D has the capacity to degrade QNC in a QNC-polluted paddy. Quinclorac (QNC) is a highly selective persistent hormonal herbicide of low toxicity. It affects plant metabolism and growth by altering plant hormones. It is principally used to control barnyard grass in rice fields . QNC has5–8–2 P25 nanoparticles were used to photocatalyze the degradation of QNC in ultrapure water irradiated by an 1,100-W xenon lamp. Degradation was complete after 40 min; however, complete degradation took 130 min when paddy water was used (2 P25 is limited to the UV region (320 to 400 nm), and UV light accounts for only 5% of the solar spectrum. Thus, most investigations into photocatalytic degradation of pollutants use irradiation from high-power xenon lamps or UV lamps. These techniques require a lot of electrical energy, which makes large-scale experiments unfeasible. Electrokinetic soil cleansing  degraded 33.5% of QNC after 21 days and the strain was effective in alleviating the harmful effects of QNC on tobacco plant growth  and C. cellulans strain DSM 43879 (GenBank accession no. NR_119095) are clustered in the same branch of the 16S rRNA phylogenetic tree, with homology of 99.93%. Therefore, strain D was identified as a C. cellulans strain.The 16S rRNA sequence of strain D was uploaded to NCBI for nucleotide sequence comparison, and a phylogenetic tree was constructed. The 16S rRNA gene phylogenetic tree of strain D is shown in Strain D had little effect in degrading QNC in strongly acidic (pH < 4) or strongly alkaline (pH > 10) environments, as shown in −1 is shown in −1; strain D degraded 33.8% of the QNC. QNC was the only carbon source for strain D and thus greatly influenced degradation by strain D. The percent degraded increased as the QNC concentration increased within the range of 20 to 50 mg · liter−1. When the concentration became too great, the percent degraded decreased. We explain this as being due to the high physiological toxicity of QNC, which inhibits the reproduction and metabolism of strain D. Lv 2SO4 > NH4NO3 > NH4Cl > urea > peptone. Thus, (NH4)2SO4 was selected as the optimal N source. Strain D showed the greatest degradation of QNC (38.3% in 21 days) when (NH4)2SO4 was used as the N source. When all other parameters were unchanged and the nitrogen source was (NH4)2SO4, the degradation rate for strain D could be increased by 5.9% to 15.8%. In addition, (NH4)2SO4 is an excellent nitrogen fertilizer that is suitable for general soil and crops. It promotes vigorous growth of branches and leaves, improves fruit quality and yield, and increases crop resistance to damage, and it can be used as base fertilizer, top dressing, or seed fertilizer; it is widely used in rice production.The type and content of nutrients affect the degradation ability of microorganisms. By optimizing the N source, degradation by strain D could be increased. The effect of different N sources on the degradation of QNC is shown in Yeast extract is rich in protein, amino acids, peptides, nucleotides, B vitamins, and trace elements. It can be used to supplement N and C sources, and it contains vitamins and micronutrients that promote the growth of microorganisms . The addTemperature importantly affects the growth and survival of microorganisms. The effect of temperature on QNC degradation by strain D was mainly to alter the growth, reproduction, and metabolism of the bacteria. The optimal temperature for bacteria that degrade QNC has been found to be in the range of 25°C to 35°C. The degradation of QNC by strain D at different temperatures, when all other culture variables were unchanged, is shown in −1; inoculation quantity, 7%; yeast extract content, 0.1%; nitrogen source, (NH4)2SO4; culture temperature, 30°C; culture period, 21 days. The degradation of QNC by strain D and the strain D semilogarithmic growth curve when conditions were optimal are shown in Optimal culture conditions were as follows: initial pH, 6; initial QNC concentration, 50 mg · literm/z 138.0548) and 3-ethylpyridine (m/z 108.0805). The degradation products were not quantified due to a lack of standards. These degradation products have not been reported in previous studies of QNC degradation, and the degradation pathways and enzymes involved are still under analysis. We suspect that these compounds are probably new metabolites of QNC that was degraded by strain D. Further tests are needed to verify our hypothesis.The QuEChERS  method was used to extract QNC and its degradation products from the MSM at different stages of culturing. QNC and its degradation products were detected by high-performance liquid chromatography–quadrupole time of flight mass spectrometry (HPLC–Q-TOF MS). Two possible QNC metabolites were detected. The mass spectrum and chromatogram of the detected QNC and the two degradation products are shown in −2 · a−1) and 2 times the recommended dose (750 g · hm−2 · a−1) were applied in soil remediation pot experiments that simulated the rice paddy. In these experiments, some soil was sterilized and QNC-degrading strain D bacteria were added so that we could investigate field bioremediation of QNC. Degradation of QNC in the soil in each treatment group over 28 days is shown in −2 · a−1. Experiments using sterilized soil  (−2 · a−1) (−2 · a−1)   and B, r2 · a−1)  and D. A2 · a−1) . When th2 · a−1)  and D, tSince it was discovered that QNC is naturally degraded principally by microorganisms, many kinds of QNC-degrading bacteria have been screened. However, researchers have mainly investigated degradation characteristics, and there have been no studies of the bioremediation of QNC-contaminated rice fields by QNC-degrading bacteria; therefore, there is no groundwork for the future practical application of QNC-degrading bacteria. In this study, we used highly effective QNC-degrading bacteria and simulated rice fields to investigate the bioremediation of QNC-contaminated rice fields. The results of our experimental work show that QNC-degrading strain D bacteria will adapt to the rice field environment and synergistically degrade QNC in conjunction with naturally occurring rice field soil bacteria. The remediation reduces the adverse effects of QNC on rice plant physiology and growth and indicates that strain D has the potential for widespread application in remediating rice paddies.4·7H2O, 0.2 g CaCl2, 1.0 g KH2PO4, 1.0 g Na2HPO4, 0.05 g FeCl3, 1.0 g NH4NO3, 1,000 ml distilled water); rice paddy soil and rice seeds were all acquired from China Rice Research Institute.The following materials were used in the study: QNC standard , QNC wettable powder , liquid LB medium , solid LB medium , and liquid MSM  were used for the reaction. The amplification reaction components were 1 μl DNA, 8 μl BigDye, 1 μl primer (3.2 pmol · liter−1), and 10 μl sterile deionized water. The reaction protocol was as follows: predenaturation at 96°C for 1 min; 25 cycles of denaturation at 96°C for 10 s, annealing at 50°C for 5 s, and extension at 60°C for 4 min; and storage at 4°C. Amplification products were submitted to Shanghai Shangya Biotechnology Co., Ltd., for sequencing. The sequencing results were uploaded to the NCBI database, and the homology comparison was performed by BLAST in GenBank (http://www.ncbi.nlm.nih.gov). MEGA7.0 software was used for phylogenetic analysis, and the phylogenetic tree was constructed using the neighbor-joining method.Solid LB medium was inoculated with the selected strain and cultured at 30°C for 48 h for observation of the morphological characteristics of the colony. The Gram positivity or Gram negativity of the strain was determined using Gram stain, and bacterial morphology was observed under a scanning electron microscope. Physiological and biochemical kits  were used for physiological and biochemical identification. eriology  was refe600] of 1.0) for later use.Strain D was inoculated into 100 ml liquid LB medium, incubated at 30°C and 180 rpm for 48 h, and centrifuged at 8,000 rpm for 5 min, and the upper layer of LB medium was decanted. The precipitated strain was washed twice in sterile physiological saline solution, and the pellet was resuspended in liquid MSM 2SO4, NH4NO3, and NH4Cl were added to separate cultures at 30°C to observe the effects of the N source. The medium was cultured at 20°C, 25°C, 30°C, 35°C, or 40°C to observe the effects of temperature. Samples were taken at 3, 7, 12, 15, 18, and 21 days after inoculation to determine the concentration of QNC and to calculate the degradation rate using the following equation: QNC degradation rate (%) =  × 100. Each treatment was performed in triplicate. The control was the treatment without inoculation.We investigated the effects on QNC degradation of pH, initial QNC concentration, inoculum amount, yeast extract content, N source, and temperature as follows. The pH of the culture solution was adjusted to 4, 6, 7, 8, or 10, and the solution was cultured at 30°C. The liquid MSM was constituted with QNC concentrations of 20, 50, 100, 200, or 500 mg · liter−1, and the pH was adjusted to 6. The N source, (NH4)2SO4, was added with 0.1% yeast extract powder and 7% bacterial suspension. The medium was cultured at 30°C and 180 rpm. Samples were taken at 3, 7, 12, 15, 18, and 21 days after inoculation to determine the QNC concentration and to calculate the degradation rate. The OD600 value of the bacterial suspension was determined using an UV spectrophotometer . Each treatment was performed in triplicate. The control was the treatment without bacteria.Liquid MSM was prepared with a QNC concentration of 50 mg · liter−1; injection volume, 5 μl. MS parameter settings were as follows: electrospray ionization; positive- and negative-ion full-scan modes; mass scan range, m/z 50 to 1,200; mass scan rate, 4 spectra · s−1. The parameters set for positive- and negative-ion scanning were as follows: drying gas temperature, 350°C; drying gas flow rate, 8 liter · min−1; sheath gas temperature, 280°C; sheath gas flow rate, 11 liter · min−1; atomizing gas pressure, 40 psi; cone fragmentation voltage, 130 V; skimmer voltage, 40 V; radiofrequency voltage, 750 V; positive-ion mode: capillary voltage, 4,000 V; nozzle voltage, 500 V; negative-ion mode: capillary voltage, 3,500 V; nozzle voltage, 1,000 V.Strain D was cultured under optimal conditions for 21 days, and samples were taken at 3, 7, 12, 15, 18, and 21 days after inoculation. The QuEChERS method was used to extract QNC and its degradation products. The degradation products of QNC were detected by HPLC–Q-TOF MS . The HPLC parameters were set as follows: XSelect HSS T3 column ; column temperature, 45°C; mobile phase, aqueous solution containing 0.1% formic acid (vol/vol) (solvent A) and acetonitrile (solvent B) for HPLC; gradient elution, 0 to 5 min, 10% to 40% solvent B; 5 to 11 min, 40% to 95% solvent B; 11 to 15 min, 95% solvent B; 15 to 15.1 min, 95% to 10% solvent B; 15.1 to 21 min, 10% solvent B; flow rate, 300 μl · min−2 · a−1 (the manufacturer-recommended dose) and one with an effective ingredient concentration of 750 g · hm−2 · a−1 (2 times the manufacturer-recommended dose), were prepared for use. Rice seeds were sterilized and soaked to accelerate germination and were cultivated to the two-leaf–one-heart stage in an artificial incubator. Rice seedlings of uniform growth were selected for transplanting.Paddy soil was dried and sieved (2 mm), and a portion of the soil was sterilized. Strain D was inoculated into liquid LB medium, cultured at 30°C and 180 rpm for 48 h, washed twice in physiological saline solution, and resuspended in sterilized water to yield a bacterial suspension for later use. For pesticide preparation, two quantities of QNC mother liquor, one with an active ingredient concentration of 375 g · hm−2 · a−1). For treatment nT-1, normal soil was sprayed with the manufacturer-recommended dose of QNC to which the bacterial suspension had been added and evenly mixed to a concentration of 3.38 × 108 CFU · g−1. For treatment nCK-2, normal soil was sprayed with 2 times the manufacturer-recommended dose of QNC (750 g · hm−2 · a−1). For treatment nT-2, normal soil was sprayed with 2 times the manufacturer-recommended dose of QNC to which the bacterial suspension had been added and evenly mixed to a concentration of 3.38 × 108 CFU · g−1. For treatment sCK-1, sterilized soil was sprayed with the manufacturer-recommended dose of QNC. For treatment sT-1, sterilized soil was sprayed with the manufacturer-recommended dose of QNC to which the bacterial suspension had been added and evenly mixed to a concentration of 3.38 × 108 CFU · g−1. For treatment sCK-2, sterilized soil was sprayed with 2 times the manufacturer-recommended dose of QNC. For treatment sT-2, sterilized soil was sprayed with 2 times the manufacturer-recommended dose to which the bacterial suspension had been added and mixed evenly to a concentration of 3.38 × 108 CFU · g−1. Each treatment was performed three times. After treatment, random multipoint sampling was used at 0, 3, 7, 14, 21, and 28 days to collect rice plants and soil samples. Soil samples were analyzed to detect QNC residues. The plant height, root length, stem dry weight, stem fresh weight, root dry weight, and root fresh weight of rice plant samples collected at 7 days and 21 days were measured. Then SPSS 25 software was used to perform one-way analysis of variance and multiple-comparison tests.Selected rice seedlings of a variety that grows consistently and is robust were transplanted into potting buckets (25 cm by 20 cm) with 6 kg of soil in each bucket. After 7 days, 8 treatments were administered, as follows. For treatment nCK-1, normal soil was sprayed with the manufacturer-recommended dose of QNC (375 g · hmCellulosimicrobium sp. strain D was deposited in GenBank under the accession no. MW404399.The complete 16S rRNA gene sequence of


Circular RNA PVT1 (circPVT1) is significantly upregulated in various human cancers and is related to poor clinical outcome of cancer patients. However, the prognostic and clinicopathological value of circPVT1 in diverse human cancers remains controversial and inconclusive.The objective of our study is to evaluate the prognostic and clinicopathological role of circPVT1 for cancer patients.P < .001). For clinicopathological value, upregulation of circPVT1 was closely related to poor clinical parameters lymph node metastasis , late clinical stage , distant metastasis , and chemoresistant .PubMed, Embase, Web of Science, and Cochrane Library were searched for eligible studies by October 1, 2020. The correlation between circPVT1 expression, and overall survival (OS) and clinical parameters was assessed by pooled hazard ratios (HRs) and odds ratios (ORs) with 95% confidence intervals (CIs). Subgroup analyses, heterogeneity, and publication bias were conducted to further enhance reliability. Twelve studies (1282 patients) were selected for this meta‐analysis, including 11 on prognosis and 10 on clinicopathological parameters. Elevated expression of circPVT1 was associated with a worse OS in cancer patients (HR, 2.009; 95% CI, 1.667‐2.408, 1.892; High expression of circPVT1 is correlated with unfavorable prognosis of cancer patients, indicating that circPVT1 can function as a potential prognostic biomarker in human cancer. If HRs and 95% CIs were not directly provided, they were extracted from Kaplan‐Meier curves according to Tierney's method.Two researchers (ZJL and LW) extracted the following data from eligible studies: first author name; publication year; country; type of cancer; sample size; detection method; cut‐off value; prognostic parameters of circPVT1 including HRs, corresponding 95% CIs and 2.4http://markummitchell.github.io/engauge-digitizer/) according to Tierney's method.I2 statistics and Cochran's Q test were used to identify the heterogeneity of the included studies. If I2 ≤ 50% and P > .05, a fixed‐effect model test was utilized to analyze the results. Otherwise, a random‐effect model test was applied for analysis.P < .05 was considered to be statistically significant. Publication bias was qualitatively determined using funnel plot analysis, and quantified by Egger's test and Begg's test. Subgroup analysis and sensitivity analysis were conducted to evaluate the source of heterogeneity and stability of the results.Statistical analysis was performed using STATA 12.0 software. The correlation between circPVT1 expression and clinicopathological characteristics was evaluated by ORs and 95% CIs. The HR and 95% CI were directly extracted from the original publications. If the study only provided Kaplan‐Meier survival curve, HR and 95% CI were synthesized by Engauge Digitizer 11.2 software , non‐small cell lung cancer (NSCLC), breast cancer (BCa), head and neck squamous cell carcinoma (HNSCC), hepatocellular carcinoma (HCC), colorectal cancer (CRC), osteosarcoma, papillary thyroid carcinoma (PTC), and gastric cancer (GC). The circPVT1 expression was measured by qRT‐PCR in all studies. Eleven studies were performed to analyze OS. Three included studies directly provided HR and 95% CI data while remaining eight studies only provided Kaplan‐Meier survival curves. Meanwhile, 10 studies were performed to analyze the clinicopathological parameters. The NOS score of 11 studies was ≥7, indicative of high quality of the majority of the studies included in our research (Table 3.3P < .001). A fixed‐effects model was selected since no heterogeneity was reported  Figure , sample 3.4.1P = .005; I2 = 56%; PH = 0.103), bone neoplasms , digestive system neoplasms , and other neoplasms   Figure .3.4.2P < .001; I2 = 22.5%, PH = 0.271) and small sample size (n < 95) , upregulation of circPVT1 was significantly related to worse OS of cancer patients  , or mean value was used  or other cutoff values were used , there was close relationship between high circPVT1 expression and poor OS in patients of multiple cancers  or indirectly from Kaplan‐Meier survival curves in the literature   Figure .3.5P = .042; I2 = 77.5%; PH = 0.000), late clinical stage , distant metastasis , and chemoresistant . However, no correlation between circPVT1 expression and age, gender, histological grade, tumor size, and T stage was detected in our results. Specially, we found two studies of Chen et al and Kong et al on GC might cause oscillation to our results. After omitting the two studies, we detected a significant correlation between increased expression of circPVT1 and tumor size  and T stage ; and a more significant correlation between high circPVT1 expression and lymph node metastasis  and distant metastasis . Similarly, there was still no correlation between circPVT1 and age, gender, and histological grade.The correlation between circPVT1 and the clinicopathological parameters is shown in Table 3.6P = .213 and Egger's test: P = .073 also indicated there was no evident publication bias in publications reported OS (Figure We conducted funnel plot analysis, Egger's, and Begg's tests to assess potential publication bias for OS. The funnel plot of OS revealed no evident asymmetry, indicative of no obvious evidence of publication bias for OS Figure . MeanwhiS Figure . The senS Figure .4YAP1 via sponging miR‐30a‐5p in GC in both vivo and vitro.CircPVT1, as an oncogenic circRNA, has been found to be upregulated in various human cancers, such as GC, CRC, BCa, and osteosarcoma.In our study, we demonstrated that upregulation of circPVT1 had a significant correlation with poor OS of human cancers. As for the subgroup analysis, the results showed that all subgroup analyses by tumor type, sample size, cutoff value, and data‐obtained method did not have a significant impact on the prognostic value of circPVT1 in human cancer. Regarding the types of tumors, the results suggested that upregulation of circPVT1 was significantly associated with worse OS in bone neoplasms, lung neoplasms, and digestive system neoplasms, respectively. In terms of the association between circPVT1 expression and clinicopathological features, we demonstrated that high circPVT1 expression was remarkably related to lymph node metastasis, late clinical stage, distant metastasis, and chemoresistant. Interestingly, a significant correlation between circPVT1 expression and tumor size, T stage, lymph node metastasis, late clinical stage, distant metastasis, and chemoresistant was detected after omitting the two studies on GC. Therefore, further studies are needed to investigate the relationship between circPVT1 expression and clinical features in GC and other cancer types. In conclusion, the results of the current meta‐analysis indicate that circPVT1 is a promising prognostic and clinical biomarker for various human cancers.However, several limitations should be taken into consideration in this meta‐analysis. Firstly, most eligible studies in the meta‐analysis were conducted in China and there may exist ethnic bias, thus weakening the representativeness of the study results. Meanwhile, only studies published in English were enrolled in the meta‐analysis, and studies with negative results may not have been published, which may contribute to selection bias and publication bias. Secondly, the number of studies in some major tumor types, such as bone neoplasms, was limited, which may influence the results of subgroup meta‐analysis based on cancer types. Also, there was only one study researching the prognostic value of circRNA in some cancer types, such as HNSCC, PTD, and HCC, so it was difficult to identify the correlation between circPVT1 expression and clinical outcome in those cancers. Thirdly, several studies only provided the survival curve, so the indirectly extracted HRs and 95% CIs may cause some errors and lead to potential source of bias. Fourthly, the cutoff value for distinguishing high or low circPVT1 expression levels was inconsistent, which may account for potential heterogeneity. Finally, the sample size of patients in this meta‐analysis was relatively small, so more studies and large samples would be necessary to better explore the correlation between circPVT1 and prognosis and clinical characteristics in patients of various cancer types.5In conclusion, upregulation of circPVT1 is significantly correlated with worse OS in human cancers. This study indicates that circPVT1 can function as a promising prognostic and clinical biomarker, and targeting circPVT1 may be a novel therapeutic strategy for human cancer. Due to the limitations in this meta‐analysis, future experimental research and clinical trials are required to verify the prognostic and clinicopathological value of circPVT1 in human cancers.The authors declare that they have no competing financial interests.Conceptualization, Z.L., L.L.; Data Curation, Z.L., X.T., L.W., L.L.; Formal Analysis, Z.L., X.T., L.W., L.L.; Investigation, Z.L., X.T., L.W., L.L.; Methodology, Z.L., X.T., L.W., L.L.; Project Administration, Z.L., X.T., L.W., L.L.; Resources, Z.L., X.T., L.W., L.L.; Software, Z.L., X.T., L.W., L.L.; Validation, Z.L., L.L.; Visualization, Z.L., X.T., L.W., L.L.; Writing‐Original Draft, Z.L., X.T., L.L.; Supervision, L.L.; Writing‐Review & Editing, L.L.All authors had full access to the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Not applicable.


Near 100% of diffractive efficiency for diffractive optical elements (DOEs) is one of the most required optical performances in broadband imaging applications. Of all flat DOEs, none seems to interest researchers as much as Two-Materials Composed Diffractive Fresnel Lens (TM-DFL) among the most promising flat DOEs. An approach of the near 100% of diffractive efficiency for TM-DFL once developed to determine the design rules mainly takes the advantage of numerical computation by methods of mapping and fitting. Despite a curved line of near 100% of diffractive efficiency can be generated in the Abbe and partial dispersion diagram, it is not able to analytically elaborate the relationship between two optical materials that compose the TM-DFL. Here, we present a theoretical framework, based on the fundaments of Cauchy's equation, Abbe number, partial dispersion, and the diffraction theory of Fresnel lens, for obtaining a general design formalism, so to perform the perfect material matching between two different optical materials for achieving the near 100% of diffractive efficiency for TM-DFL in the broadband imaging applications. The so-called DFL is generally referred to p = 1. According to the basic diffractive optics theory of DFL presented by Dale A. Buralli12, as shown in Fig. i is the i-th FZ radius, 0 is the design focal length, 0 is the design wavelength, i is the spacing between the i-th FZ and the i + 1-th FZ, h is the FZ height, D is the FZ diameter, and rmax is the outermost FZ radius.Usually, exploiting the 2pπ phase (where p is a positive integer) as the Equal Optical Path Difference is the way to configure a diffractive Fresnel lens (DFL)R, fG, and fB presenting the focal points focused by the red, green, and blue light respectively.Due to the dispersion, generally the parallel incident white light with different wavelengths, ranging from 400 to 700 nm, is focused by DFL at different focal points on the optical axis. The optical power varies linearly with the wavelength of the incident light, as shown in Fig. 12, the diffraction focus and efficiency of a light source with a single wavelength can be determined by the following equations.0 is the design wavelength, ƒ0 is the design focal length, 0) is the refractive index at λ = λ0.Furthermore, according to the above-mentioned theory of DFL19) is calculated by Eqs.  of the optical materials of PMMA , an, an19) i by Eqs.  for the 0 = 0.587 µm, and α = 1. Equation ,,7), 100%Consequently, the total diffractive efficiency from m = 1 to m = 3 comes up with 20. In their research result, a blazed grating, composed of two optical materials, including glass (Schott BaF52 glass) and PC (polycarbonate), was proposed to improve the diffractive efficiency for the diffracted order at m = 1 (i.e. α = 1) by exploiting the refractive index difference Δn(λ) = n1(λ) − n2(λ) being proportional to wavelength λ. When the refractive index difference satisfies Δn(λ)/λ=constant at all wavelengths λ in the spectrum of visible light, all the incident light energy in the higher diffracted order is all transferred into the first diffracted order, as to achieve the objective of 100% diffractive efficiency. Furthermore, B. H. Kleemann mentioned the design concepts for the blazed grating composed of two optical materials in 200821 and particularly named such structure as Common depth EA–DOEs. In contrast to the term “Common depth”, a singlet DFL composed of two optical materials is called Two-Materials Composed Diffractive Fresnel Lens (TM-DFL) in this study.A solution to make up for the energy loss in the first diffracted order was first presented by Kenneth J. Weible in 199920, two transparent optical materials of glass22 and PC23, as shown in Fig. 2(λ) and n2(λ0) of the second material.According to Kenneth’s research by Eqs. , and 8)20, two tAlthough TM-DFL can improve the diffractive efficiency of the conventional SM-DFL, the requirement for Δn(λ) 24 indicated that in the case of SM-DFL composed with materials existing in nature, the detuning factor α in Eq.  to satisfy the requirement for 25, the so-called nanocomposites are produced by adding a proper volume fraction of nanoparticles with a diameter smaller than 5 nm, such as Diamond, ZrO2, TiO2, ITO (indium tin oxide), and AZO  (presented as green stars), to the existing polymeric materials, such as PMMA (poly(methyl methacrylate)), COP (cyclic olefin copolymer), PC (polycarbonate), and PS (polystyrene) (presented as blue stars), to adjust the optical parameters of materials nd, vd, and Pg,F. Though such new nd, vd, and Pg,F (presented as orange points) do not exist in nature, they can be tailored within a certain range (presented as pink area) in both the Abbe diagram and partial dispersion diagram, as respectively shown in Fig. 25. For instance, by adding TiO2 nanoparticles with different volume fraction to the polymeric material PC, the distribution of nd-vd can extend to cover a region in 17 < vd < 28 and 1.6 < nd < 1.85 while the distribution of PgF-vd covers a region in 17 < vd < 28 and 0.58 < PgF < 0.65.To overcome the above-mentioned problems, Daniel Werdehausen in 201926 proposed a design method of mapping and fitting based on the numerical computation for matching the material refractive index of TM-DFL to achieve the light diffractive efficiency higher than 99.9%. According to DOEs’ phase profiles change across the different dispersion regimes26, the material parameters of material 1(Mat.1) are first selected and set at nd,1 = 1.8, vd,1 = 60, and Pg,F,1 = 0.55. By the method of mapping, the material parameters nd,2, vd,2, and Pg,F,2 of material 2(Mat. 2) are discretely varied to calculate the distribution of the average diffractive efficiency d,2, vd,2) and  respectively. The subscript Mat.1 in both function d,1, vd,1, and Pg,F,1 of Mat.1 being fixed as a constant.Furthermore, the same research team in 2020d,2, vd,2, Pg,F,2 of Mat. 2 in a large region to calculate and set up both diffractive efficiency map of d,2, vd,2, Pg,F,2 of Mat. 2 matches to the selected nd,1, vd,1, and Pg,F,1 of Mat.1 for achieving the near 100% diffractive efficiency. Hereafter, the black dotted curved line in d,2, vd,2, and Pg,F,2 can be determined by comparing the producible nanocomposites for completing the optimal match of parameters in two nanocomposite materials.The major feature of the mapping method is to discretely modulate nd,1 = 1.8, vd,1 = 60, and Pg,F,1 = 0.55. To acquire the general relationship between a, b, c, and the material parameters of MAT.1, ten of the Abbe characteristic curves with different values of nd,1, ranging from 1.5 to 2.0, are used to fit to obtain the nd,1 dependence of a, b, and c as below.Furthermore, applying the mathematical fitting to the Abbe characteristic curve, a natural exponential function is obtained, as below.26.However, the fitting to the partial dispersion characteristic curve, is not discussed26 confronts the following issues.The methods of mapping can obtain a single Abbe characteristic curve and partial dispersion characteristic curve through a big volume of numerical computations at a time while losing efficiency and accuracy.d,1 in a special form of a natural exponential function with 1/3 power. In addition to the fitting accuracy or error, it is not a general equation for the analytical evaluation of the system. The so-called “analytical evaluation” refers to analyze the variance in the entire system caused by the parameter variation without using massive numerical computations. In other words, it is a qualitative and quantitative way to get insight into the general physical behaviors of a system by theoretical formulas.The fitting method can describe the Abbe characteristic curve as Eq.  which isd,1 of Mat.1 without any further relation with vd,1 and Pg,F,1 of Mat.1.Three coefficients a, b, and c of the Abbe characteristic curve merely contains nd,1, vd,1, and Pg,F,1) and  between two materials.There is no fitting applied to the partial dispersion characteristic curve for the analytical evaluation on the relationship among parameters, i.e.  comp comp9) c0 is the design wavelength. Further, “1” in the Δn is the refractive index of air. That is, the incident light directly contacts the air after going through DFL. Since the refractive index of air is almost irrelevant to 0. Therefore, TM-DFL is used to effectively overcome the efficiency issue caused by the refractive index of air. For this purpose, Eq. . First, nd, nF, nC, ng and λd, λF, λC, λg are substituted into Eq.  of transparent optical materials in the spectrum of visible light can be calculated by Cauchy's Eq. .16\documCoefficients A, B, and C are solved as below.d, vd, Pg,F, and wavelengths of Fraunhofer d, F, C, and g spectral lines.Coefficients A, B, C of Cauchy's equation in Eqs. –25) cle cle25) c0 = λd into Eqs. 0 = λd inBy expanding Eq. , it obtad,2 of Mat.2 be defined as belowd,2, nF,2, and nC,2 are the refractive indices of Mat.2 at the wavelengths of the Fraunhofer d, F, and C spectral lines. Substituting Eq. /.For TW-FDL, Eqs. –31) rel rel31) rn in Eq. , let thed,1 is the Abbe number of Mat.1. Accordingly, both formulas 35\documed,2 in Eq. 35) can a2 in Eq.  will be g,F,2 of Mat.2 be defined as below.g,2, nF,2, and nC,2 are the refractive indices of Mat.2 at the wavelengths of the Fraunhofer g, F, and C spectral lines. Substituting Eq. 39) depicg,F,2 in formula 39) can ad2 and Pg,F,2 in formula d2 and Pgd2 and Pg,F,2 against the light wavelength λd, λF, λC, and λg because it is no reason to change the definition of Fraunhofer line.Clearly, It is no necessary to apply the same process of partial differential to both n26, formulas , . In other words, the mapping and fitting methods cause non-negligible errors. Regarding the partial dispersion characteristic curve depicted on the partial dispersion diagram in Fig. 26.In contrast, in our studies, the Abbe and partial dispersion characteristic curves are obtained by our formulas  and 39)39) respeIn summary, a theoretical formula-based analytical method is proposed in our studies to improve the disadvantages of the numerical computational-based mapping and fitting method. More definitely, the theory of Cauchy's equation, Abbe number, partial dispersion, and the diffractive theory of Fresnel lens are blended into optically connecting two different nanocomposite materials in TM-DFL for achieving the near 100% diffractive efficiency with all wavelengths in the visible spectrum at the first diffracted order. In addition to perfectly matching optical parameters between two materials without numerous computations, it also satisfies the objective of general analysis for TM-DFL in both quantitative and qualitative evaluations. The major features of the optical behavior of TM-DFL in our study are elaborated below.d,2 at nd,1 = 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, and vd,1 = 50, 40, 30. When vd,2 is fixed, it shows a feature: the larger nd,1, the larger nd,2. When nd,1 is fixed, nd,2 is split into a subset of lines at vd,1 = 30, 40, 50, for showing another feature: the less vd,1, the larger nd,2. Apparently, the Abbe characteristic curves of Mat.2 is nothing to do with Pg,F,1 because Pg,F,1 is not included in formula  in formula  nd,1 = 2, 1.75, 1.5, vd,1 = 30, and Pg,F,1 = 0.7, 0.55, 0.35, are done and shown in Fig. g,F,2  depends on Pg,F,1 and vd,1 only, but not depend on nd,1.The sum of terms in the square brackets in Eq.  is alway formula , under cg,F,2 in formula  being a simple form used to replace the term of Pg,F,2), show the data of Pg,F,2 at different row i which is calculated by formula /Δvd,2(i), show the local slope by directly dividing ΔPg,F,2(i) = Pg,F,2(i) − Pg,F,2(i + 1) by Δvd,2(i) = vd,2(i) − vd,2(i + 1). As a result, the quantitative calculations in Table g,F,2 when the equality of ΔPg,F,2(i)/Δvd,2(i) = S(i) = constant is satisfied at all i = 1, 19.Let’s move back to the partial dispersion characteristic curve P formula  with the formula . Another formula  with thed in Eq. . Furtherd,2, Pg,F,2, vd,2 in Mat.2 can match up with the predetermined materials nd,1, Pg,F,1, and vd,1 in Mat.1 to satisfy the near 100% diffractive efficiency.Further, let’s check out how well the material parameters n1 , Pg,F,2 respectively. Then, the detuning factor 1, n1into Eq. . Finallyinto Eq. , the difuchy Eq. . For theinto Eq. , then n2g to Eq. . Finallyd,2(vd.2) and the partial dispersion characteristic curve Pg,F,2 are calculated according to formula  = λd/) where nd,1 ≡ n1 (λd) and nd,2 ≡ nd,2, to guarantee the near 100% diffractive efficiency at the first diffracted order and all wavelengths of the incident light in the visible spectrum. Interestingly, there exists a singularity of h at vd,2 = 60 where vd,2 = vd,1 and nd,2 = nd,1. Consequently, the optical behavior of TM-DFL is categorized into three regions as below.d,2 = vd,1 = 60 and nd,2 = nd,1 = 1.8. In other words, both FZ height h and focal length f0 approach to infinity when TM-FDL is composed of two same optical materials,Transparent region: As shown in Fig. d,2 < vd,1, h > 0, nd,2 < nd,1, and Pg,F,2 < Pg,F,1Focusing region: As shown in Fig. d,2 > vd,1, h < 0, nd,2 > nd,1, and Pg,F,2 > Pg,F,1Divergent region: As shown in 7(b) and 7(c), TM-DFL is equipped with the divergent power when vIn general, for the conventional DFL, the diffractive efficiency is determined by the FZ height h, the refractive index difference Δn, and the incident light wavelength λ while the optical focusing power is determined by the Δn and the curvature 1/R of the surface relief of DFL. Let’s take up the example used in Fig.  formula  and 39)d,2(vd.2)d,2 and Pg,F,2 is elaborated. Also, physical behaviors of nd,2 and Pg,F,2 are illustrated and proved, including (1) the independence of Pg,F,1 in nd,2, (2) the independence of nd,1 in Pg,F,2, (3) the linearity and constant slope of Pg,F,2, (4) beyond 0.05% of the theoretical error in the calculation of diffractive efficiency, and (5) the optical behavior of convergence and divergence. We believe that our new approach will be an effective and precise way to achieve a near 100% diffractive efficiency for the design of TM-DFL.In our studies, we develop a theoretical framework to obtain a general formalism for the design of TM-DFL in broadband imaging applications. Unlike the existed approach of the numerical computation based methods of mapping and fitting, the optical theories related to Cauchy's equation, Abbe number, and partial dispersion, as well as the diffraction theory of Fresnel lens, have been perfectly blended into a new foundation for working out a TM-DFL with a precise material matching that can theoretically achieve a near 100% diffractive efficiency. The derivation of Equations for the calculations of n