Gegen-Qinlian decoction (GQD) is a classic traditional Chinese medicine (TCM) formula. GQD is effective against colon or liver-related diseases including ulcerative colitis, non-alcoholic fatty liver, and type 2 diabetes. In this study, a liquid chromatography-tandem mass spectrometry method was developed, validated, and then applied to reveal the tissue distribution and integrated pharmacokinetic properties of major effective constituents of oral GQD in mice. The established method was quick, sensitive, and accurate enough to analyze GQD constituents in plasma and tissue homogenate samples quantitatively. According to their concentrations in the portal vein, systemic circulation, liver and colon samples of the mice after oral administration of GQD, the concentration-time curves of the constituents were respectively plotted. The results showed that daidzein, baicalin, and baicalein had relatively high exposure levels in the livers, while puerarin, berberine, epiberberine, coptisine, palmatine, jatrorrhizine, magnoflorine, glycyrrhizic acid, and glycyrrhetinic acid were enriched in the colons. Given that these constituents have significant biological activity, they could be regarded as the major effective constituents of GQD in treating colon or liver-related diseases, respectively. In addition, the integrated pharmacokinetic properties of GQD were studied. The GQD “integrated constituent” reached peak concentration at 4.0 h in the portal vein, the systemic circulation, the livers, and the colons, with half-lives of 1.5–4.1 h and mean retention time of 4.5–6.3 h, respectively. Furthermore, the concentration of the GQD “integrated constituent” in the colons was approximately 10 times higher than that in the livers, both of which were much higher than that in the systemic circulation, indicating its accumulation in these tissues, especially in the colons. In conclusion, the tissue distribution and integrated pharmacokinetic properties of oral GQD were revealed in the study. The results of the tissue distribution study would contribute to identifying the major target tissues and effective constituents of GQD, while the results of the integrated pharmacokinetic study would help to explain the pharmacokinetic properties of oral GQD as a whole. Gegen-Qinlian decoction (GQD) is a classic traditional Chinese medicine (TCM) formula, which is composed of four TCMs including Pueraria Lobatae Radix (PR), Scutellaria Radix (SR), Coptidis Rhizoma (CR), and Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle (GR) according to the ratio of 8: 3: 3: 2  is one of the basic pharmacokinetic parameters to establish a dosage regimen of drugs. However, it was not convictive enough to formulating the dosing regimen of GQD based on the T1/2 value of any single constituent or single type of constituents. A strategy of “integrated pharmacokinetic study of multiple constituents of TCM” was put forward to resolve the common problem faced by TCMs  of each constituent. Finally, the pharmacokinetic parameters of the “integrated constituent” are calculated according to its concentration-time (C-T) curve. The integrated pharmacokinetics of classical TCM formulas including Huanglian-Jiedu decoction , a rapid and sensitive quantitative analysis method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. The method was fully validated with plasma matrix and partially validated with liver homogenate matrix. The integrated pharmacokinetics of the GQD constituents in blood  and major target tissues  were then comparatively studied in mice. Based on the obtained data, the tissue distribution and the integrated pharmacokinetic properties of GQD were revealed.PR (batch number 190910), SR (batch number 191207), CR (batch number 190820), and GR (batch number 191118) were all purchased from Shanghai Kangqiao Herbal Pieces Co., Ltd. . According to the Pharmacopeia of the People’s Republic of China (2020 edition), the crude drug were identified as the dried root of Pueraria lobata (Willd.) Ohwi, Scutellaria baicalensis Georgi, Coptis chinensis Franch., and Glycyrrhiza uralensis Fisch. respectively. The voucher specimens were deposited in Shanghai University of Traditional Chinese Medicine.The crude drug of The reference compounds of berberine hydrochloride, baicalein, baicalin, wogonin, daidzein, puerarin, glycyrrhizic acid, glycyrrhetinic acid, liquiritin, isoliquiritin, and jatrorrhizine hydrochloride were purchased from Shanghai Yuanye Biological Co., Ltd. . The reference compounds wogonoside, daidzin, palmatine hydrochloride, coptisine hydrochloride, mycophenolic acid, naringin, and berberrubine were purchased from Dalian Meilun Biotechnology Co., Ltd . The reference compound liquiritigenin was purchased from Chengdu Must Bio-technology Co., Ltd . The reference compounds magnoflorine, demethyleneberberine, and epiberberine were purchased from National Institutes for Food and Drug Control . The purity of all the reference compounds was greater than 98%.Methanol and acetonitrile were the products of Burdick and Jackson . Ammonium formate, formic acid, and acetic acid were purchased from Thermo Fisher Scientific Inc. . All the materials were HPLC grade.Clean grade male and female Kunming mice (weighted at 22–24 g) were used in this study. The mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. , which has a production license number of SCXK (Beijing) 2016-0011. The mice were raised in the experimental animal center of Shanghai University of traditional Chinese medicine, which has the experimental license of SYXK (Shanghai) 2020-0009. The ambient temperature of the feeding room was maintained at 22–24°C, with a 12-h dark/light cycle. Before the experiment, all animals were fasted for 12 h but could drink freely. The animal experiments were approved (PZSHUTCM211129015) by and conducted in accordance with the guideline of the Institutional Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine.Stock solutions of the reference compounds were prepared in dimethylsulfoxide at the concentration of 1.0 mg/mL. A series of standard working solutions of the reference compounds, including those of quality control (QC) samples, were prepared by diluting the stock solutions with methanol. The internal standards (ISs), naringin and mycophenolic acid, were dissolved in methanol (1.0 mg/ml) and then diluted with methanol to obtain the working solutions that contained 20 μg/ml naringin and 150 ng/ml mycophenolic acid, respectively. All the solutions were stored at 4°C and were brought to room temperature (about 22°C) before use.Blood of the mice  was mixed fully with heparin and 5% volume of ascorbic acid solution . Then the mixed solution was centrifuged at 4°C for 10 min at the speed of 8,000 rpm to obtain plasma. The plasma samples for calibration curves and QCs were prepared by adding 2 µl the corresponding mixed working solutions of the reference constituents to 38 µl blank plasma.Sequentially, 10 µl mixed working solution of the ISs, 20 µl ammonium formate aqueous solution (100 mM), and 120 µl acetonitrile were added to 40 µl the plasma samples . After shaking for 3 min, the mixtures were centrifuged for 10 min at 4°C at the speed of 14,000 rpm. 180 µl supernatant was collected and then dried with nitrogen flow at 40°C. The residue was dissolved in 90 µl solution composed of methanol, water, and acetic acid at the volume ratio of 70: 29.9: 0.1. The solution was mixed by shaking for 1 min, and then centrifuged for 10 min at 4°C at the speed of 14,000 rpm. The supernatant was collected and injected in the LC-MS/MS system for analysis.The liver and the colon homogenates were prepared with 10 times (V: W) the volume of ascorbic acid solution (200 μg/ml). Then the homogenates were pretreated as plasma to obtain the tissue samples for LC-MS/MS analysis.The LC-MS/MS analysis was performed using an ACQUITY UPLC  equipped with an autosampler, and an API QTRAP 6500 + mass spectrometer  equipped with an electrospray ionization source.18 column , a product of Waters technology (Shanghai) Co., LTD . The mobile phase was composed of phase A  and phase B (methanol). The elution gradient program was set as below: 0 min, 10% B; 1 min, 40% B; 5 min, 60% B; 10 min, 70% B; 12 min, 90% B; 18 min, 90% B; 18.1 min, 10% B; 20 min, 10% B. The flow rate was 0.3 ml/min. The column temperature and autosampler temperature were maintained at 40 and 4°C, respectively. The injection volume was 2 μl.The chromatographic separation was performed using an ACQUITY BEH CThe working parameters for the mass spectrometer were set as follows: ion-source temperature 500°C; spray voltage 4500 V (+) or 4500 V (-), respectively; atomization gas pressure 50 psi; auxiliary gas pressure 50 psi, and curtain gas pressure 35 psi. Naringin and mycophenolic acid were used as the positive and negative IS, respectively. Quantification was performed using the multiple reactions monitoring mode. The parameters including declustering potential, collision energy, collision cell exit potential, and transitions for the analytes were listed in The calibration plasma samples were prepared by adding the mixed working solutions of the constituents to blank plasma at eight concentrations. The linear ranges of the constituents were listed in Full and partial methodological validation were performed respectively for the plasma and the liver homogenate samples in terms of specificity, calibration curves, lower limit of quantification (LLOQ), precision and accuracy, carry over, recovery, matrix effect, dilution linearity, and stability .PR was decocted separately for 20 min, and then decocted together with SR, CR, and GR at the weight ratio of 8: 3: 3: 2 for another 1 h. The aqueous extract was filtered through four layers of gauze. Then the residue was extracted again with 10 times volume of water for 1 h. The filtrates obtained from the two decoctions were combined and vacuum-dried at 60°C to obtain the powder of GQD extract.The herbal pieces were extracted with 10 times volume of boiling water for twice. For the first time, the herbal piece of The contents of 17 constituents in the GQD extract were measured. Briefly, the extract powder was dissolved in the water solution of methanol  at the concentration of 1 mg/ml. The solution was treated with ultrasound for 60 min and then centrifuged at 14, 000 rpm for 10 min. The obtained supernatant was diluted and mixed with the solution of the ISs, and then injected into a LC-MS/MS system for quantitative analysis. The instrumentation and conditions was mostly the same as those described in section “2.3.3”. However, the elution gradient program was set as below: 0 min, 10% B; 1 min, 10% B; 5 min, 40% B; 11 min, 60% B; 15 min, 70% B; 24 min, 90% B; 26.1 min, 90% B; 28 min 10% B. The mass spectrometry parameters of the constituents and ISs were listed in Fifty-four Kunming mice were randomly divided into nine groups according to body weight and gender . Each group was orally administered with water solution (0.2 ml/10 g body weight) of the GQD extract powder at the dosage of 6.1 g/kg body weight. The dosage of GQD extract powder equals to 18 g/kg herbal pieces of GQD, which is equivalent to its clinical dosage. At each designed time point  after oral administration, six mice were anesthetized with ether, and blood was collected from the portal vein  and then the systemic circulation  of the mice to prepare plasma samples, and the livers and the colons were removed to prepare respective homogenate. After pretreatment, the concentrations of 19 major constituents of GQD in each sample were measured by the validated LC-MS/MS method (see “2.3”). Then the C-T curves of the constituents in the plasma and the tissue homogenates were respectively drawn and their pharmacokinetic parameters were respectively calculated.0–24 h values of the constituents were summed up to obtained the ∑AUC0–24 h value  was performed to obtain the pharmacokinetic parameters including peak concentration (Tmax), time to reach peak concentration (Cmax), area under the concentration time curve (AUC0–24 h), elimination half-life (T1/2), and mean retention time (MRT). It should be noted that the pharmacokinetic parameters of a GQD constituent was calculated based on its average concentration in six mice at each time point. The results were expressed as mean ± SD.A non-compartmental analysis using the WinNonlinPR, 1.0–5.0 ng/ml for four constituents  of SR, 0.3–1.0 ng/ml for eight constituents  of CR, 3.1 ng/ml for two flavonoids (liquiritin and liquiritigenin) and 25.0–30.0 ng/ml for two saponins (glycyrrhizic acid and glycyrrhetinic acid) of GR. As shown in There was no endogenous interference of the GQD constituents and the ISs in the blank plasma matrix . The LLOThe LLOQs  were mosPR, puerarin had the highest content, and the content of daidzin was much higher than daidzein. Among the four constituents of SR, the content of baicalin was the highest, followed by wogonoside, both were much higher than baicalein and wogonin, respectively. Among the seven constituents of CR, the content of berberine was the highest, followed by several other alkaloids such as epiberberine, and the content of demethyleneberberine was much lower than above alkaloids. In terms of the GR, the content of glycyrrhizic acid was the highest, followed by liquiritin and isoliquiritin, while glycyrrhetinic acid was not detected.A total of 992 g herbal pieces were decocted with a yield of 33.9% to obtain 336 g of GQD extract powder. The total and extracted ion chromatograms of GQD constituents in water solution were shown in PR, baicalin and wogonoside derived from SR, berberine and epiberberine derived from CR, glycyrrhetinic acid and liquiritigenin derived from GR, had relatively high exposure levels (Cmax and AUC0–24 h) in the portal vein of the mice. These eight constituents could thus be considered as the major absorbed constituents of GQD after oral administration. Among these constituents, daidzein and puerarin were absorbed the fastest with Tmax at 0.25–0.50 h; wogonoside, epiberberine, and liquiritigenin had Tmax at 2.0 h, while baicalin, berberine, and glycyrrhetinic acid was the slowest in terms of absorption, with the Tmax at 4.0 h. Among these constituents, berberine had the longest T1/2, which was at 8.0 h; the T1/2 of epiberberine, glycyrrhetinic acid, and liquiritigenin were within 4.0–6.0 h, while the T1/2 of puerarin, daidzein, baicalin, and wogonoside were within 2.0–3.0 h.The complete C-T curves of 18 GQD constituents (except demethyleneberberine) were obtained from the portal vein samples of the mice . The phaPR, baicalin and wogonoside derived from SR, berberine and epiberberine derived from CR, and glycyrrhetinic acid derived from GR had relatively high exposure levels in the systemic circulation of the mice. Therefore, these seven constituents were the major GQD constituents entering the systemic circulation. Among these constituents, daidzein and puerarin were absorbed the fastest with Tmax at 0.25–0.50 h; the Tmax of berberine was 1.0 h; the Tmax of epiberberine was 0.08 h, but actually the C-T curve of epiberberine showed double peak, with the time to reach the second peak at 1.0 h; the Tmax of baicalin, wogonoside and glycyrrhetinic acid was all at 4.0 h. Among these constituents, the T1/2 of daidzein is 1.50 h, the T1/2 of puerarin, baicalin, and wogonoside was within 3.0–4.0 h, while glycyrrhetinic acid, berberine, and epiberberine were all about 6.0 h.Except for demethyleneberberine, berberrubine, liquiritigenin, and glycyrrhizic acid, the complete C-T curves of 15 GQD constituents were obtained in the systemic circulation . The phaPR, baicalein, baicalin and wogonin derived from SR, berberine, epiberberine, and berberrubine derived from CR, glycyrrhetinic acid and liquiritigenin derived from GR had relatively high exposure levels in the livers of the mice. Therefore, these ten constituents were the major GQD constituents distributed in the livers. Among these constituents, daidzein, puerarin, and liquiritigenin were absorbed the fastest with Tmax at 0.25–0.50 h; the Tmax of baicalin was 2.0 h; the Tmax of baicalein, wogonin, berberine, epiberberine, berberrubine and glycyrrhetinic acid was all 4.0 h. In general, the T1/2 values of the GQD constituents in the livers were quite different. The constituents of SR had the shortest T1/2: baicalin was 1.0–2.0 h, baicalein and wogonin were 2.0–3.0 h; the T1/2 of the constituents of PR were longer than that of SR: puerarin and daidzein have T1/2 within 3.0–4.0 h; the T1/2 of liquiritigenin was 3.0–4.0 h, glycyrrhetinic acid was 6.0–7.0 h; the constituents of CR had the longest T1/2: berberine was about 10.0 h, while epiberberine and berberrubine were up to 15.0–19.0 h.Complete C-T curves of 19 GQD constituents were obtained in the liver samples . The phaPR, baicalin, baicalein and wogonoside derived from SR, berberine, epiberberine, palmatine, coptisine, jatrorrhizine, and magnoflorine derived from CR, glycyrrhetinic acid and glycyrrhizic acid derived from GR had relatively high exposure levels in the colons of the mice. Therefore, these thirteen constituents were the major constituents of GQD distributed in the colons. Among these constituents, daidzein, baicalin, and wogonoside were absorbed the fastest with Tmax at 1.0 h; the Tmax of glycyrrhizic acid was 2.0 h; the Tmax of puerarin, baicalein, berberine, epiberberine, palmatine, coptisine, jatrorrhizine, magnoflorine and glycyrrhetinic acid were all at 4.0 h. In general, the T1/2 values of the GQD constituents in the colons were quite different. The constituents of PR had short T1/2: the T1/2 of puerarin and daidzein were within 1.0–4.0 h. The T1/2 of baicalin and wogonoside were up to 15.0–19.0 h, but baicalein and wogonin were within 2.0–3.0 h. The T1/2 of the alkaloid constituents derived from CR were mostly within 3.0–5.0 h. The T1/2 of glycyrrhizic acid was within 3.0–4.0 h, while the T1/2 of glycyrrhetinic acid was within 4.0–5.0 h.In the colon samples, complete C-T curves of all the 19 GQD constituents were obtained . The phaAs shown in The C-T curves of the GQD “integrated constituent” were shown in 1/2 after oral administration or the constituent with concentration that were 1/10-1/20 of its Cmax could be determined. In addition, the items including intraday and interday precision and accuracy, recovery, matrix effect and stability of each constituent were good, and there was no significant residue of the constituents. Pharmacokinetic analysis showed that the concentrations of most constituents were within their linear ranges of the established method. Because the method had good dilution linearity, individual samples with concentrations higher than the ULOQs could also be accurately determined after dilution. The previously reported methods could be used to simultaneously measure the concentrations of only 8-12 GQD constituents , the systemic circulation (1581.3 ng/ml) and the livers (2871.7 ng/g), suggesting that the colon might be the most important target tissue of puerarin. Puerarin is extensively metabolized into 66 metabolites after oral absorption  of berberine in the livers can reach more than 200 times the exposure level in the systemic circulation  and the colons (1834.8 ng/g) was higher than that in the systemic circulation (195.5 ng/ml), indicating that glycyrrhetinic acid was accumulated in the livers and the colons. Studies have shown that there are glycyrrhetinic acid-specific receptors on hepatocytes (GR constituent in GQD, but the concentration of glycyrrhizic acid in the portal vein, the systemic circulation, and the livers was relatively low, because most of glycyrrhizic acid could be metabolized by intestinal bacteria into glycyrrhetinic acid (Glycyrrhizic acid, glycyrrhetinic acid, liquiritin, and liquiritigenin are the representative constituents of atocytes , which matocytes  and cholatocytes  effects.nic acid . Surprisnic acid .Integrated pharmacokinetic studies showed that the GQD “integrated constituent” reached peak concentration at 4.0 h in the portal vein, the systemic circulation, the livers, and the colons, with half-lives of 1.5–4.1 h and mean retention time of 4.5–6.3 h, respectively. In addition, the constituents of GQD were accumulated in the livers and the colons, which were associated with the pharmacological effects of GQD: it exerts systemic/absorptive effects on reducing blood sugar and lipid absorption in the liver, and it has a local effect on intestinal diseases in the colon. Furthermore, the concentration of the “integrated constituent” in the colons was much higher than that in the livers, suggesting that inflammatory intestinal diseases is likely to be the main indication of oral GQD.1/2 values in the tissues. However, the decline in the C-T curves of the “integrated constituent” was obvious. Therefore, the influence on the calculation of the T1/2 values of the “integrated constituent” was limited. The integrated pharmacokinetics of GQD in rat plasma has been reported (. 1.6–11.4 h (A deficiency of the pharmacokinetic study was that after 24 h of oral administration of GQD, some constituents in the colons and the livers were not completely eliminated, which might affect the calculation of their Treported . Howeverreported  vs. 1.6–6–11.4 h  or 7.0–76–11.4 h ].Integrated pharmacokinetics was proposed in 2008 and it promoted the studies on the pharmacokinetic properties of multicomponent complexes such as TCMs . HoweverThe established method was quick, sensitive, and accurate enough to analyze GQD constituents in plasma and tissue homogenate samples quantitatively. The entire C-T curves of 18, 15, 19, and 19 constituents were respectively obtained from the portal vein, systemic circulation, liver, and colon samples of the mice after oral administration of GQD. Daidzein, baicalin, and baicalein had relatively high exposure levels in the livers, while puerarin, berberine, epiberberine, coptisine, palmatine, jatrorrhizine, magnoflorine, glycyrrhizic acid, and glycyrrhetinic acid were enriched in the colons. Given that these constituents have significant biological activity, they could be regarded as the major effective constituents of GQD in treating colon or liver-related diseases, respectively. In addition, the integrated pharmacokinetic properties of GQD were studied. The GQD “integrated constituent” reached peak concentration at 4.0 h in the portal vein, the systemic circulation, the livers, and the colons, with half-lives of 1.5–4.1 h and mean retention time of 4.5–6.3 h, respectively. Furthermore, the concentration of the GQD “integrated constituent” in the colons was approximately 10 times higher than that in the livers, both of which were much higher than that in the systemic circulation, indicating its accumulation in these tissues, especially in the colons.In conclusion, the tissue distribution and integrated pharmacokinetic properties of major effective constituents of oral GQD in mice were revealed in this study. The results of the tissue distribution study would contribute to identifying the major target tissues and effective constituents of GQD, while the results of the integrated pharmacokinetic study would help to explain the pharmacokinetic properties of oral GQD as a whole.


Salvia officinalis L. (sage) in spring (cutting 1) and summer (cutting 2). Based on the results analysis of variance, the effects of WDS, PUT, and cutting time were significant for the dry weight, leaf area index (LAI), EO content, EO yield, chlorophyll (Chl) t, carotenoid, Na, and K of sage. According to regression results, the response of EO content, EO yield, non‐photochemical quenching (NPQ), spad, Chl a, Chl t, K, and K/Na to WDS can be expressed by a quadratic model, indicating that they would attain their maximum in 75.5%, 34.86%, 38.33%, 84.13% 60%, 70%, 50.40%, and 40.28% available soil water depletion (ASWD), respectively. The response of dry weight, LAI, EO content, EO yield, Fv/Fm, spad, ΦpsII, Chl a, Chl b, Chl t, carotenoid, K, and K/Na to PUT can be expressed by a quadratic model, showing that they would attain their most under 0.98, 1.14, 1.34, 1.16, 1.27, 1.18, 1.17, 1.25, 1.17, 1.27, 1.31, 1.21, and 1.19 mM of PUT, respectively. These findings suggest that, probably, the functions and structures of the photosynthetic system were further enhanced with PUT, thereby they can be promoting primary electron transfer in PSII. Also, stomatal and photosynthetic activity improved with increasing K levels with PUT.A 2‐year (2017–2018) field experiment was performed to specify if the foliar application of putrescine (PUT) under optimum and water‐deficit stress (WDS) conditions would favorably affect leaf gas exchange, greenness, chlorophyll fluorescence parameters, pigments, sodium (Na), potassium (K), as well as yield and content of the essential oil (EO) relationships in  A 2‐year experiment was conducted in water stress and putrescine (PUT) foliar application. Endogenous PUT, EO content, Pn, Gs, Na, and K were higher in spring than in summer. The response of Chl b & Fv/Fm to irrigation regime can be expressed by a linear model. Dry weight, LAI, EO yield, Fv/Fm, and NPQ were higher in summer than in spring. Putrescine and harvest time affected for Fv/Fm. Thirty‐year mean annual temperature and rainfall are 22°C and 232.6 mm, respectively  were extracted via the hydrodistillation method in a Clevenger device with double‐distilled water (1000 ml). The collected surplus of aqueous EO was dried over anhydrous sodium sulfate. Then, the weight of pure EO was determined, and its percentage was computed.2.32 concentration (Ca) was 380 μM/mol, and photosynthetic photon flux density was 1250 μM/m2/s. For estimating the leaf greenness, GREENNESS INDEX‐502 was used on the same leaves mentioned previously (Konica Minolta).An Li‐6400XT Portable Photosynthesis System (Li‐Cor Inc.) was utilized to determine the net photosynthetic rate (Pn) and stomatal conductance (gs) of plants per plot after each cutting time. The chamber was corrected to 25°C (temperature), ambient CO2.42/s , nonphotochemical quenching (NPQ), photochemical quenching (qP) was determined applying a modulated fluorescence chlorophyll meter . Leaves were dark‐adapted via leaf clips (15 min). After dark adaptation, the highest photochemical performance (Fv/Fm), actual photochemical performance (ΦPSII) = (Fm'‐Fs)/Fm', qP = (Fm'‐Ft)/(Fm'‐Fo'), and NPQ =1‐ (Fm'‐Fo')/(Fm‐Fo) were computed as well, and the light severity applied to calculate the yield of ΦPSII and NPQ was 1,200 μmol/mRoháček, .2.5To estimate the chlorophyll and carotenoid, the procedure of Lichtenthaler  was used2.6Endogenous (endo) PUT extraction and thereafter HPLC measurement were performed following the procedure of Lütz et al. . First, 2.7+ and K+ contents were determined by flame photometry (Jenway ‐Flame Photometer Models PFP7). To estimate the area index, the leaf area meter (Delta‐T Devices Ltd.) was employed after using treatments in the end of each cutting time.For estimating sodium and potassium, the procedure of Ahanger et al.  was used2.8p > .05) was examined with polynomial orthogonal contrasts. Pearson's correlation coefficients were specified applying the CORR method.The procedure as type3 in MIXED method of SAS v. 9.4 (SAS Institute) was applied to analyze data. The significant effects of WDS, PUT, cutting time, and their two‐ and three‐way interactions were considered fixed effects, while years, replicates ×years and years ×WDS × PUT ×cutting time were considered chance effects. The PDIFF choice of least square means adjusted for the Tukey‐Kramer was applied for mean comparisons. The interactions among experimental factors were separated by slicing procedure. The significance of linear and quadratic regression models (33.12) and LAI (0.83) were obtained in 20% ASWD. Indeed, there was a descending trend in dry weight and LAI by incrementing severity of water‐deficit stress (WDS)  under 0.98 and 1.14 mM of PUT, respectively  Figure . The resy Figure . There wd LAI 0.8 were obt3.22) under 75.5% and 34.86% ASWD, respectively , suggesting that EO content and EO yield would reach their maximum (1.39% and 279.62 g/m2) under 1.34 and 1.16 mM of PUT  and Gs (0.057 mmol/m2 s‐1) were obtained in 20% ASWD. Indeed, there was a descending trend in Pn and Gs by incrementing severity of WDS  was observed for 80% ASWD Figure . The res3.4The result showed that the main influence of WDS and PUT was on Chl a, Chl b, Chl t, and carotenoid. Also, the significant effect of cutting time was on Chl a, Chl t, and carotenoid (Supplementary Information −1 FW) in 60% and 70% ASWD, respectively  was observed for 80% ASWD  was obtained in 20% and 40% ASWD, respectively  under 1.25, 1.17, 1.27, and 1.31 mM of PUT, respectively  under 63.96% ASWD and minimum (13.40 mg/g DM) under 1.20 mM of PUT, respectively Figure . The res4p < .01) and EO yield  (Supplementary Information p < .01) and Gs  (Supplementary Information Slight water‐deficit stress (WDS) 34.86% ASWD) would be required for achieving the maximum EO content of sage. In the present study, the maximum EO yield of sage was produced under potential yield conditions in 75.5% ASWD. The lowest dry weight and LAI of sage were produced under 80% ASWD. Analogous to our results, the dry weight diminished with increasing WDS where the EO yield of sage was the highest under 60% ASWD  and Gs  (Supplementary Information p < .05), Fv/Fm and Pn , and Fv/Fm and Gs   and K/Na  (Supplementary Information p < .01) (Supplementary Information p < .01), Chl b , Chl t , and carotenoid  (Supplementary Information p < .01)  and Gs  (Supplementary Information p < .01) (Supplementary Information p < .01) Chl t , and carotenoid  , K , and K/Na  (Supplementary Information p < .01) and Gs  (Supplementary Information According to regression results, the maximum and minimum Na concentration was observed in 63.96% ASWD and 1.20 mM of PUT, respectively. Also, K and K/Na would reach their maximum by applying 1.21 and 1.19 mM of PUT, respectively. Instead, there were positive and negative correlations among endo PUT and Na (−0.43, 5The study indicated that the maximum EO yield of sage was produced under potential yield conditions in 75.5% ASWD. Greenness index, Chl, and carotenoid increased by the intensity of WDS. The lowest LAI, Pn, and Gs were observed under 80% ASWD. But, the highest Fv/Fm was obtained under 80% ASWD. The reduced photosynthesis can be due to stomatal factors and nonstomatal limitations including the stagnation in PSII activity and electron transport. Regression results showed that the NPQ increased under 38.33% ASWD. There was a negative correlation between NPQ and K and K/Na. Indeed, environmental stresses make the electron transfer chain saturated and increment proton accumulation, whereby NPQ would increase. There was a negative correlation between NPQ and Chl and carotenoid. So, it could be concluded that excess excitation energy is mostly used to produce Chl and carotenoids compared to the production of NPQ under WDS conditions in sage. There was a negative correlation between NPQ and EO content. Also, NPQ and EO were higher in cutting 2 and cutting 1, respectively. Nonetheless, the dissipation of surplus photosynthetic energy is mainly accomplished by the biosynthesis of highly reduced compounds such as isoprene and secondary metabolites. PUT showed that Fv/Fm and ΦpsII improvement, however, did not affect NPQ. These findings propose that, probably, PUT optimized energy broadcast and improved the structure and function of the photosynthetic system, and thereby it can be promoting primary electron transfer in PSII. PUT incremented the accumulation of Chl a, Chl b, Chl t, and carotenoid, indicating that PUT improved the transport of the photosynthetic matter. The maximum concentration of endo PUT was obtained with 2.25 mM of PUT. There were positive correlations between K/N and endo PUT, Pn, and Gs. Indeed, stomatal and photosynthetic activity improved with increasing K levels with the application of PUT.App S1Click here for additional data file.


To analyze the value of multiparameter magnetic resonance (mpMRI) in the diagnosis of pancreatic cancer with vascular invasion from two aspects: morphology and function, so as to provide a reliable diagnostic basis for preparing the clinical treatment plans. Ktrans, Kep, Ve values from DCE. Independent samples t-test was used to compare the parameters of pancreatic cancer with vascular invasion group (16 cases) and nonvascular invasion group (15 cases). ROC curve was used to analyze the efficacy of each parameter in diagnosing pancreatic cancer vascular invasion.  Totally 31 case data of pancreatic cancer patients diagnosed in our hospital from January 2020 to March 2021 were enrolled in this study. All patients underwent multiparameter magnetic resonance imaging  before surgery, and then all patients underwent pancreatic cancer surgery. Two experienced radiologists analyzed these obtained images according to the image reports and combined them with the pathological results. Taking pathological results as gold standard, the sensitivity, specificity, and accuracy of quantitative parameters derived from T2WI, DKI, DCE, T2WI + DKI, T2WI + DCE, and T2WI + DKI + DCE for the diagnostic capabilities of pancreatic cancer vascular invasion were calculated using diagnostic laboratory methods. Kappa consistency test was used to estimate the consistency of two radiologists' diagnosis and analysis. The images obtained by DKI sequence were input into the postprocessing software MITK-Diffusion v2014.10.02, The images obtained from DCE sequence were processed by the Tissue 4D software on the Siemens syngo via workstation to calculate and analyze each tumor ROI's MD, MK values from DKI, and Ktrans, Kep, and Ve were significantly different between the vascular invasion group and the nonvascular invasion group (p < 0.05). Each parameter's AUC of ROC curve was 0.773, 0.829, 0.794, 0.802, and 0.846 (p < 0.05). Take MD = 2.285 × 10−3 mm/s2, MK = 0.72, Ktrans = 0.103, Kep = 0.337, and Ve = 0.353 as thresholds; the sensitivity of these parameters to diagnose vascular invasion of pancreatic cancer was 73.33%, 75%, 87.5%, 68.8%, and 68.8%. The specificity of them was 75%, 80%, 60%, 86.7%, and 86.7%, respectively.  The sensitivity, specificity, and accuracy of T2WI were 62.5%, 53.5%, and 58.1%; those of DKI were 56.3%, 60.0%, and 58.1%; those of DCE were 68.8%, 60.0%, and 64.5%; those of T2WI + DKI were 68.8%, 66.7%, and 67.7%; those of T2WI + DCE were 75.0%, 66.7%, and 71.1%; those of T2WI + DKI + DCE were 81.2%, 73.3%, and 77.4%, respectively. These two diagnostic radiologists analyzed image data with good consistency, Kappa = 0.834. MD, MK,  The combined analysis of T2WI + DKI + DCE can improve the specificity and accuracy of diagnostic efficiency of vascular invasion of pancreatic cancer and provide an important diagnostic basis for pancreatic cancer's preoperative treatment. Pancreatic cancer has a high mortality rate, and the five-year survival rate does not exceed 5%. According to the location of the lesions, it can be divided into pancreatic head cancer, pancreatic body cancer, and pancreas cancer, of which more than 50% are pancreatic head cancer . PancreaThe data of 31 patients were diagnosed as pancreatic cancer with vascular invasion by surgery and pathology in our hospital from January 2020 to March 2021 and successfully completed MRI examinations. MR images including T1WI, T2WI, DKI, and DCE-MRI were retrospectively analyzed. There were 16 cases in the group and 15 cases in the no vascular invasion group. Among them, there were 19 males and 12 females, aged 25-75 years, with an average of 54.6 ± 12.9 years. Inclusion criteria are as follows: (1) no related treatment for pancreatic cancer before MRI examination, (2) good coordination of breath-hold and no motion artifacts in the image, and (3) postoperative pathological biopsy-confirmed pancreatic cancer. This study was approved by the Medical Ethics Committee of the hospital.b-value = 0, 250, 500, 750, 100, 1500, and 2000 s/mm2; NEX = 2; FOV = 330 mm × 258 mm; matrix = 192 × 192; slice thickness = 6.5 mm; layer spacing = 1.3 mm; 15 directions were applied for each b-value diffusion sensitive gradient field, and scanning time is 10 min 42 s. Corrected T1-vibe cross-sectional was scanned before contrast agent injection: FOV = 260 mm × 260 mm, slice thickness = 3.6 mm, TR = 5.08 ms, TE = 1.74 ms, average number of times = 8, matrix = 138 × 192, voxel size = 1.9 mm × 1.4 mm × 3.6 mm, and flip angle = 2° and 15°. Dynamic contrast-enhanced MRI parameters are as follows: flip angle = 15°, TR = 4.24 ms, TE = 1.66 ms, matrix = 138 × 192, voxel size = 1.9 mm × 1.4 mm × 3.6 mm, slice thickness = 3.6 mm, and 40 scan periods in total with 7 s/period. Bolus injection of contrast machine Ounaiying after 3 scans, dose = 0.1 mmol/kg, flow rate = 2.5 ml/s, bolus injection immediately after the contrast machine injection 20 ml normal saline flush tube.All patients underwent scanning on a 1.5 T superconducting MR system  with a 32-channel phased-array body coil. Patients were asked to fast for 4 hours before examination. Conventional scanning sequence parameters are as follows: axial T1WI positive and negative phase GRE sequence: TR = 262mms, TE = 2.62 ms, FOV = 400 mm × 320 mm, matrix = 256 × 256, slice thickness = 6 mm, slice interval = 1.2 mm, and NEX = 1; T2WI sequence: TR = 7520 ms, TE = 96 ms, FOV = 400 mm × 320 mm, matrix = 256 × 256, slice thickness = 6 mm, and layer spacing 1.2 mm. Diffusion kurtosis imaging are as follows: TR = 6200 ms; TE = 78 ms; Two senior radiologists experienced in abdominal MRI diagnosis have independently read conventional T2WI, DKI, and DCE-MRI images, respectively, without knowing the pathological results. The blood vessels are surrounded by tumor tissue >1/2 diameter; vessels are embedded, occluded, not shown, or seen in filling defects, and the surrounding with collateral formation is regarded as vascular invasion .(1)DKI Postprocessing. Save the desired MRI images in DICOM format. The images obtained by DKI sequence were input into the postprocessing software MITK-Diffusion v2014.10.02 (http://mitk.org/wiki/MITK) and calculated to generate mean diffusivity (MD) and mean kurtosis (MK) images representing diffusion information. A radiologist with more than 8 years experience in diagnosing abdominal diseases placed a region of interest (ROI) at the largest layer of the tumor (area 7.12-9.68mm2) and measured the MK and MD values to avoid necrosis, At the cystic degeneration site, all parameters were measured 3 times, and the average value was taken.(2)DCE Image Postprocessing. Import the T1 data of the flat-sweep small flip angle and the 31 sets of data of dynamic enhancement into Siemens syngo via workstation . Motion correction and image matching were performed in steps, ROI was selected, and the Tissue 4D analysis software was used to analyze the quantitative data of DCE-MRI, and the pharmacokinetic model was the Tofts two-compartment model. First, combined with T2WI, dynamic enhancement from 31 groups of T1WI was performed. In the image, the scan phase with the most obvious early enhancement and the best contrast with the surrounding tissue was selected as the phase for subsequent analysis, and a radiologist with more than 5 years experience in diagnosing abdominal diseases manually drew the ROI on the dynamic enhanced image, select the largest layer of the tumor and at least 3 or more layers of tissue above and below the tumor for ROI measurement, and try to avoid the surrounding blood vessels. Then, the Ktrans, Kep, and Ve maps in the ROI tissue were calculated, and the ROI was copied to the corresponding parameter pseudo-color map, and the corresponding Ktrans, Kep, and Ve values were further measured.Ktrans, Kep, and Ve values of each tumor foci. Two independent samples t-test was used to compare the difference of each parameter value between the vascular invasion group and the nonvascular invasion group, and the ROC was drawn. ROC curve was used to analyze the diagnostic efficacy of each parameter for vascular invasion of pancreatic cancer.Statistical analysis was performed using SPSS17.0 software. Taking the pathological results as the gold standard, the sensitivity, specificity, and accuracy of T2WI, DKI, DCE, T2WI + DKI, T2WI + DCE, and T2WI + DKI + DCE in diagnosing pancreatic cancer vascular invasion were calculated by diagnostic experiments. The Kappa consistency test was used to evaluate the consistency of the image data analyzed by the two readers. The obtained images were input into the corresponding software to calculate and analyze the MD, MK, Two diagnostic imaging physicians analyzed image data in good agreement, Kappa = 0.834.In all 31 pancreatic cancer cases, there was vascular invasion: T2WI: 25 cases, overestimated 15 cases; DKI: 25 cases, overestimated 16 cases; DCE: 23 cases, overestimated 12 cases; T2WI + DKI: 20 cases, overestimated 9 cases; T2WI + DCE: 19 cases, overestimated 7 cases; T2WI + DKI + DCE: 19 cases, overestimated 6 cases; no vascular invasion: T2WI: 14 cases, underestimated 6 cases; DKI: 14 cases, underestimated 5 cases; DCE: 15 cases, underestimated 6 cases; T2WI + DKI: 15 cases, underestimated 5 cases; T2WI + DCE: 14 cases, underestimated 4 cases; T2WI + DKI + DCE: 14 cases, underestimated 3 cases .The sensitivity, specificity, and accuracy of T2WI, DKI, DCE, T2WI + DKI, T2WI + DCE, and T2WI + DKI + DCE for the diagnosis of vascular invasion of pancreatic cancer were, respectively, 62.5%, 53.5%, 58.1%, 56.3%, 60.0%, 58.1%, 68.8%, 60.0%, 64.5%, 68.8%, 66.7%, 67.7%, 75.0%, 66.7%, 71.1%, 81.2%, 73.3%, and 77.4% . The senKtrans, Kep, and Ve were higher and statistically significant (p < 0.05).The comparison results are shown in Ktrans, Kep, and Ve between the vascular invasion group and the nonvascular invasion group (p < 0.05); with MD value of 2.285 × 10−3 mm/s2, MK value of 0.72, Ktrans value of 0.103, Kep value of 0.337, and Ve value of 0.353 as the thresholds, the sensitivities for diagnosing vascular invasion of pancreatic cancer were 73.33%, 75%, and 75%, respectively. The specificity was 75%, 80%, 60%, 86.7%, 86.7%, respectively. See There were significant differences in MD, MK, Pancreatic cancer is becoming the second leading cause of cancer-related death, with high mortality due to a lack of early detection methods and the inability to successfully treat patients once diagnosed . TherefoKtrans, Kep, and Ve, which can quantitatively analyze the functional state of the microcirculation of tumor tissue. At present, there have been studies on the application of DCE-MRI in the identification of benign and malignant pancreatic masses [Ktrans, Kep, and Ve in the vascular invasion group were higher than those in the normal group. Ktransrepresented the diffusion rate of the contrast agent from the intravascular to the extracellular space outside the blood vessel, and Kep represented the return rate of the contrast agent from the extracellular space into the blood vessel, both of which are related to the permeability of capillaries, andKtransis also affected by the amount of blood perfusion in the tissue and the surface area of capillaries. The occurrence of tumors is accompanied by angiogenesis to meet the requirements of oxygen, nutrient requirements, and metabolic waste removal [Ktrans and Kep, and Ve represents the volume percentage of the extracellular space occupied by the extracellular space. The more poorly differentiated tumor, the greater the cell atypia, the tighter the arrangement, and the more immature new blood vessels [Ve value of pancreatic cancer with vascular invasion group, which is inconsistent with the results of this study. It is speculated that it may be related to the small sample size of this study, and the sample size will be further increased in future studies to demonstrate.DCE-MRI is the application of rapid MRI sequence to continuously collect images before, during, and after intravenous injection of contrast agent, showing the information in the process of contrast agent entering the target organ or tissue blood vessel, passing through the capillary bed and finally being removed, and the degree of signal enhancement. It reflects the physical and physiological properties of the target organ or tissue, including tissue perfusion, capillary surface area, and extravascular-extracellular space . Using ac masses , but the removal . Because vessels , correspb values and three mutually perpendicular scanning directions; the MK value is calculated from the average apparent kurtosis coefficient (AKC) in each direction, the larger the MK value, the more complex the target tissue structure, so it can be used to measure the tissue structure complexity [Ktrans, Kep, and Ve between the vascular invasion group and the nonvascular invasion group (p < 0.05). The AUC values of the ROC curve of each parameter were all greater than 0.750, and some even reached more than 0.8. The above results further indicate that DCE-MRI combined with DKI has great potential in early, noninvasive, and overall prediction of whether pancreatic cancer is accompanied by vascular invasion.The human body is composed of different tissues, and the cell types, cell density, and blood supply of each tissue are different. The diffusion of water molecules is non-Gaussian . The DKImplexity . MalignaThere are still some shortcomings in this study: first, the sample size is relatively small. Because pancreatic cancer is insidious and highly malignant, most patients cannot be surgically removed when it is discovered. Therefore, it is relatively difficult to collect pancreatic cancer with surgical and pathological results. To follow-up, we will continue to conduct research in this area and further verify the above results in a large sample; secondly, due to the influence of various factors such as imaging methods, parameters, and image postprocessing, the data obtained by DCE-MRI may vary among institutions, which is also a common problem faced by current DCE-MRI research. Therefore, it is urgent to develop a general DCE-MRI standardized software to meet daily clinical work or organize multicenter research in order to make the obtained study results more reliable and stable.To sum up, the combined application of T2WI + DKI + DCE could improve the diagnostic performance of pancreatic cancer with vascular invasion and provide a basis for early diagnosis and treatment of pancreatic cancer. MD and MK values derived from DKI and DCE-MRI parameters could quantitatively evaluate pancreatic cancer with vascular invasion. Therefore, in practice, rational use of the morphological features and functional information of multiparameter magnetic resonance imaging can improve the accuracy of pancreatic cancer vascular invasion diagnosis and has a high diagnostic value for pancreatic cancer with vascular invasion.


Helicobacter pylori infection. Proton pump inhibitors, such as omeprazole, are the most widely used drugs to treat this illness. The aim of the study was evaluate cytogenetic effects of omeprazole in stomach epithelial cells of patients with gastritis in presence and absence of H. pylori, through cytogenetic biomarkers and catalse and superoxide dismutase analysis.Gastritis is a superficial and prevalent inflammatory lesion that is considered a public health concern once can cause gastric ulcers and gastric cancer, especially when associated with H. pylori (n = 22) and (6) patients with gastritis and H. pylori on OME therapy (n = 22).The study included 152 patients from the Gastroenterology Outpatient Clinic of Hospital Getúlio Vargas, Teresina—Brazil, that reported continuous and prolonged omeprazole use in doses of 20, 30 and 40 mg/kg. The participants were divided into groups: (1) patients without gastritis (n = 32); (2) patients without gastritis but with OME use (n = 24); (3) patients with gastritis (n = 26); (4) patients with gastritis undergoing OME therapy (n = 26); (5) patients with gastritis and H. pylori infection, indicating clastogenic/aneugenic effects, chromosomes alterations, gene expression changes, cytotoxicity and apoptosis.OME induced cytogenetic imbalance in the stomach epithelium through the formation of micronuclei ; bridges ; buds ; binucleated cells ; (groups 2 and 3 > 1); picnoses , groups 2 and 5 > 1, 3; group 4 > 1, 2, 3, 5); cariorrexis  and karyolysis . The OME cytogenetic instability was associated with The cytogenetic changescan be attributed to several mechanisms that are still unclear, including oxidative damage, as observed by increased catalase and superoxide dismutase expresion. Positive correlations between antioxidant enzymes were found with micronuclei formation, and were negative for picnoses. Thus, the continuous and prolonged omeprazole use induces genetic instability, which can be monitored through cytogenetic analyzes, as precursor for gastric cancer. Helicobacter pylori (H. pylori) co-infection, which increase hydrochloric acid and pepsin secretion by the gastric mucosa [Gastric lesions, such as gastritis, can damage the gastric wall and/or duodenal epithelium, producing ulcers and even cancer , 43, 49.c mucosa , 43. Proc mucosa , 64, 88,c mucosa , 46, 70.PPIs cause several adverse effects, and there are reports that its prolonged use promotes cardiovascular and renal alterations, anemia, thrombocytopenia, gastric polyps and carcinoma , 94, 107Drug metabolic products can induce genetic mutations, breaks and/or chromosome rearrangements. There are reports of OME metabolites cytotoxicity, hepatoxicity and carcinogenicity . CytochrAllium cepa) [Saccharomyces cerevisiae and murine Sarcoma 180 cells [H. pylori co-infection, through micronucleus test and expression of catalase and superoxide dismutase.In non-clinical studies, OME toxicogenic effects were identified in plant cells (um cepa) , as well80 cells . Thus, bThe present study was a controlled cross-sectional research, approved by the Research Ethics Committees (CEP) of UNINOVAFAPI (no. 1.521.307), Federal University of Piauí—UFPI (no. 1.607.441) and Ethics Committee of Hospital Getúlio Vargas . All participants agreed to participate voluntarily and signed the Free and Informed Consent Form (FICF), in accordance with resolution 466/12 of the National Health Council.H. pylori infection, were examined after endoscopy and urea test. Participants were grouped according to the criteria: WG—patients without gastritis (n = 32); WG + OME—patients without gastritis with OME use (n = 24); G—patients with gastritis (n = 26); G + OME—patients with gastritis and OME use (n = 26); G + HP—patients with gastritis and H. pylori (n = 22) and G + HP + OME—patients with gastritis and H. pylori, using OME (n = 22).One hundred and fifty-two patients from the Gastroenterology Outpatient Clinic of Hospital Getúlio Vargas, Teresina—PI (2017–2019) were enrolled in this study. The participants had been undergone to upper digestive endoscopy and reported prolonged OME use (or not) in doses of 20 and 40 mg/kg. Medical reports about the presence or absence of gastric diseases, including gastritis and The study included patients: (1) with or without gastritis; over 18 years old and legally responsible; (2) that signed the informed consent form; (3) that have prolonged OME useor not, by medical recommendation or self-medication, or reported no OME use;. The following participants were excluded: (1) over 70-year-old; (2) current illnesses that required surgical treatment, chemotherapy or radiotherapy.After Informed Consent Form signed, questionnaires were applied according to Carrano and Natarajam  based onExfoliated cells from the oral epithelium of patients undergoing endoscopy were obtained by scraping the inside of the cheek with a cytobrush. Cells of gastric epithelium (region of the body and antrum of the stomach) were collected at the time of endoscopy. All samples were placed in tubes with sodium phosphate buffer (PBS) , properly identified, and transported in dry ice to the Laboratory of Toxicological Genetics of the Federal University of Piauí, for immediately processing and tests. Peripheral blood samples were collected with heparin, and transported similarly to the other samples. The urea test was performed by the hospital’s medical team and the results were released together with the medical report.The Micronucleus Test was performed according to Thomas et al. , with sog, 20 min) and the supernatants used for catalase (CAT) activity assay. The reaction medium was prepared with H2O2 (18 mL) plus 1 M Tris HCl Buffer, 5 nM EDTA pH 8.0 (1.0 mL) and H2O (0.8 mL). Then, 980 μL of the reaction medium and 20 μL of the 10% erythrocyte homogenate was placed in the quartz cuvette. Finally, the reading was performed in a spectrophotometer for 6 min at 230 nm. The blank was made by reading the relative absorbance at 230 nm with only 1 mL of the reaction medium [g, 20 min) and the supernatants used for superoxide dismutase (SOD) activity assessment. SOD activity was tested using the reduction rate of cytochrome C by superoxide radicals, using the xanthine-xanthine oxidase system as a source of superoxide anion (O2−) [2− with cytochrome C. For protein concentration, the method of Lowry et al. [From the peripheral blood samples collected, 10% erythrocyte homogenates were prepared (50 mM sodium phosphate buffer pH 7.4), which were centrifuged  followed by the Bonferroni test as a post hoc test. The data were analyzed using the GraphPad Prism 6.0 software , the experimental groups were compared with the control group and with each other. Pearson's correlations were performed using the IBM SPSS Statistics 23 statistical program. P < 0.05 was defined as statistically significant.H. pylori coinfection, reported family history of cancer and absence of other hereditary diseases.After applying the questionnaire depicting nutritional aspects, socio-cultural, health and lifestyle information for each patient, the investigated population was characterized Table . The patH. pylori in patients with gastritis (5.09 ± 1.71) also contributed to these effects, which in OME therapy presented increased MN formation (8.22 ± 1.74), in relation to all study groups , clastogenic and/or aneugenic effects were observed through MN formation in WG + OME 3.62 ± 1.81) and gastritis , in relation to patients that didn’t have gastritis and were not OME user (1.62 ± 0.83). Patients with G + OME (5.091 ± 1.71) also had more MN than those with gastritis and no OME use. The presence of .81 and gH. pylori in patients with gastritis (3.69 ± 2.80) did not increase buds in comparison to those with gastritis without OME use (2.13 ± 1.80), but when in therapy with OME (13.73 ± 2.25), an increase of buds was detected, indicating effects on expression and genes  Fig. . Increass 3.69 ± .80 did nH. pylori (3.65 ± 2.01), it was not seen an increase in nucleoplasmic bridges in relation to the group without gastritis and no OME use. However, in patients with H. pylori and OME use (13.59 ± 3.99) increased nuclear abnormalities were detected  and with gastritis (12.69 ± 2.96) when compared to the group without gastritis and OME use (2.03 ± 1.42). In patients with gastritis (3.73 ± 1.82) and gastritis and .52 and wH. pylori , when compared to patients without gastritis and no OME use (2.33 ± 0.91). Moreover, similar results were found in patients with gastritis, H. pylori and no OME use (5.01 ± 1.27). Likewise, in patients with gastritis with/without OME use (4.23 ± 1.25), an increase in binucleated cells was observed . In addition, cytotoxicicity was also observed in patients with gastritis 7.11 ± 3.97), as well as in patients with gastritis and positive for .97, as wH. pylori (7.84 ± 3.16) and in H. pylori positive patients and OME therapy , when compared to patients without gastritis and are not using OME   and in patients with gastritis and OME use (18.46 ± 4.32), as well as in patients that are not using OME, but positive for H. pylori  or with H. pylori , when compared to patients without gastritis and no OME use (150.00 ± 49.02). The presence of H. pylori in patients with gastritis and on OME therapy  also induced an increase in cariorrexis  in relation to patients without gastritis and no OME use (149.90 ± 46.32). However, these effects were also observed in patients with gastritis (324.20 ± 179.40) and with gastritis, H. pylori (258.00 ± 122.70) and no OME therapy  in stomach epithelium cells of patients without gastritis (202.20 ± 69.65), and with gastritis and no ori 209, 0 ± 78.06ori 209, 0 ± 78.06H. pylori, they presented catalase increases in relation to patients without gastritis and were not taking OME  can cause cytogenetic changes in stomach epithelial cells by aneugenic and/or clastogenic effects through micronuclei formation. The OME effects on micronuclei formation in human cells are still unwell described. However, there are in silico studies suggesting that OME can lead to chromosomal changes, as well as contribute to micronuclei formation , also prH. pylori infection, indicate a risk of genotoxicity, with greater significance in relation to negative patients [H. pylori infections can cause chronic gastritis, peptic and duodenal ulcers, adenocarcinoma and gastric lymphoma [Investigations in gastric epithelial cells of patients with gastritis and with positive patients , as welllymphoma . There alymphoma , 96. MN lymphoma , internalymphoma , double-lymphoma , 35. It lymphoma , 79.According to Amieva and Peek , there aH.pylori virulence is determined by the presence of the cytotoxin associated with the CagA gene, VacA cytotoxin and SabA adhesion proteins. The VacA cytotoxin induces membrane channel formation, cytochrome C release and modulates antigen presentation [2 or urea/ammonia) that guides the bacteria to bind the mucosa [According to Raza et al. , the H.entation . SabA ace mucosa . SabA exe mucosa , 54, 102DNA damage can be induced by OME through its secondary metabolites sulfone, sulfite and hydroxy-omeprazole , 85, as H. pylori infection, in and/or OME therapy), such as nucleoplasmic buds and bridges. Tthe buds are the result of DNA amplification or repair [In addition to micronuclei formation, other cytogenetic cahnges were observed in the stomach epithelial cells of patients with/without gastritis  . In stomdivision . H. pylo release , as well release . These e release , 90, 105Moreover, OME has hepatotoxic effects as a result of apoptosis stimulated by tumor necrosis factor alpha (TNF-α), as well as by alterations of liver enzymes AST and ALT , 37, 98.H. pylori infection, as previously observed in other nuclear alterations. Although apoptosisis one of the mechanisms associated to acute gastric injury [In this study, OME induced apoptosis in stomach epithelial cells due to nuclear fragmentation (cariorrexis) and nuclear dissolution (karyolysis), as seen in patients without gastritis and with gastritis in OME use and/or therapy, and also in patients with c injury , OME hasc injury , colorecc injury , 67, andc injury , 68, 72.2O2 degradation through dismutation reactions, mainly in peroxisomes, and it has been considered as an important oxidative biomarker [2O2. In this regard, to act effectively in maintaining cellular integrity and function, SOD depends on the balance between SOD, GPx and CAT [According to previous studies, drugs that induce oxidative damage may increase the levels of endogenous antioxidant enzymes such as catalase and superoxide dismutase , 47, 80.iomarker , 77. Sup and CAT , 77.H. pylori infection. H. pylori infection can also increase reactive oxygen and nitrogen species in the stomach [H. pylori, and also increase antioxidant enzymes such as SOD and CAT, and glutathione reductase (GSH) [In this study it was possible to detect an increase in antioxidant defenses for these enzymes, especially in patients with  stomach . Moreove stomach  independse (GSH) , 41. Druse (GSH) , 47, 81,se (GSH)  and reguse (GSH) .OME is one of the drugs that can induce oxidative stress , which c2O2 when it binds to protein C283, which contains CACT and C136 for generating beta oxidation of fatty acids [S. cerevisiae, in addition to cytogenetic damage in murine Sarcoma 180 cells [During metabolism, OME can generate sulfone, sulfite and hydroxy-omeprazole, compounds that can generate more oxidative damage , 15, 28.ty acids . OME can80 cells , 75.Among these mechanisms, our study points out that cytogenetic changes can be induced by oxidative effects that lead to micronuclei formation and other nuclear alterations indicative of cytotoxicity. Corroborating these analyzes, positive and negative statistical correlations were observed between micronuclei and CAT/SOD concentrations, and between pyknosis, respectively. Proton pump inhibiting drugs (PPIs), such as OME, may have genotoxic and/or carcinogenic effects  through H. pylori infection, and OME use and/or therapy, it was possible to point out that OME induces cytogenetic changes due to (1) clastogenic and/or aneugenic effects that induce micronuclei formation; (2) altered gene expression, chromosomal rearrangements and fusion of chromosomal ends; (3) cytotoxicicity by increased picnoses and binucleated cells and (4) apoptosis by increasing karyorexis and karyolysis. Several mechanisms, not yet elucidated, can be attributed to these OME cytogenetic effects, but oxidative effects can also be involved, as observed by increased concentrations of endogenous antioxidant enzymes such as catalse and superoxide dismutase, which have also been associated to increased micronuclei and picnoses. These data point out the risks regarding long term OME use/therapy, as well as the monitoring of cytogenetic changes and oxidative damage, as an important strategy for the genetic instability prevention.In this study, in stomach epithelial cells of patients without gastritis and with gastritis, especially those with


Polygonatum cyrtonema Hua polysaccharide (PCP 85−1−1) was separated by fractional precipitation and was purified by anion exchange and gel permeation chromatography. The average molecular weight (Mw) of PCP 85−1−1 was 2.88 × 103 Da. The monosaccharide composition implied that PCP 85−1−1 consisted of fucose, glucose, and fructose, and the molar ratio was 22.73:33.63:43.65. When 2% PCP 85−1−1 was added to tobacco shreds, the ability of moisture retention and moisture-proofing were significantly enhanced. The moisture retention index (MRI) and moisture-proofing index (MPI) increased from 1.95 and 1.67 to 2.11 and 2.14, respectively. Additionally, the effects of PCP 85−1−1 on the aroma and taste of tobacco shreds were evaluated by electronic tongue and gas chromatography–mass spectrometry (GC-MS). These results indicated that PCP 85−1−1 had the characteristics of preventing water absorption under high relative humidity and moisturizing under dry conditions. The problem that traditional humectants are poorly moisture-proof was solved. PCP 85−1−1 can be utilized as a natural humectant on porous carbohydrates, which provides a reference for its development and utilization.Porous carbohydrate materials such as tobacco shreds readily absorb moisture and become damp during processing, storage, and consumption (smoking). Traditional humectants have the ability of moisture retention but moisture-proofing is poor.  The structure of tobacco shreds contains many capillaries, hydrophilic colloids, and crystalline components, which are representative of porous carbohydrate materials. It is susceptible to environmental conditions such as temperature and relative humidity (RH). Polyol compounds, such as glycerol, propylene glycol, and sorbitol , have been widely used in the tobacco industry to improve the moisture retention of tobacco shreds ,2,3. ThePolysaccharides are generally strong hydrophilic compounds and possess excellent moisture absorption and retention capacities. Polysaccharides and natural extract humectants generally have good hydrophilic properties, thus providing new ideas and directions for improving the water-holding capacity of cigarettes . HoweverPolygonatum genus, including Polygonatum sibiricum Red, Polygonatum cyrtonema Hua, and Polygonatum kingianum Coll. et Hemsl. are widely used for medicine and food [Polygonatum cyrtonema Hua is distributed throughout the temperate Northern Hemisphere in places such as China, Japan, Korea, India, Russia, Europe, and North America [Polygonatum cyrtonema Hua contains multiple components, such as alkaloids, flavones, steroid saponins, lignins, amino acids, and Polygonatum cyrtonema Hua polysaccharides (PCP) [In China, plants of the and food . Polygon America . At pres America ,10,11. Pes (PCP) .Polygonatum cyrtonema Hua polysaccharides are believed to be one of the most important active compounds of Polygonatum cyrtonema Hua. Studies have shown that they have an antioxidative [Polygonatumcyrtonema Hua were mainly fructooligosaccharides (FOS) with fructose as the structural backbone. He et al. [Polygonatumcyrtonema Hua. Structural analysis suggested that the fructan consisted of a (2 → 6) linked β-D-Fruf residues backbone with an internal α-D-Glcp residue and two (2 → 1) linked β-D-Fruf residue branches. Therefore, in this study, PCP 85−1−1 was purified by graded alcohol precipitation, and its physicochemical properties, moisture retention, and moisture-proofing abilities were further studied.xidative , anti-baxidative  and antixidative  ability.xidative , improvexidative ,18, and xidative . Accordixidative ,21,22, fe et al.  found twe et al.  also obtPolygonatum cyrtonema Hua polysaccharides on porous carbohydrate materials are scarce. In this study, we investigated the influence of Polygonatum cyrtonema Hua polysaccharides on the adsorption and desorption properties of tobacco shreds. Structural characteristics of Polygonatum cyrtonema Hua polysaccharides also were analyzed. Additionally, the effect of the Polygonatum cyrtonema Hua polysaccharides on the changes in taste and aroma components of tobacco shreds after smoking were analyzed by electronic tongue and gas chromatography–mass spectrometry (GC-MS).However, the reports on the moisture retention and moisture-proofing functions of Polygonatum cyrtonema Hua polysaccharides by fractional precipitation is shown in Polygonatum cyrtonema Hua polysaccharides were mainly low molecular polysaccharides. The purity of PCP 20 was the highest, at 62.44 ± 1.64% d. As shown in a, 5.32 ± 0.54% c, 2.47 ± 0.61% b, and 15.43 ± 1.21% d, respectively (p < 0.05); the purity was 62.44 ± 1.64% d, 56.64 ± 3.57% a, 57.77 ± 1.58% b, and 58.18 ± 2.25% c, respectively; the contents of the protein were 3.98 ± 0.0075 c mg/g, 3.15 ± 0.10 a mg/g, 3.83 ± 0.015 c mg/g, and 3.45 ± 0.11 b mg/g, respectively; and the polyphenol contents were 5.54 ± 0.21 d mg/g, 2.76 ± 0.059 c mg/g, 2.28 ± 0.036 b mg/g, and 1.61 ± 0.001 a mg/g, respectively (p < 0.05). The yield of PCP 85 was different from a previous study finding that Zhang et al. [v/v), while PCP 85 was obtained with an ethanol concentration of 85% (v/v) in this study.The yield of g et al.  obtainedPolygonatum cyrtonema Hua polysaccharides, hyaluronic acid, and sorbitol at RH 81% are shown in f, 3.38 ± 1.02% a, 24.96 ± 1.06% d, 26.60 ± 2.33% e, 19.43 ± 1.08% b, and 21.41 ± 0.31% c, respectively ). Comparative moisture absorption properties of the polysaccharides, hyaluronic acid and sorbitol were as follows: hyaluronic acid > PCP 40 > PCP 20 > PCP 85 > PCP 60 > sorbitol (p < 0.05). The results showed that PCP 20 and PCP 40 had the best moisture absorption properties, whereas PCP 60 and PCP 85 had the best moisture-proofing properties. This results of PCP 40 (26.60%) and PCP 85 (24.14%) are better than Chou [Pholiota nameko by fractional precipitation showed PNP-40 (14.37%) and PNP-80 (18.00%) moisture absorption at 81% RH after 24 h. However, PCP 60 (19.43%) was lower than PNP-60 (21.48%). The content of uronic acid in PNP-60 was speculated to be the reason for its better moisture absorption ability. Although hyaluronic acid has an efficient moisturizing effect, compared with hyaluronic acid, natural polysaccharides have moisture absorption properties as well as a variety of physiological activities, such as anti-oxidant activity [The moisture absorption values of han Chou , who fouactivity . The appb, 80.10 ± 0.99% a, 85.59 ± 0.85% b, 85.37 ± 0.78% b, 88.75 ± 1.14% d, and 86.59 ± 1.01% c, respectively ). Comparative moisture retention properties of polysaccharides, hyaluronic acid, and sorbitol were as follows: PCP 60 > PCP 85 > PCP 20 > hyaluronic acid > PCP 40 > sorbitol. Among the polysaccharides, PCP 60 had the best moisture retention (p < 0.05). Hyaluronic acid is a common humectant that shows good moisture retention performance [Sargassumhorneri polysaccharides in dry silica gel after 48 h is about 53% [2O to unify the massive moisture contents. The chain of polysaccharides can also mutually interweave with the lattice, which plays a very strong role in guaranteeing moisture content [As shown in formance . It has bout 53% . Togethe content .Polygonatum cyrtonema Hua polysaccharides can be utilized as a natural humectant on porous carbohydrates (tobacco shreds).The above results show that PCP 60 and PCP 85 had the characteristics of preventing water absorption under high relative humidity and moisturizing under dry conditions. In conclusion, Polygonatum cyrtonema Hua (PCP85-1-1) were obtained.Based on the yield and the moisture absorption and moisture retention activity, the fraction (PCP 85) with the best activity was selected and further purified through anion exchange and gel permeation chromatography to characterize the structure. PCP 85 was initially separated in a DEAE–Sepharose Fast Flow cellulose column A. The frPolygonatum cyrtonema Hua polysaccharides with a molecular weight of 2–5 kDa. These studies showed PCP was a low-weight natural polysaccharide. However, in these reports, Polygonatum cyrtonema Hua polysaccharides were a fructan. The monosaccharides of PCP 85 were different from the previous study, which were determined by HPLC-RID analysis. As shown in Polygonatum cyrtonema Hua.As shown in −1 was attributed to the O–H stretching vibration [−1 and 2884.30 cm−1 were assigned to the C–H stretching vibration. The absorption peak near 1643.89 cm−1 was the stretching vibration of C=O, and the absorption peak at 1414.08 cm−1 was the variable angle vibration of C–O, which is considered a characteristic absorption peak for plant polysaccharide structure [−1 represented the stretching vibration of the C–O–C glycosidic bond in the furan ring [−1 and 871.40 cm−1 showed that PCP 85−1−1 contained a fructose ring skeleton containing β- glycosidic bonds [The FT-IR spectra of the polysaccharide sample are shown in ibration , showingtructure . These tran ring . The absic bonds .d, and the moisture content values of the tobacco shreds with 1% propylene glycol and glycerol were 38.38 ± 0.25% bc and 37.18 ± 0.68% ab, respectively. The moisture content of the tobacco shreds with 1% PCP 85−1−1 was 36.03 ± 0.12% a ), and that with 2% PCP 85−1−1 was 35.55 ± 0.22%. When 1% PCP 85−1−1 was added to the tobacco shreds, the moisture resistance was higher than that of propylene glycol and glycerol (p < 0.05). These results showed that the addition of PCP 85−1−1 could slow down the moisture adsorption of tobacco shreds. Moreover, the moisture content values of the tobacco shreds with PCP 85−1−1 were all lower than those of control and blank, implying that PCP 85−1−1 was capable of improving the resistance of tobacco shreds to moisture. Ai et al. [According to the moisture absorption and moisture retention characteristics of the polysaccharides in vitro, the ideal moisture retention/moisture-proofing humectants (PCP 85−1−1) for tobacco shreds were selected, and moisture-proofing tests were carried out. The moisture of the tobacco shreds was very sensitive to the ambient humidity . The adsi et al.  also repp < 0.05). Additionally, 2% PCP 85−1−1 had the highest moisture retention effect with an equilibrium moisture content of 12.12% (p < 0.05). Similar results were obtained in other reports. Huang et al. [Wedelia prostrate could effectively improve the moisture retention of tobacco shreds and reduce the moisture dissipation of tobacco shreds. However, the equilibrium moisture content of the polysaccharide from Wedelia prostrate was 9.28%, which was lower than the tobacco shreds with PCP 85−1−1. These results revealed that PCP 85−1−1 accelerated moisture adsorption and an increase in moisture adsorption capacity of the tobacco shreds by PCP 85−1−1, probably due to the hydrophilicity of PCP 85−1−1, which favors water adsorption through hydrogen bonds [The moisture retention curves of PCP 85−1−1 are shown in g et al.  concludee is the same, lower v correlates with higher MPI and MRI [We used the Page model to analyze adsorption and desorption data, evaluate the half-life average moisture adsorption/desorption rate (v) as a kinetic indicator, and calculate MPI and MRI as a comprehensive index. The half-life average moisture adsorption/desorption rate represents the average value at which the change in moisture reaches the intermediate point . Tobacco shreds with low v values in both dry and humid environments are considered more stable. According to Equations (6) and (7), when M and MRI .2) of different tobacco shred samples for both desorption and adsorption processes were all above 0.950, and the mean relative percentage deviation modulus (E) ranged from 0.7181% to 1.6381%. E values lower than 10% suggest an adequate fit for practical applications [As shown in ications . Thus, tp < 0.05). Additionally, 2% PCP 85−1−1 had the maximum MRI and MPI values of 2.11 and 2.14, respectively. These results further explain that PCP 85−1−1 has a bilateral moisturizing ability. The improvement of the moisture retention capacity with the addition of PCP 85−1−1 could be explained by the hydrogen bonding effect at low RH. However, the improvement of the moisture-proofing ability could be explained by PCP 85−1−1 being a biological macromolecule which has certain film-forming properties. The film can block capillaries and weaken capillary adsorption [The Page model was used to further calculate the half-life average water adsorption/desorption rate v, MPI, and MRI. The results are shown in sorption ,36.p < 0.05). However, the weakening of the sweetness was not conducive to the taste. Compared with the control group, the taste of the tobacco shreds with 2% PCP 85−1−1 was improved  and did not adversely affect the taste. This result was consistent with the previous reports. Ai et al. [For consumers, the taste of cigarette smoke is very important. Therefore, we analyzed the taste change of the cigarette smoke after adding PCP 85−1−1 through electronic tongue technology. As shown in i et al.  found thTobacco shreds with different moisture contents had a different feeling from tobacco smoke. When the moisture content from the tobacco shreds was moderate, the feeling of the tobacco smoke was soft and comfortable, and the irritation was small. The combustion and cracking degree of the cigarette was executive, and the irritation and dryness of the smoke were strong under the low moisture content. However, when the moisture content was too high, the tobacco shreds had poor flammability, weak aroma, dull taste and lack of energy ,38. ZengThe content of aroma components was calculated by the internal standard method. GC-MS data  showed tThe volatile compound content of the control group was 4651.61 μg/g, higher than the cigarettes with the addition of 1% glycerol (3607.35 μg/g) and the cigarettes with PCP 85−1−1 4135.5 μg/g). However, the volatile compound content of cigarettes with PCP 85−1−1 was higher than the glycerol group. The majority of volatile components were nitrogenous compounds. Among them, nicotine had the highest content with a content of 1551.27–2472.44 μg/g. According to the research , a high 35.5 μg/gp < 0.05), and furans, phenols, and ketones increased in the tobacco shreds with PCP 85−1−1 humectant compared to the group with glycerol. The increase of these substances may improve the taste and aroma of cigarettes when smoking. Similar results were obtained in other reports. Tobacco shreds with humectants produced a large number of pyrolysis products during smoking, mainly including ketones, aldehydes, esters, alkenes, acids, phenols, and other aroma substances that contribute to tobacco flavors, such as furfural, 5-methyl furfural, 2-methyl-1,3-cyclopentadiene, phenethyl phenylacetate, and benzene compounds [The content of different types of aroma compounds was analyzed by normalized z-score . The numompounds . It showPolygonatum cyrtonema Hua was cultivated in Sanming City, Fujian Province, China. The dried rhizome of Polygonatum cyrtonema Hua tubers was ground into a fine powder using a high-speed disintegrator . The powder was sieved through 80 mesh (Aperture 0.18 mm) and stored in a dryer for the extraction experiment. The tobacco shred samples (flue-cured tobacco) were obtained from China Tobacco Yunnan Industrial Co., Ltd. . Glycerol (purity ≥ 99.0%), propylene glycol (purity ≥ 99.0%), ethanol (purity ≥ 99.7%), NaCl (purity ≥ 99.5%), dichloromethane (purity ≥ 99.5%), concentrated sulfuric acid (purity 95.0–98.0%), phenol (purity ≥ 99.0%), trifluoroacetic acid (purity ≥ 99.5%), Folin-phenol (1 mol/L), sodium nitrate (purity ≥ 99.0%), potassium bromide (purity ≥ 99.0%), and sodium dihydrogen phosphate (purity ≥ 99.0%) were purchased from Sinopharm Chemical Reagent Co., Ltd. . Gallic acid (purity ≥ 99.8%), Coomassie brilliant blue G-250 (purity ≥ 99.0%), bovine serum albumin (purity ≥ 99.0%) were purchased from Shanghai TITAN Technology Co., Ltd. . D-fructose (purity ≥ 99.0%), D-galactose (purity ≥ 99.0%), D-glucose (purity ≥ 99.0%), D-arabinose (purity ≥ 99.0%), L-fucose (purity ≥ 99.0%), L-rhamnose (purity ≥ 99.0%), D-mannose (purity ≥ 99.0%), D-xylose (purity ≥ 99.0%), and methanol (purity ≥ 99.9%) were from Sigma . Hyaluronic acid was purchased from Yuanye Biotechnology Co., Ltd. .Polygonatum cyrtonema Hua powder was extracted at the optimal extraction temperature of 80 °C using the optimal material liquid ratio of 25 mL/g for 2 h, once. The extract was separated from Polygonatum cyrtonema Hua residue using gauze (Aperture 0.18 mm) and separated at 6000 r/min for 15 min by centrifugation . After centrifugation, the supernatant was concentrated using a rotary vacuum evaporator  at 50 °C.v/v) successfully and incubated overnight at 4 °C. The precipitate was then collected by centrifugation at 4000 r/min for 20 min, freeze-dried, and four different fractions  were obtained.PCP was obtained by fractional precipitation. Different volumes of absolute ethanol were added to the concentrated supernatant to create a series of ethanol concentrations  81%) at 25 °C for 2, 4, 6, 8, 12, 24, and 48 h. The weight of each sample was accurately weighed, and the moisture absorption rate was calculated from the weight difference before and after moisture absorption. The moisture absorption was determined using Equation (1).0 was the weight of an oven-dried sample, and tm was the weight of the sample after moisture absorption for a specific time.The moisture absorption experiment was performed with modification as described before . Polygon0 was the weight of the sample at the beginning of the moisture retention test and tm was the weight of the sample after moisture retention for a specific time.The weight of each sample was recorded at 48 h of the moisture absorption experiment, which was the first weight of sample moisture retention. The Petri dish was placed in a desiccator containing saturated sodium carbonate solution (relative humidity (RH) 43%) at 25 °C. The weight of each sample was accurately weighed and the moisture retention rate was calculated from the weight of the sample after water loss compared with the first weight of sample moisture retention . The moiFor further purification, the crude polysaccharides (PCP 85) dissolved in deionized water were subjected to DEAE—Sepharose Fast-Flow column (2.6 cm × 40.0 cm). The column was eluted with a stepwise gradient with 200 mL of distilled water followed by 0.1, 0.2, and 1 M NaCl solutions at a 3 mL/min flow rate. The eluates (10 mL/tube) were collected, and the total carbohydrate contents were detected by using the phenol–sulfuric acid method. The water fraction was further purified with a Superdex 75 pg column (1.6 × 60 cm) using distilled water as eluent at a flow rate of 1 mL/min, and fractions were collected based on the peaks of eluting profile detected by the refractive index (RI) detector.The analysis was based on the method of Wang  with app3 and 0.05 mol/L NaH2PO4  with flow rate of 0.5 mL/min and column temperature of 30 °C, and the peak was detected with waters 2414 differential refraction detector and eight-angle laser light scattering detector.Molecular weight analysis was performed with modification as previously described . Briefly−1 and 500 cm−1.For FT-IR spectroscopy, 1 to 2 mg of the polysaccharide sample was mixed with 200 mg KBr, grounded, and pressed into a 1 mm KBr pellet. IR spectra were recorded on a Fourier-transform infrared (FTIR)-spectrometer, scanning between 4000 cmAs described in Lin et al. , the tobThe balanced tobacco shreds were placed at 22 °C/RH 85% and 22 °C/RH 33% for adsorption and desorption experiments. The moisture content of tobacco shreds was calculated according to the difference between the quality of tobacco shreds before and after water absorption and the dry base weight of tobacco shreds. The final moisture content was considered the equilibrium moisture content (EMC). The initial moisture content of tobacco shreds was determined using the oven-drying method at 100 °C for 2 h . The moi0 is the initial moisture content, (%); Me is the equilibrium moisture content, (%); tM is the moisture content at a given time, (%); t is the adsorption or desorption time, (h); and n and k are model constants. The fitting parameters were estimated using non-linear regression analysis with Origin 9.0 and the fit was evaluated using R2 and E.i,exp, and Mi,pre are experimentally observed and model-predicted moisture contents, respectively; n is the number of data points.MRI (moisture retention index) was calculated as described by Lou et al.  to evalu1/2 represents the time when MR = 1/2 (h); e for different samples, (%).The half-life average moisture adsorption/desorption rate v (%/h), MPI, and MRI were calculated with Equations (5)–(7), respectively:As an instrument for rapid detection of food flavor, the electronic tongue can solve the shortcomings of artificial sensory evaluation, such as time and high cost, and can objectively evaluate food flavor . The tobAccording to the GB/T 19609-2004 in China , cigaretThe smoke was collected in artificial saliva, with a constant volume of 200 mL. Sensory analysis of artificial saliva was performed with an electronic tongue, and three parallel tests were performed for each group.Consistent with the smoking method in m/z to 300 m/z in 1 s. The mass spectrum transmission line temperature was 280 °C. The ion source temperature was 230 °C. The electronic ionization (EI) source energy was 70 eV, and the quadrupole temperature was 150 °C.Chromatographic analysis was performed using a QP2010 series GC–MS  equipped with an Agilent 19091S column (60 m × 0.25 mm × 0.25 μm). The injection volume was 0.2 μL. The injection temperature was 250 °C. All samples were injected in split mode with a split ratio of 5:1 and a flow rate of 1.0 mL/min. The initial temperature was maintained at 50 °C for 4 min and then raised to 300 °C at a rate of 4 °C/min and maintained for 10 min. The solvent delay time was 6 min. Mass spectra were acquired in full-scan mode with repetitive scanning from 30 t test using SPSS software  for mean differences among the samples by one-way analysis of variance (ANOVA). Differences between groups at p < 0.05 were considered statistically significant.Results are presented as mean ± standard error of the mean (SEM). The statistical significance was analyzed by student’s Polygonatum cyrtonema Hua polysaccharide (PCP 85−1−1) was obtained by fractional alcohol precipitation, and its structural composition, moisture absorption, and moisturizing characteristics were studied. PCP 85−1−1 was classified as homogeneous polysaccharide by the molecular weight analysis and the monosaccharide composition analysis and mainly consisted of fucose, glucose and fructose, and the molar ratio was 22.73:33.63:43.65. The FT-IR spectra characteristics of PCP 85−1−1 showed that PCP 85−1−1 contained a furan ring skeleton with β-glycosidic bonds. Moisture absorption and the moisturization test showed that PCP 60 and PCP 85 had a good two-way moisturizing effect. The ability of PCP 85−1−1 to improve the moisture retention and moisture-proofing characteristics of porous carbohydrates was analyzed. The result revealed accelerated moisture adsorption and an increase in moisture adsorption capacity of the tobacco shreds with PCP 85−1−1 under dry conditions, and PCP 85−1−1 can slow down the moisture adsorption in tobacco shreds under high relative humidity. It was concluded that PCP 85−1−1 had excellent moisture retention and moisture-proofing characteristics. Finally, the aroma analysis showed that the addition of PCP 85−1−1 improved the increase in furans, phenols, and ketones and can enrich the fullness of flue gas and improve the taste by changing the moisture content. In conclusion, Polygonatum cyrtonema Hua polysaccharides can be utilized as a natural humectant on porous carbohydrates (tobacco shreds).In conclusion,


Fibronectin glomerulopathy is a rare, familial glomerular disease characterized by mesangial fibronectin deposition in the glomeruli. It is caused by the genetic defect in fibronectin and does not involve the activation of the immune system. Therefore, glomerular immunoglobulin and complement staining is generally absent or weak. Monoclonal gammopathy (MG) is an increasing cause of renal lesion, featured by light chain (κ or λ) and/or heavy chain restriction in glomeruli. Herein, we report a case of fibronectin glomerulopathy presenting as strong IgA and C3 immunostaining in renal biopsy, concomitant with monoclonal gammopathy .A 44-year-old female was admitted to our hospital for one-month pedal edema. The serum albumin of 19.6 g/l, and the 24-h urine protein was 15.092 g. Immunofixation electrophoresis displayed monoclonal IgA. The renal biopsy showed the mesangial deposits positive for IgA (3+) and C3 (3+) and also for IgG (2+), IgM (2+), and C1q (2+) IF microscopy. In addition, the staining intensity of light chain κ was slight greater than that of light chain λ. The glomerular deposits were strongly positive by FN by immuohistochemistry. The patient was treated with bortezomib, dexamethasone in combination with cyclophosphamide and gained partial remission.We present the first FNG patient with strong IgA and C3 immunostaining in the context of monoclonal IgA κ in the circulation. Perhaps FNG, monoclonal IgA κ and immune activation are potentially interplayed and eventually induce renal injuries. Fibronectin glomerulopathy (FNG) is an autosomal dominant disorder characterized by glomerular mesangial and subendothelial fibronectin (FN) deposition, with no glomerular immunoglobulin (Ig) or complement (C). The clinical features of FNG include proteinuria, microscopic hematuria and hypertension leading to end-stage renal failure in the second to sixth decades of life, and it was first described in 1987  and, theA 44-year-old female was admitted to our hospital for one-month pedal edema. She had no remarkable medical or family history.On admission, her blood pressure was 134/84 mmHg. A physical examination revealed mild edema of the lower extremities. There were no specific findings suggestive of connective tissue diseases.Initial laboratory data showed decreased hemoglobin of 97 g/L and serum albumin of 19.6 g/l. The renal and hepatic functions were normal. Urinalysis showed proteinuria 3+ and no microscopic hematuria or casts. The 24-h urine protein was 15.092 g, with middle-molecular and large-molecular protein accounting for 62.8 and 37.2%, respectively. Plasma C3 was slightly decreased at 0.74 g/l , whereas C4 was normal at 0.23 g/l (0.16–0.35 g/l). IgG was 3.41 g/l (7.51–15.6 g/l), IgA was 8.66 g/l (0.82–4.53 g/l), and IgM was 0.66 g/l (0.46–3.04 g/l). C-reactive protein was slightly elevated at 7.5 mg/l (0–5 mg/l). Immunofixation electrophoresis displayed monoclonal IgA κ. A free light chain examination displayed a κ concentration of 595.00 mg/l (0.39–15.10 mg/l) and a λ concentration of 147 mg/l (0.81–10.10 mg/l), resulting in an increased κ to λ ratio of 4.0476 (0.461–4.000). The tests for ANA, anti-dsDNA antibodies, ANCA, anti-ENA antibodies, hepatitis B virus antigen and hepatitis C virus antibodies were all negative. Renal ultrasound revealed normal size and mild cortical hyperechogenicity.Bone marrow aspiration indicated hyperplastic anemia and 3% plasma cells. The flow cytometry of bone marrow detected 1.22% monoclonal plasma cells which were positive for CD38, CD138, CD117, CD27, CD200 and clonal κ but negative for λ.The renal biopsy obtained thirty-eight glomeruli, with two displaying global sclerosis. In other glomeruli, the mesangium was greatly expanded, with minimal hypercellularity, by massive eosinophilic, strongly PAS-positive and silver-negative materials, leading to narrowed or occlusive capillary lumina Fig. . No doubThe patient was given valsartan (80 mg qd) in combination with the BCD regimen .She was followed up for approximately 3 years and the pedal edema improved gradually. The patient reported no treatment-related adverse effects. The proportion of plasma cells decreased to 0.5%, and the serum IgA level decreased to 0.67 g/L. Monoclonal IgA κ became negative at 7 months post treatment. Two months later, the serum albumin increased from 18 to 25 g/L to 34 g/L, and the 24-h urine protein decreased from 9 to 13 g to 4 g. At her most recent examination, the serum albumin was 40.8 g/l and the 24-h urine protein was 1.54 g. However, her serum creatine increased from 54.6 μmol/l to 102.8 μmol/l  and/or heavy chain restriction in the glomeruli . For the patient, the IF intensities of κ was only slightly greater than that of λ, seemingly excluding the possibility that the renal lesion was induced by MG. However, her monoclonal immunoglobulin was IgA κ, while the glomerular fibrillary deposits stained dominantly for IgA and C3, and her renal symptoms were significantly improved by BD regimens, suggesting that the monoclonal IgA κ played some role in the renal injury. The less staining intensity difference of two types of light chains may be caused by deposited IgG and IgM which provide some λ light chain. To identify the relationship between FNG and MG, a PubMed search was conducted using the term “FNG”. Only an 88-year-old patient coexisted with FNG and MG (MIgG κ), with a single C3 deposit (2+) in the glomeruli , and theThe present patient should be especially differentiated from lupus nephritis, cryoglobulinemia, fibrillary glomerulonephritis, immunotactoid glomerulonephritis, and membranoproliferative glomerulonephritis which can manifest as fibrillar deposits with multiple positive Ig and complement immunostaining . Lupus nThere is no standard treatment regimen for FNG. Reportedly, indomethacin , mizoribIn conclusion, we reported the first FNG patient with strong IgA and C3 immunostaining in the context of monoclonal IgA κ in the circulation who partly responded to the BD regimen. Perhaps FNG, MG and immune activation are potentially interplayed and eventually induce renal injuries, but the mechanism needs to be further explored. In addition, FNG should be considered in nonamyloid lobular glomerulopathy, even when immunocomplexes are found in the glomeruli.