This study showed that NAR-NCT could improve the bioavailability of NAR.Naringenin (NAR), 4′,5,7-trihydroxydihydroflavone, has a wide range of pharmacological activities but shows poor water solubility and low bioavailability. The pharmacokinetics and bioavailability of naringenin-nicotinamide cocrystal (NAR-NCT), which offers improved solubility, were evaluated in this study. Rats were orally administered NAR, a physical mixture of naringenin and nicotinamide (NAR + NCT), and NAR-NCT. The relative bioavailability of NAR-NCT was 175.09% of NAR, C Naringenin (NAR), 4′,5,7-trihydroxydihydroflavone , is a diCocrystal is defined as the crystal of combination with active pharmaceutical ingredient and cocrystal former. It with specific physical and chemical properties can form through weak noncovalent interactions, for example, hydrogen bonding . CompareNaringenin (>96%) and nicotinamide  were provided by Bailingwei Technology . Internal standard (IS), hesperetin >98%, , was proThe cocrystal was prepared by solvent evaporation. 50 mg of NAR and 45 mg of NCT (at a molar ratio of NAR and NCT of 1 : 2) were mixed with ethyl acetate (10 mL). After ultrasonic dissolution, the mixtures were heated at 40°C for 4 hours . Differeμm) at 40°C and 0.3 mL/min. The mobile phase constituted 0.2% acetic acid-water  and methanol (B). The injection volume was 3 μL. The chromatographic program was as follows: gradient elution, 0–3 min, 60% B; 3–3.5 min, 60–90% B; 3.5–6.5 min, 90% B; 6.5–7 min, 90–60% B; and 7–10 min, 60% B.The chromatographic separation was carried out by a Shimadzu UFLC-20AD XR system . The column was Shim-Pack C18-ODS column  with a negative mode electrospray ionization (ESI) interface was equipped with Shimadzu UFLC-20AD XR. Multiple reaction monitoring was used to analyse samples. Mass spectrometry parameters are shown in Secondary mass spectrometry enhanced product ion (EPI) scan was used to obtain a high-quality MS/MS spectrum on a specific ion. This scan type was performed on NAR and IS. The product ion mass spectra about NAR and IS are recorded in μg/mL stock solution. It was diluted with methanol to obtain standard working solutions of 10.3, 30.8, 102.6, 513, 1026, 2052, 4104, 6156, and 8208 ng/mL. Hesperidin was dissolved in methanol (300 ng/mL) as an internal standard solution.NAR was dissolved in methanol to obtain a 102.6 The calibration standards of NAR at a range of 1.03 to 821 ng/mL, which were prepared from blank plasma and NAR standard working solutions.QC samples were formulated at low, medium, and high concentrations  corresponding to NAR of 3.08, 410, and 616 ng/mL, respectively, following the procedure described above. The samples were protected from light.μL) was combined with 10 μL of 300 ng/mL internal standard solution in a centrifuge tube, and the mixture was vortexed for 5 min. Then, samples were mixed with ethyl acetate (300 μL), and mixtures were vortex-mixed for 5 min. After that, mixtures were centrifuged at 8000 r/min for 2 min. All supernatants were pipetted into a new tube and dried at 40°C under flowing nitrogen. And then, residues were reconstituted with methanol (100 μL) and vortex-mixed for 1 min.Plasma .To demonstrate that substances being measured were intended analytes and endogenous substances and other metabolites do not interfere with the assay, method specificity was established firstly. This analysis involved blank plasma samples (different sources), the mixture of NAR standard solution and blank plasma, and plasma samples after oral administration (X2) to obtain the calibration curve.Six blank rat plasma samples were combined with the NAR standard working solutions to give final NAR plasma concentrations of 1.03, 10.3, 51.3, 103, 205, and 821 ng/mL. As described in The lowest concentration of analyte that can be reliably quantified in the sample was the LLOQ. Six standard plasma samples containing NAR were analysed as described in At the LLOQ, the relative standard deviation  was ≤20%, the relative error  was required to be within ±20%.NAR plasma standard samples were prepared at the LLOQ, LQC, MQC, and HQC concentrations , according to The acceptance criteria were RSD (%) was ≤15%, RE (%) was limited to ±15% for the intra- and interday, except at the LLOQ, where RSD (%) should be ≤20% and RE (%) within ±20%.μL) was added to the mixture, vortexed for 5 min, and added ethyl acetate (300 μL). After that, the samples were centrifuged at 8000 r/min for 2 min. All supernatant was transferred to a new tube. All of them were concentrated to dryness under flowing nitrogen, and the residue was redissolved in methanol (200 μL) to prepare it for analysis. The peak area of NAR (As) was recorded.NAR plasma standard samples were prepared at the LLOQ, LQC, MQC, and HQC concentrations . Then, internal standard solution (10 μL) from different sources were mixed with ethyl acetate (300 μL). The samples were vortexed for 5 min, then centrifuged for 2 min at 8000 r/min, and then all supernatant was removed for analysis. Aliquots 10 μL of the standard solutions  and 10 μL of 300 ng/mL internal standard solution were added. All of them were concentrated to dryness under flowing nitrogen, and the residues were redissolved in methanol (200 μL) for analysis. The peak area of NAR (As′) was recorded.Blank plasma samples (100 The extraction recovery (%) of NAR was obtained by substituting As and As′ into the following equation:The recovery of the IS was investigated similarly.μL, at concentrations of 10.3, 30.8, 4104, and 6156 ng/mL) were combined with the internal standard solution  in a centrifuge tube. The mixture was vortexed (5 min), dried at 40°C under flowing nitrogen, and redissolved in methanol (200 μL). The supernatant was then analysed. The peak area of NAR (Bs) was recorded.The standard solutions (10 μL), from different sources, were each mixed with ethyl acetate (300 μL), and the mixture was vortexed (5 min) and centrifuged . All supernatant of each sample was removed to a new tube, and standard solution  was added to the internal standard solution . The sample was dried at 40°C under flowing nitrogen. Methanol (200 μL) was used to reconstitute in the residue. The peak area of NAR (Bs′) was recorded.Blank plasma samples (100 The matrix effect (%) on NAR was calculated according to the following formula:The ME of the IS was investigated.Analyte's stability in the matrix at the LLOQ, LQC, MQC, and HQC under different conditions was investigated. The evaluated conditions were 24 h short-term stability at room temperature (25 ± 3°C), freeze-thaw cycles three times, long-term stability under −80°C storage 21 days, and room temperature in an automatic sampler for 24 h.The acceptance criteria for the RSD (%) was ≤15%, RE (%) was limited to ±15%, except at the LLOQ, where RSD(%) was ≤20% and RE(%) within ±20%.n = 6). Accuracy of the concentration determined following dilution was required to be between 85% and 115%, and the precision should be within 15%.The dilution reliability of NAR in plasma must be examined because of the high concentrations  of some drugs in the plasma. The plasma sample  was diluted with blank plasma 10 times and analysed according to Eighteen SD rats  were obtained from National Institutes for Food and Drug Control . The room environment was about 22 ± 2°C, the humidity of 50 ± 10%, light and dark recycle for 12 h. Before the experiment, the rats can be given water freely but were fasted for 12 h. All experiments were following the National Institutes of Health Guide for the Care and Use of Laboratory Animals .There were three groups for oral administration. The first group was administered NAR (30 mg/kg). The next group was administered NAR + NCT, in which the molar ratio of NAR and NCT was 1 : 2, in terms of NAR 30 mg/kg. The last group was administered NAR-NCT (57 mg/kg), which was equal to the NAR dose of 30 mg/kg. These were ground with 0.5% sodium carboxymethyl cellulose, at a dose of 1 mL/100 g. At 0.05, 0.083, 0.167, 0.333, 0.5, 1, 2, 4, 8, 10, 12, and 24 h after administration, 0.5 mL serial blood samples were obtained from the ocular vein into centrifuge tubes. The samples were centrifuged at 10000r/min , and after that, all supernatant of each sample was removed to store at −80°C.Noncompartmental modelling was evaluated for pharmacokinetic parameters through DAS Software .The following equation was used to calculate the relative oral bioavailability  in rats:The NAR-NCT cocrystal was prepared and characterized, which can improve solubility in water. The DSC endothermic peak of the NAR-NCT was 134.68°C. The characteristic peaks in the DSC, XRPD, and IR spectra of NAR-NCT were different from the naringenin-nicotinamide cocrystal reported in the literature . It is sThe stability of NAR-NCT was investigated at 60°C, at a relative humidity of 90 ± 5%, under light of 5000 Lx for 10 days and after 6 months at room temperature. In terms of appearance, NAR-NCT showed no difference under the above conditions. A comparison of the XRPD spectra  showed tμL of ethyl acetate were analysed. Results showed that ethyl acetate (300 μL) had the best extraction effect. Therefore, ethyl acetate (300 μL) was used to the extracted solvent for pretreatment.Protein precipitation and liquid-liquid extraction methods were tested for the removal of interfering species from the sample. Organic solvents, such as methanol, acetonitrile, diethyl ether, ethyl acetate, and n-hexane were investigated, and the results are shown in The blank plasma sample showed no signals interfering with the measurement of the analyte. NAR and IS were well separated from endogenous substances and metabolites, and no interference was observed. The chromatograms are shown in Y = 0.11X + 0.0914, and r = 0.9995. The standard error of slope and intercept was 0.002 and 0.005, respectively. LLOQ was 1.03 ng/mL for the NAR plasma sample.The calibration curve showed good linearity, at the range of 1.03–821 ng/mL, The results of precision and accuracy during intraday and interday were as follows: both RSD (%) values were ≤8.5%, while both RE (%) values were at the range of −6.2% to 4.7% and −2.6% to 2.8%, respectively .The extraction recoveries were 69.0% to 76.5%, and RSD (%) values were ≤10.2%. The matrix effects were 107.7% to 131.2%, and RSD (%) values were ≤7.7% .The plasma samples were stable for 24 h at room temperature (25 ± 3°C) with the RE% value of 0.9% to 10.3%. After three freeze-thaw cycles, NAR also remained stable (RE%: 1.6% to 4.8%). The sample exhibits 21 days of long-term stability (RE%: −6.9% to 0.2%) at −80°C, and the ready-to-inject sample was stable for 24 h in an autosampler at 25°C (RE%: −8.4% to 4.4%). These results are recorded in After the plasma QC samples were diluted with blank plasma, the RE ranged from −8.1%% to 1.9%, and the RSD was 4.7% . These rThe plasma concentrations of NAR were determined by LC-MS/MS. The concentrations were analysed with DAS Software. The main pharmacokinetic parameters are recorded in max of NAR + NCT was greater by a factor of 2.06, and Tmax changed from 0.49 h to 0.11 h. Although NAR + NCT accelerated drug absorption, it did not improve drug metabolism. The values of CL and t1/2 showed that the elimination rates of the two groups were similar. As a result, the relative bioavailability was 77.23% of NAR.Compared with NAR, the Cmax was increased by a factor of 8.43, and Tmax was advanced from 0.49 h to 0.09 h. In contrast to NAR + NCT, the CL of NAR-NCT decreased from 91.1 L/h/kg to 49.1 L/h/kg, and t1/2 increased from 5.37 h to 8.24 h, showing the rapid absorption and slow elimination of this formulation. The relative bioavailability of NAR-NCT was 175.09% of NAR, compared with 80.89% reported in the literature [max increased and Tmax decreased. After the dissolution of NCT, the amorphous NAR, with high free energy, slowly converted to a metastable state and then to the lowest energy NAR configuration . The time required for this process resulted in slower NAR elimination and increased AUC in the body.A comparison between NAR-NCT and NAR showed that Cterature . This reterature . NCT wasmax, respectively. After 0.5 h, a longer interval of blood collection was set. The rats could be allowed to drink water freely in longer blood sampling intervals. Therefore, the process of blood sampling did not have a serious impact on the pharmacokinetics of NAR.The peak time of NAR, NAR + NCT, and NAR-NCT is 0.49 h, 0.11 h, and 0.09 h, respectively, so NAR has reached the peak at 0.5 h. And the last points of the pharmacokinetic study in the three groups were less than 1/10 of COnly the drug-time curve of NAR-NCT showed the double peaks, which was consistent with enterohepatic circulation  and exteWe investigated the differences in pharmacokinetic characteristics and the relative bioavailability of NAR after oral administration of NAR, NAR + NCT, and NAR-NCT in rats. These results show that NAR-NCT was a viable method for improving the bioavailability of NAR. These results provide important references for the application and research of NAR in the future.


P = 0.05). The overall survival (OS) at 12, 24, and 36 months was 96.2%, 90.1%, and 79.5%, respectively. TP53 disruption was associated with an increased risk of death (P = 0.01). Data on 162 patients ≥65 years treated with ibrutinib were analyzed and compared with 165 patients ≥65 years treated with BR. Factors predicting for a longer PFS at multivariable analysis in the total patient population treated with BR and ibrutinib were age  and treatment with ibrutinib . In a post hoc analysis of patients in advanced stage, a significant PFS advantage was observed in patient who had received ibrutinib (P = 0.03), who showed a trend for OS advantage (P = 0.08). We arrived at the following conclusions: (a) BR is a relatively effective first‐line regimen in a real‐world population of unfit patients without TP53 disruption, (b) ibrutinib provided longer disease control than BR in patients with advanced disease stage.Limited information is available on the efficacy of front‐line bendamustine and rituximab (BR) in chronic lymphocytic leukemia (CLL) with reduced renal function or coexisting conditions. We therefore analyzed a cohort of real‐world patients and performed a matched adjusted indirect comparison with a cohort of patients treated with ibrutinib. One hundred and fifty‐seven patients with creatinine clearance (CrCl) <70 mL/min and/or CIRS score >6 were treated with BR. The median age was 72 years; 69% of patients had ≥2 comorbidities and the median CrCl was 59.8 mL/min. 17.6% of patients carried TP53 disruption. The median progression‐free survival (PFS) was 45 months; TP53 disruption was associated with a shorter PFS ( Bendamustine and Rituximab was a relatively effective first‐line regimen in real‐world untreated CLL patients with reduced renal function or coexisting conditions without TP53 disruption.In a matched‐adjusted indirect comparison with a cohort of CLL patients treated upfront, ibrutinib provided longer PFS than bendamustine and rituximab in those with advanced stage. The endpoints for this analysis were the PFS and OS.For the purpose of this analysis, we retrieved pertinent data by reviewing clinical charts, electronic medical records, and related databases in patients ≥65 years enrolled in the GIMEMA ERIC study who had received BR in first line and in patients ≥65 years of a US study who had received single agent ibrutinib.2.4The analyses were performed following the intention‐to‐treat principle as previously described.33.1IGHV gene configuration, 17.6% carried 17p‐ and/or TP53 mutation (data available in 83.4% of the patients).One hundred fifty‐seven patients treated at 31 centers (24 GIMEMA centers and 7 ERIC centers) were included. The demographic data at baseline have been outlined in Table 2 in 45.8% of the patients, 70 mg/m2 in 33.1% of the patients and 50‐70 mg/m2 in 21.1% of the patients. A dose reduction >10% of the planned dose of bendamustine was recorded in 24.2% of cases; treatment delay occurred in 22.9% of patients.One hundred and fourteen of 157 patients (73.1%) received the planned number of cycles; treatment was discontinued as a result of toxicity in 18% patients (n = 28), withdrawal of consent in 2% (n = 3), or other reasons in 7% (n = 11). The number of cycles actually administered to patients who discontinued treatment was ≥4 in 71% of cases. The starting dose of bendamustine was 90 mg/mSecond‐line treatment was offered at symptomatic progression according to local guidelines.3.2TP53 mutations as compared to patients without del(17p)/TP53 mutations . Other variables had no significant impact on the ORR (Table S2).The ORR to BR was 91.7%. The probability of attaining a response was significantly lower in patients with del(17p)/TP53 mutations, ECOG performance status and by the presence of 2 or more comorbidities, as shown in Table ‐unmutated status were of borderline significance. Age (cut‐off 65 years) and other clinicobiologic parameters had no impact on PFS. del(17p)/TP53 mutations represented the only adverse parameter in multivariable analysis , with ais Table ).TP53 mutation .A second‐line treatment was administered to 3.2% (95% CI 1.2%‐6.9%), 8.3% (95% CI 4.3%‐13.9%), and 21.5% (95% CI 13.1%‐31.1%) of patients at 12, 24, and 36 months, respectively . A shorter TTNT was associated with del(17p)/TP53 mutation (P < 0.01) and lack of response to treatment (P < 0.01) were associated with a shorter OS in univariate as well as in multivariate analysis , 90.1% (95% CI 85.0%‐95.5%), and 79.5% (95% CI 70.0%‐90.5%), respectively, with a median OS that was not reached with a 26‐month median follow‐up  and 13 months (95% CI 10‐14), respectively. As shown in Table Data on 165 and 162 older patients without del(17p)/P < 0.01).Factors associated with a longer PFS in multivariate analysis in the combined patient population treated with BR and ibrutinib were represented by age as a continuous variable and by treatment with ibrutinib Table . Age wasP = 0.03), which also showed a trend for an advantage in terms of OS  and OS (P = 0.66) in these patients , the ibrutinib cohort n = 96 patients), and the BR cohort (n = 46 patients) were comparable in terms of age (cut‐off 70 years) and interval between diagnosis and first‐line treatment (cut‐off 36 months), as shown in Table  patientss Figure  and (B))4This is the first robust report of the efficacy and safety of BR in unfit patients treated outside clinical trials, as these patients were usually enrolled in trials using chlorambucil and an anti CD20 monoclonal antibody.Even though this study is not based on a registry reporting the efficacy of BR and ibrutinib in all the incident CLL cases and accepting the limitations of a retrospective analysis, to ensure accuracy in data collection we encouraged each participating center to include all patients who started BR and we performed consistency checks on each case report form. Furthermore, we included, in addition to PFS, objective measures of efficacy such as OS and TTNT. Because bone biopsy was not mandatory, we were not able to assess complete response.Our BR cohort resembled closely patients with CLL treated in the daily clinical practice in terms of age, creatinine clearance, performance status and comorbidities.The proportion of patients who completed the planned therapy in our analysis (73.1%) is in line with the data form the MABLE trial that compared chlorambucil and rituximab with the BR regimen in a fludarabine‐ineligible CLL population.Grade 3‐4 infections in this study (11%) occurred at a similar frequency as in clinical trials (7.7%‐19%).TP53 disruption and, interestingly, failure to respond to BR was an independent dynamic prognostic factor for OS, a finding probably reflecting the lack of effective salvage treatment for a proportion of patients during the study period. The IGHV‐unmutated configuration was associated with a shorter PFS, that was of borderline significant probably as a consequence of the limited number of patients assessed.With a median PFS of 45.1 months and a projected OS rate at 24 and 36 months of 90.1% and 79.5%, respectively, our data show that BR is an effective front‐line treatment option for an unfit patient population treated outside of clinical trials. The only baseline characteristics with a significant negative impact on the efficacy endpoints in this study were represented by the In our analysis PFS is similar to that observed in the MABLE trial (median 39.6 months).It is worth noting that chlorambucil and rituximab produced an 87% ORR with an OS rate at 48 and 60 months of 86.1% (95% CI: 79.4‐93.5) and 81.2% (95% CI: 72.4‐91.2), respectively, in elderly patients treated outside clinical trials.Because there is evidence that treatment continuation and efficacy with ibrutinib may be inferior outside of clinical trials,Although the two groups were treated in different time periods with consequent difference in follow‐up time, we performed an indirect comparison trying to minimize the effect of the heterogeneity of patient populations.First, we documented that in the total study population (excluding patients with 17p‐) age and treatment with ibrutinib were associated with a longer PFS in multivariate analysis.When restricting our analysis to patients with advanced stage (Rai III‐IV) with comparable baseline characteristics, we were able to show that ibrutinib was associated with a longer PFS and a trend of longer OS as compared with BR, whereas no difference was observed in patients with early intermediate stage. Though caution should be exercised when interpreting these data, as they were obtained in quite a small number of patients treated outside of clinical trial, it is noteworthy that similar findings were reported in a randomized prospective study.TP53 aberrations and IGHV‐unmutated configuration, (b) there are no appreciable differences in terms of efficacy and safety between BR in clinical trials that enrolled older patients with CLL and this real world experience on unfit patients with reduced renal function and with coexisting conditions and, (ci) and ibrutinib provided more durable disease control than BR in the front‐line setting in patients treated outside of clinical trials, especially in patients with advanced disease stage.Taken together our data show that (a) BR is a safe and effective first‐line regimen in a real‐world cohort of unfit CLL patients, with the exception of patients with unfavorable genetic characteristics (ie del(17p)/While providing evidence that BR is an effective first‐line regimen in unfit patients, these data reinforce the notion that ibrutinib has an established place in the front‐line treatment of older patients with CLL.Antonio Cuneo: advisory board and lecturing for Janssen, Gilead, Abbvie, Roche.Anthony R. Mato: consultancy for TG Therapeutics (in addition DSMB), Abbvie, Pharamacyclics, Johnson & Johnson, Regeneron, Astra Zeneca, and Celgene and research funding from TG Therapeutics, Abbvie, Pharamacyclics, Johnson & Johnson, Regeneron, Portola, DTRM, and Acerta.Gian Matteo Rigolin: lecturing for Abbvie, Gilead and research funding from Gilead.Luca Laurenti advisory board and lecturing for Janssen, Gilead, Abbvie, Roche and Astra ZenecaJohn N. Allan Advisory board/Consultant for Sunesis, PCYC, Abbvie, Genentech and research funding from Janssen, GenentechJohn M. Pagel: Consultancy for Astrazeneca, Pharmacyclics, and Gilead.Constantine S. Tam honorarium and research funding from Janssen.Paul M. Barr: consultancy for from Abbvie/Pharmacyclics, Gilead, Janssen, TG therapeutics, AstraZeneca, Celgene, Morphosys, Seattle Genetics.Alan P. Skarbnik: consultancy for Abbvie, Pharmacyclics, Celgene, Kite, AstraZeneca, Genentech, Seattle Genetics; Speakers Bureau for Abbvie, Pharmacyclics, Celgene, Kite, Gilead Sciences, Jazz Pharma, Beigene, AstraZeneca, Genentech, Seattle Genetics, Verastem, Novartis; Stock Ownership in COTA Healthcare.Nirav N. Sha: honoraria, travel support, and research funding from Miltenyi Biotec, honoraria and travel support from Incyte, and Celgene; advisory boards for Kite, Celgene, and Cellectar; research support for clinical trials from BMSChaitra S. Ujjani: research support from Pharmacyclics, Astra Zeneca and Abbvie; consulting for Astra Zeneca and Abbvie.Lindsey Roeker: ASH grant funding for work outside of this manuscript, minority ownership interest in AbbVie and Abbott LaboratoriesGianluca Gaidano: Advisory Boards for Janssen, Abbvie, Astra‐Zeneca, SunesisMichael Doubek: Honoraria and Research grants from Roche, AbbVie, AOP Orphan, Janssen‐Cilag, Gilead and Amgen.Lucia Farina: advisory board for Janssen; lecturing for Abbvie.Marta Coscia: advisory board and lecturing from Janssen for Gilead, Abbvie, and esearch funding from Janssen and Karyopharm TherapeuticsRobin Foà34: Editorial boards and/or speaker's bureau for Janssen, AbbVie, Amgen, Novartis, Roche, Pfizer.Paolo Ghia: honoraria from AbbVie, Adaptive, ArQule, BeiGene, Celgene/Juno, Dyname, Gilead, Janssen, Sunesis and research funding from AbbVie, Gilead, Janssen, Novartis.AC, ARM, GMR, AP, MV, RF and PG designed the study and interpreted the data. AC, ARM, GMR, MG, LL, JNA, JMP, DMB, BTH, AW, NL, CST, RJ, FL, PMB, MS, APS, JJP, ARS, SJS, NNS, CSU, LR, EMO, AB, LT, MS, MM, AT, FI, GG, MD, LF, SM, FDR, MC, FRM, JdlS, AMP, IF, GC and PG collected data. AP and RC performed statistical analyses. AC and GMR wrote the manuscript. All the authors reviewed the manuscript for important intellectual contents and approved the final version of the manuscript.Fig S1Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.


Arterial stiffness is important in the development of albuminuria. The brachial–ankle pulse wave velocity (baPWV) acts as an indicator of arterial stiffness and may be associated with cardiovascular disease morbidity and mortality. The urine albumin-to-creatinine ratio (UACR) is a metric used to diagnose albuminuria and has also been shown to be associated with cardiovascular disease. Here, we aim to elucidate the relationship between the baPWV and UACR in the Chinese community.A community-based cohort of 3669 subjects was selected for the analysis. The BaPWV and UACR were measured from each subject. UACR ≥ 30 mg/g was defined as pathological albuminuria.P = 0.08). However, in participants with baPWV ≥ 1269 cm/s, the prevalence of microalbuminuria and macroalbuminuria increased with increasing baPWV 100 cm/s .The mean baPWV was 1536.59 ± 305.89 cm/s, and the median UACR value was 6.11 mg/g . A threshold-effect analysis was conducted, and the results showed that the cut-off value for the baPWV was 1269 cm/s. In subjects with baPWV values lower than 1269 cm/s, the prevalence of microalbuminuria and macroalbuminuria was not significantly associated with the baPWV (odds ratio 0.77, 95% confidence interval 0.57–1.03, These findings suggest that, in this Chinese community-based cohort, elevated baPWV is independently associated with pathological albuminuria with a cut-off value of 1269 cm/s as determined by threshold-effect analysis. Pathological albuminuria, including macroalbuminuria and microalbuminuria, is used as an early indicator for renal damage, especially in patients with diabetes mellitus and hypertension . DespiteThe gold standard for measuring urine albumin is 24-h urine collection. However, this method is inconvenient and, therefore, is less widely used than spot urine collection for nephropathy screening. The urine albumin-to-creatinine ratio (UACR) is a commonly used marker for diagnosing albuminuria . A UACR Pulse wave velocity is an index for arterial stiffness and is also independently associated with cardiovascular events. Although carotid-femoral pulse wave velocity (cfPWV) is considered the noninvasive gold standard for measuring arterial stiffness , baPWV iSome studies have shown that arterial stiffness is associated with albuminuria in diabetic and hypertensive participants: the higher the baPWV, the higher the albuminuria prevalence , 14. The9540 residents residing in the Gucheng and Pingguoyuan communities of the Shijingshan district in Beijing, China, aged 40 years or older were recruited by calling patients from medical records of community health centers and from people responding to recruitment posters between December 2011 and April 2012 for the initial atherosclerosis cohort. From May to July 2014, 3823 (64.1%) out of 5962 participants with gene chip data who were invited for follow-up responded and attended an onsite visit. Participants without UACR or baPWV measures in 2014 were excluded from the study. Participants with ankle–brachial indexes (ABIs) below 0.9 were also excluded, because it is difficult to measure baPWV precisely in people with peripheral artery disease. Thus, 3669 subjects remained in the analysis. The study protocol was reviewed and approved by the ethics committees of Peking University First Hospital and Peking University, and each subject provided written informed consent. All investigators were trained at the Peking University First Hospital.Demographic and lifestyle information, including age, gender, and smoking and drinking habits, were obtained using standard questionnaires. Current smoking was defined as smoking one cigarette per day for at least 6 months prior. Current drinking was defined as drinking once per week for at least 6 months prior. Medical histories  as well as medication histories  were also attained by questionnaire.2). Venous blood samples were obtained after 12–15 h of fasting, which separated within 30 min of collection. Each participant’s total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, triglyceride, and fasting blood glucose (FBG) concentrations were measured using the Roche C8000 Automatic Analyzer . The serum creatinine  was measured using the same instrument by an enzymatic method. The estimated glomerular filtration rate (eGFR) was estimated based on the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation as 141 × minα × max−1.209 × 0.993Age × 1.018, where Scr is serum creatinine (mg/dL), κ is 0.7 for females and 0.9 for males, α is − 0.329 for females and − 0.411 for males, min indicates the minimum of Scr/κ or 1, and max indicates the maximum of Scr/κ or 1.Seated brachial BP was assessed three consecutive times in each participant using an Omron HEM-7117 electronic sphygmomanometer with standard calibrations after a 5 min resting period, and the results were averaged. The body mass index (BMI) of each participant was calculated as the subject’s weight (in kg) divided by his or her height squared  The UACR was defined as the ratio of urine albumin to urine creatinine in units of mg albumin per g creatinine. Normal albuminuria was defined as UACR < 30 mg/g, microalbuminuria was defined as UACR > 30 mg/g and < 300 mg/g, and macroalbuminuria was defined as UACR ≥ 300 mg/g.A BP-203RPE III instrument  was used to measure the baPWV from each subject. After the participant rested in the supine position for at least 5 min, a trained investigator wrapped cuffs around both arms and legs and recorded the pulse waveforms from the cuffs simultaneously. The baPWVs were automatically calculated by the instrument, and the higher one of the two bilateral baPWVs was recorded for subsequent analyses.n (%). Normally distributed variables were compared using a t test, skewed distributed variables were compared using a Kruskal–Wallis test, and categorical variables were compared using the Pearson Chi-square test or Fisher’s exact test. Multivariate logistic regression models were used to investigate the association between baPWV and pathological albuminuria after adjusting for age, gender, current smoking and drinking habits, BMI, hypertension, diabetes mellitus, dyslipidemia, anti-hypertensive agents, anti-diabetic agents, lipid-lowering agents, and self-reported history of CVD. A threshold-effect analysis was also performed. All statistical analyses were performed using Empower(R)  and R software (www.R-project.org). A two-sided P < 0.05 was considered significantly significant.Normally distributed continuous variables were expressed as mean ± standard deviation (SD). For skewed distributed variables, the median of the interquartile range was used. Categorical variables were reported as 2. 15.66% of the participants were current smokers and 14.11% of them were current drinkers. Among the participants, 12.27% of the participants had self-reported histories of CVD, 45.71% had hypertension, 19.84% had diabetes mellitus, and 75.69% had dyslipidemia. 25.70% were taking anti-hypertensive agents, 9.08% were taking anti-diabetic agents, and 9.62% were taking lipid-lowering agents. According to the criteria, 8.15% of the participants had eGFRs > 90 mL/min/1.73 m2, 82.93% had an eGFRs in the range of 90 mL/min/1.73 m2 to 60 mL/min/1.73 m2, and 8.92% had eGFRs<60 mL/min/1.73 m2.The baseline characteristics and albuminuria classifications of all study participants are shown in Table The median UACR value was 6.11 mg/g (IQR 4.17–10.68 mg/g). According to the definition of albuminuria, 92.40% of the participants had normal albuminuria, 6.76% had microalbuminuria, and 0.84% had macroalbuminuria. The average baPWV was 1536.59 ± 305.89 cm/s. Participants with microalbuminuria and macroalbuminuria were older and had higher BMIs, were more likely to be males and current drinkers, and more often had histories of hypertension, diabetes mellitus, dyslipidemia, and CVD.P < 0.001] with every 100 cm/s increase in baPWV  = 1.14, 95% confidence interval (CI) 1.09–1.20, P = 0.008). However, in participants with baPWV of 1269 cm/s or higher, the rate of microalbuminuria and macroalbuminuria increased with the baPWV increasing 100 cm/s . Furthermore, a log-likelihood ratio test was performed, and the results indicated statistically significant differences between two slopes below and above a baPWV of 1269 cm/s (P = 0.017). An interaction test was performed with participants with baPWVs ≥ 1269 cm/s using the modification factors mentioned above, as shown in Table Given the non-linear relationship between baPWV and pathological albuminuria, a threshold-effect analysis was also conducted. The threshold analysis was adjusted for gender, age, BMI, smoking and drinking status, eGFR classification, and histories of hypertension, diabetes mellitus, dyslipidemia, CVD, anti-hypertensive agents, anti-diabetic agents, and lipid-lowering agents Table . The cutIn our cross-sectional study, we found that baPWV was associated with pathological albuminuria in a Chinese community-based cohort. A threshold-effect analysis revealed that the cut-off point for the baPWV was 1269 cm/s. When baPWV was lower than 1269 cm/s, baPWV was not associated with pathological albuminuria. However, when baPWV was higher than 1269 cm/s, the risk of having microalbuminuria and macroalbuminuria increased as the baPWV increased. To our knowledge, this is the first study validating an association between baPWV and pathological albuminuria with a threshold.Microalbuminuria and macroalbuminuria are markers for target renal damage in diabetic and hypertensive patients. It occurs via a mechanism involving increased vascular permeability related to endothelial damage. As the morbidity of chronic kidney disease is increasing worldwide , which iThe prevalence of pathological albuminuria varies across regions and populations. In the general Chinese population, the prevalence of pathological urine albumin was 9.4% . HoweverBaPWV is a metric to estimate arterial stiffness and reflects the stiffness of the aorta and peripheral arteries. It is more readily applicable in the clinic than cfPWV, since it is automated and easier to measure. The mean baPWV in our study was 1536.59 ± 305.89 cm/s, which is lower than those in Xu’s study, which reported an average baPWV of 2154.50 ± 502.97 cm/s in the populations with an ABI < 0.9 and 2049.49 ± 456.65 cm/s in populations with ABI ≥ 0.9 . The difP < 0.001). Moreover, these relationships were stronger among patients with diabetes, hypertension, and macroalbuminuria compared with those without these conditions [Our findings were consistent with those of another community-based cross-sectional study of 2127 community residents of Han ethnicity in Beijing, China aged 60–95 years . The parnditions . In thisnditions . HoweverBesides, Matsui et al. found that in hypertensive patients, the arterial stiffness reduction measured by baPWV was associated with the improvement of renal damage evaluated by UACR change, and the effect was independent of home and office blood pressure reduction. Therefore, reducing arterial stiffness may be an important strategy for preventions of albuminuria progression and cardiovascular events . TherefoGenerally, a baPWV of 1400 cm/s is deemed as the cut-off value to screen patients, especially middle-aged patients, including both males and females . Our finThe mechanism of the relationship between baPWV and UACR remains unclear. Albuminuria is considered an early marker of systemic microvascular damage. Some researchers believe that baPWV is an indicator of arterial stiffness, which, in turn, contributes to endothelial dysfunction and increases the permeability of the glomerular basement membrane, leading to albuminuria . FurtherOur study was the first study to demonstrate conclusively that baPWV has a threshold effect with pathological albuminuria after adjusting for risk factors in a community-based Chinese cohort. Our findings revealed a cut-off value for baPWV that may be used to screen patients at high risk of developing pathological albuminuria and may provide a specific target for drug therapy, such as indicating whether renin–angiotensin–aldosterone system (RAAS) inhibitors should be prioritized. However, there were several limitations in our study. The main limitation was it was a cross-sectional study, and further follow-up is required to validate the cause–effect association. Second, we did not ask participants with hypertension if they were taking RAAS inhibitors. However, after adjusting for the use of anti-hypertensive agents, the relationship between baPWV and pathological albuminuria remains. Another limitation is that baPWV was used instead of cfPWV, which is the gold standard. However, as several studies have demonstrated that baPWV and cfPWV are equally related to cardiovascular risk factors, we chose baPWV as a substitute , 31. In Based on our findings, we concluded that increased baPWV is independently associated with pathological albuminuria in a Chinese community-based cohort. A threshold-effect analysis revealed a baPWV cut-off of 1269 cm/s, above which patients have elevated risk of microalbuminuria and macroalbuminuria. This finding supports the theory that arterial stiffness plays an important role in the formation of albuminuria. These results should raise awareness among clinicians regarding the elevated risk of albuminuria in patients with high baPWVs.


On March 15, 2020, after a life of 74 years, the Sanitary Medical Doctor Ronaldo Santos do Amaral died in Brasilia, Federal District, succumbing after a long battle in a fight against a severe chronic disease. He was born in Salvador, Bahia, on November 22, 1945. Although he came from a simple and hardworking family, he aspired to become a medical doctor despite a very difficult social and economic context. In the mid-1960s, he succeeded in entering the Faculty of Medicine of Bahia University and graduated in the beginning of 1970s. After a brief stint as clinical doctor in small cities in the interior of Bahia, including São Francisco do Conde-his parents’ homeland-he embarked on what would ultimately become his great mission-Public Health.In 1973, he joined the former Fundação de Serviços de Saúde Pública (FSESP), and soon after, the former Superintendência de Campanhas de Saúde Pública (SUCAM)-institutions of the Ministry of Health-to work in the region of the Municipality of Ilhéus, Brazil, participating mainly in the control of malaria and the bubonic plague-important regional endemics at that time.He was selected to attend the Master’s Course at the School of Public Health in Mexico. After completing this course in the early 1980s, he was invited to work at the headquarters of SUCAM, in Brasília, working in the divisions for the control of yellow fever and dengue and then nationally coordinating the schistosomiasis controlHe represented Brazil in numerous international meetings in countries in South America and other regions like Mexico, Guatemala, Dominican Republic, Panamá, United States, England, Switzerland, and China.He retired from FUNASA after making excellent contributions to the control of endemic diseases in the country.Dr. Ronaldo developed many technical-scientific activities that resulted in the publication of scientific articles, either as an author or as a co-author, among which we highlight:An analysis of the impact of the Schistosomiasis Control Programme in Brazil. Memorias do Instituto Oswaldo Cruz, v. 101, 2006.La esquistosomiasis mansoni en el Brasil: epidemiologia y control. Revista Salud Pública, BAYER, v. 15, 2000.Evolução e situação atual do controle da Esquistossomose no Brasil. Revista da Sociedade Brasileira de Medicina Tropical, v. 27, n.III, 1994Criadouros de Aedes aegypti encontrados em alguns bairros da cidade do Rio de Janeiro-RJ/Brasil, em 1984-1985. Cadernos de Saúde Pública (FIOCRUZ), Fiocruz, v. IV, n.3, 1988.Aplicação espacial de inseticidas em Saúde Pública. Cadernos de Saúde Pública (FIOCRUZ), FIOCRUZ, v. IV, n.2, 1988.Duas ameaças de um mosquito: Febre Amarela e Dengue. Revista a Saúde no Brasil, 1983.Investigação comparativa sobre a prevalência de Geohelmintíases em relação com elementos de Saneamento Básico das Habitações, na população menor de 15 anos, na cidade de El Grullo no Estado de Jalisco-México. Revista Baiana de Saúde Pública, Bahia-BA, v. 2, 1982.Dr. Ronaldo was President of the Brazilian Association of Sanitarians in the 1990s and fought hard to establish a state career for sanitarians at the federal level. Unfortunately, this objective has not been achieved so far.In his final days, he left us a great lesson in courage. He calmly rejected any palliative measures such as blood transfusion and hemodialysis, accepting the inexorable evolution of the underlying disease that would lead to his death.


Malnutrition often occurs in acute stroke patients receiving enteral tube feeding (ETF). Unless malnutrition is improved, their clinical outcome is poor. However, strategies to improve malnutrition in these patients have not been established. Branched-chain amino acids (BCAA) may enhance protein synthesis and attenuate inflammation. Our study aimed to investigate whether a leucine enriched BCAA dietary supplement (LEBDs) could quickly increase serum levels of albumin (Alb) or transthyretin (TTR) and decrease high-sensitivity C-reactive protein (CRP) in the development of severe malnutrition within a few days after stroke onset compared to standard BCAA dietary supplement (SBDs).We retrospectively included acute stroke patients who: 1) were admitted between August 2016 and July 2017; 2) underwent ETF for 7 days or longer after admission, and 3) underwent blood examination of Alb, TTR, and CRP on admission, the fifth day and the seventh day. We defined severe malnutrition as severe hypoproteinemia: decrease of TTR to less than 15 mg/dl on the 5th day. In LEBDs and SBDs groups, patients started to receive a dietary supplement containing leucine of 1.44 and 0. 72 g twice a day on the fifth day, respectively. We evaluated Alb (g/dl), TTR (mg/dl), and CRP (mg/dl) on admission, the fifth day, and the seventh day.Twenty-nine patients met our inclusion criteria:15 in LEBDs and 14 in SBDs. In LEBDs and SBDs groups, the median Alb was 3.5 and 3.3 g/dl, TTR was 12.7 and 10.7 mg/dl, and CRP was 1.02 and 0.673 mg/dl on admission, respectively. In LEBDs, the median Alb and TTR decreased to 2.6 g/dl and 11.9 mg/dl, and CRP increased to 5.337 mg/dl on the fifth day. On the 7th day, TTR increased, and CRP decreased, although Alb did not improve. In SBDs, the median Alb and TTR decreased to 2.6 g/dl and 9.7 mg/dl, and CRP increased to 4.077 mg/dl on the fifth day. On the 7th day, Alb, TTR, and CRP did not improve.In acute stroke patients receiving leucine enriched BCAA dietary supplement, quick improvements in transthyretin and CRP were observed. Malnutrition often occurs in severe acute stroke patients who are unable to take foods orally due to dysphagia or disturbed level of consciousness and who must receive enteral tube feeding (ETF) –3. MalnuFor the retrospective observational study, we included acute stroke patients who: 1) were admitted between August 2016 and July 2017, 2) started ETF on the second day and continued to undergo ETF for 7 days or longer after admission, 3) underwent blood examination on admission, the fifth day and the seventh day, and 4) started to receive LEBDs or SBDs to improve severe malnutrition on the fifth day. We defined severe malnutrition as rapid progression of hypoproteinemia: decrease of TTR to less than 15 mg/dl, as TTR is a rapid turnover protein [We excluded from our analysis patients who: 1) died due to very severe stroke within 7 days after its onset, 2) could take foods orally on admission, or 3) did not undergo a thorough blood examination.We calculated the target calories for patients according to the Harris-Benedict Equation (HBE) .We started to administer enteral nutrients on the second day of admission. Enteral nutrients (EN) we used to be IMPACT (Nestle Japan Co. Ltd.), PEPTAMEN AF (Nestle Japan Co. Ltd.), MEIN  or RENALEN MP . IMPACT includes contents that consist of protein of 14 g (arginine-enhanced), containing leucine of 0.975 g, lipid of 7.0 g, containing middle-chain triglyceride (MCT) of 1.5 g, and carbohydrates of 33.5 g against 250 kcal per 250 ml of one pack. PEPTAMEN AF includes contents that consist of protein of 19 g, containing leucine of 2.03 g and whey peptide, lipid of 13.2 g, containing MCT of 6.7 g, and carbohydrates of 26.4 g against 300 kcal per 200 ml of one pack. MEIN consists of protein of 10 g, containing leucine of 0.96 g and whey peptide, lipid of 5.6 g, containing MCT of 1.2 g, and carbohydrates of 29.8 g, containing palatinose, against 200 kcal per 200 ml of one pack. Renalen MP consists of protein of 14 g, containing leucine of 1.32 g, lipid of 11.2 g, containing MCT of 2.3 g, and carbohydrates of 64.0 g, containing palatinose, against 400 kcal per 250 ml of one pack. Palatinose is a disaccharide that consists of one glucose and one fructose, and it has a low glycemic index. Enteral nutrients except for RENALEN MP were protein-rich nutrients. Leucine enriched BCAA dietary supplement (LEBDs)  consists of protein of 8 g, containing 1.44 g of leucine, and sugar of 18.5 g, against 200 kcal per 100 ml of one pack, and standard BCAA dietary supplement (SBDs)  consists of protein of 7.5 g, containing leucine of 0.72 g and sugar of 29.3 g, against 200 kcal of one pack . Differences between LEBDs and SBDs were compared using a Fisher’s exact test for categorical variables and a Wilcoxon rank-sum test for non-parametric data. For a comparison of paired variables, we used a Wilcoxon signed-rank test for non-parametric data. We performed a two-sided test for unpaired variables, a one-sided test for paired variables, and considered a probability of less than 0.05 statistically significant. We used the JMP (version 15.1) program to perform the statistical analysis.During the study period, we treated 800 acute stroke patients in our institution. Among patients who underwent ETF on the second day, the TTR level on the fifth day was less than 15 mg/dl in 29 patients, and the 15 patients started to receive LEBDs and the 14 patients started to receive SBDs on the 5th day. There were no differences in patients’ characteristics between the two groups. Their median age was older than 80 years. In LEBDs and SBDs groups, the median GCS on admission was 9 and 9.5, respectively, and they suffered from impaired consciousness.p <  0.0001) and the median TTR decreased to 11.9 mg/dl (p <  0.001), and CRP increased to 5.337 mg/dl (p <  0.05) on the fifth day. We added the LEBDs  to baseline EN. On the 7th day, TTR increased (p <  0.001) and CRP decreased (p <  0.05) although Alb did not improve  and the median TTR decreased to 9.7 mg/dl (p < 0.05), and the median CRP increased to 4.077 (p < 0.05) on the fifth day. We added the SBDs  to baseline EN. On the 7th day, Alb, TTR and CRP did not improve , TTR was 12.7 and 10.7 mg/dl (ns), and CRP was 1.02 and 0.673 mg/dl (ns) on admission, respectively (Table 2), respectively. In 12 of the 15 patients, eGFR was less than 60 (mL/min/1.73 m2). The 12 patients suffered from renal dysfunction on admission (Table 2). The 5 patients suffered from renal dysfunction on admission  and estimated glomerular filtration rate (eGFR) on admission were 0.78 (mg/dl), and 56.4  of the 15 patients discharged within 17 days, and they were transferred to comprehensive rehabilitation centers after their TTR and CRP were improving. Two of the 15 patients died on the eighth day and the twenty-first day, respectively. Median hospitalization in LEBDs was 12 days. In SBDs, 12 (85.7%) of the 14 patients discharged within 17 days, and they were transferred to comprehensive rehabilitation centers, although neither TTR nor CRP were not improving. One of the 14 patients died on the tenth day. Median hospitalization in SBDs was 10 days  in the 29 patients . Their TWe supposed that the LEBDs had a quick effect on improvement of hypoproteinemia and cessation of inflammation in the 15 patients. After administration of the LEBDs, the TTR level rose soon, and the CRP level decreased soon, probably because leucine enriched BCAA played critical roles in the regulation of energy homeostasis, nutrition metabolism, gut health, immunity as they acted as potential anti-inflammatory mediators –28, furtOur study had several limitations. Our study included a small number of patients. Our study was not a prospective randomized controlled study but a retrospective observational cohort study of routine medical care. We, therefore, did not have a placebo-controlled group without BCAA supplement, and we compared LEBDs group with SBDs group. The LEBDs contained MCT or n-3 fatty acids; no information, however, was provided in SBDs. Our patients started to use different types of enteral nutrients as the baseline. All patients were likely of Japanese ancestry; therefore, not representative of the general population. Our study investigated short-term outcomes of blood examinations, in-hospital death, and hospitalization days but did not study the long-term effect. To confirm our results, therefore, future prospective studies are required in patients with the standardization of enteral nutrients.In acute stroke patients receiving leucine enriched BCAA dietary supplement, quick improvements in transthyretin and CRP were observed compared to patients with standard BCAA dietary supplement. The results warrant the further clinical application of the LEBDs in acute patients with malnutrition.


We characterize sampling and interpolating sets with derivatives in weighted Fock spaces on the complex plane in terms of their weighted Beurling densities. The ed using , 1.3) u u\documeositions  , , \documeensities  as in  in  is calleion, see , 20, 22.We recall the following fact from potential theory, .Thus , gives ((z).Thus . \documeThe following lemma helps profit from Riesz decomposition in estimates involving derivatives.(i)(ii)Let By Leibniz rule:Finally, we note that Cauchy bounds extend to weighted derivatives.Let For each Throughout this section we assume that We start by showing that interpolation can be solved locally.Let We consider n-th derivative of two composite functions can be computed by means of Faà di Bruno’s formula and the Bell polynomials . As in  we constThe first step is to construct the non-analytic interpolant. Fix that, by  and .Let Now we consider the set Step 2. We show that if (Q.1)(Q.2)(Q.3)To prove the claim, for each The first two conditions, together with ((Q.1) and (Q.2). To check (Q.3), we let f, ((Q.3).For each Applying  to the lude from  that\docStep 3. We show that (Q.1), (Q.2) and (Q.3) for (Q.1), (Q.2) and (Q.3) with respect to (Q.1), (Q.2) and (Q.3) with respect to Let The constructed sequences satisfyTo see that interpolates with the right values on We have thus shown that Step 4. We show that z and radius r, counted with multiplicities, satisfies The number of points of the set The modification of sampling sets to reduce their multiplicity is based on the following perturbation lemma.Let We apply Theorem We can now modify a sampling set to reduce its maximal multiplicity without altering its sampling property.Let Since For fore, by ,5.12ThusSuppose that e, e.g., , Prop. 3 that in , the spaB and use the mean value property for harmonic functions to concludeR). Hence, (Towards the sufficiency of the density conditions, let ondition  is verifSuppose that e, e.g., , Prop. 1Conversely, suppose that Fϕ~2. By , \docume combine  and (6.1


Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that primarily affects the joints. Overweight and obesity can aggravate disease activity and clinical outcome in patients with RA. However, the role of bariatric surgery in inducing weight loss in the treatment of RA has not been confirmed. In this 12-month prospective cohort study, RA patients with obesity who were referred to our hospital were included. Thirty-two patients were classified into the bariatric surgery group according to the patient’s decision after a comprehensive assessment of surgery indications, and 33 patients received only pharmacotherapy for RA. At the 12-month follow-up, the response rates of ACR20, ACR50 and ACR70 were 75.0% vs. 51.5%, 53.1% vs. 39.4% and 31.3% vs. 21.2% in the bariatric surgery and non-surgery groups, respectively ; the mean DAS28-ESR, DAS28-CRP and cDAI scores were 1.5 ± 0.9 vs. 2.4 ± 1.4, 1.2 ± 0.9 vs. 2.2 ± 1.7 and 9.5 ± 6.8 vs. 15.8 ± 12.5, respectively, in surgical patients compared to non-surgical patients . Compared to baseline, after 12 months, a significant reduction was observed in the use of leflunomide, biological agents, combination treatments, and NSAIDs in both groups (p < 0.05 or p < 0.01). However, there was no difference in medication use between the 2 groups either at baseline or at the 12-month follow-up . Compared to non-surgical patients, in RA patients with obesity, weight loss after bariatric surgery was associated with lower disease activity. Medication tapering for RA in patients who underwent bariatric surgery was not superior to that in non-surgical patients. Although there is a complicated pathogenesis for RA, several factors have been identified that represent the possible causes for the onset and development of RA, including female sex, genetic predisposition and socioeconomic status3. Recent studies have demonstrated that an increase in body mass index contributes to a higher risk of developing RA6.Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that primarily affects the joints and results in severe pain and swelling, progressive damage and disability, which ultimately lead to physical malfunction and even early death7. Overweight and obesity can aggravate disease activity and clinical outcomes via changing histological features and the inflammatory gene signature of the synovial membrane in patients with RA, especially among female patients8. Furthermore, RA patients generally take immunosuppressive therapy, which has numerous adverse effects, including susceptibility to infection and weight gain9. RA patients with obesity have a worse response to therapy, including treatment with conventional synthetic disease modifying antirheumatic drugs (csDMARDs), such as methotrexate, sulfasalazine, hydroxychloroquine and leflunomide, biologic DMARDs (bDMARDs), including tumour necrosis factor-α inhibitors and non-TNFα inhibitors, or targeted synthetic DMARDs (tsDMARDs)11. Assessment and improvement of weight indicators should be an important part of the routine care of RA patients in clinical practice12.The global prevalence of obesity and associated diseases has increased considerably in recent decades13. After substantial weight loss from bariatric surgery, RA patients had further decreased disease activity, lower serum inflammatory markers, less RA-related medication use, and lower RA cause-specific mortality14. However, other factors, such as improved physical activity and metabolic changes after bariatric surgery, may also have contributed to these postsurgical improvements14. On the other hand, studies have demonstrated that weight loss is also associated with worsening disability, possibly due to its association with chronic illness and the development of age-related or disease-related frailty16. However, the role of weight loss in the treatment of RA has not been confirmed since the efficacy has not been studied in a controlled trial with a comparison group. This prompted us to conduct an investigator-initiated, 12-month study to determine whether bariatric surgery is effective in improving the clinical outcomes in active RA patients with obesity compared with similar RA patients who did not undergo bariatric surgery. This study also evaluated other aspects of the RA-related medication tapering profile of bariatric surgery in RA patients with obesity.Weight loss through lifestyle and dietary modification has been shown to improve the clinical manifestations of RAAll 80 RA patients with obesity were initially screened, and 65 eligible patients were enrolled in the study. According to the patient’s decision and surgery indications, 32 patients underwent bariatric surgery for obesity and received pharmacotherapy for RA, and 33 patients received only pharmacotherapy. In the bariatric surgery group, 6 patients discontinued treatment before week 12 because of adverse events, and 1 patient discontinued treatment due to a protocol violation and was lost to follow-up. In the non-surgery group, 5 patients discontinued treatment due to adverse events (n = 1), protocol violation (n = 3), or loss to follow-up (n = 1) before the final evaluation at week 12. Finally, 81.3% (26/32) and 84.8% (28/33) of patients in the bariatric surgery and non-surgery groups, respectively, completed the 12-month evaluation. The study profile and patient disposition are shown in Fig. In the bariatric surgery and non-surgery groups, the mean ages were 51.8 ± 11.8 years and 57.6 ± 16.4 years, respectively, which were significantly different (p = 0.04); 90.6% and 91.0% of patients were women; 12.5% and 15.2% had smoking habits; the RA durations were 10.3 ± 4.8 years and 8.5 ± 4.9 years; and body weights were 111.8 ± 11.8 kg and 108.5 ± 9.5 kg, respectively. All patients had active disease, which was reflected by the number of tender joints (TJCs) and swollen joints (SJCs), erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) values, and the PtGA, PyGA, and high DAS28 and cDAI scores. There were no statistically significant differences among the 2 groups for any of the aforementioned variables (p > 0.05) except for PtGA (p = 0.03). In the bariatric surgery group, the surgery type included Roux-en-Y gastric bypass 19/32 (59.4%) and laparoscopic sleeve gastrectomy 13/32 (40.6%). The comorbidities of the recruited patients included osteoarthritis (75.0% vs. 66.7%), hypertension (40.6% vs. 33.3%), type 2 diabetes (28.1% vs. 24.2%), congestive heart failure (12.5% vs. 6.1%), coronary artery disease (3.1% vs. 9.1%), and chronic obstructive pulmonary disease (6.3% vs. 6.1%); these variables were not significantly different between the bariatric surgery and non-surgery groups (p > 0.05). The baseline demographic and clinical characteristics of the included patients are detailed in Table 2 at baseline, 31.9 ± 10.2 kg/m2 at 4 months, 29.6 ± 11.9 kg/m2 at 8 months, and 26.9 ± 13.5 kg/m2 at 12 months of follow-up  with a corresponding reduction of 61.4 ± 32.3%, 75.2 ± 34.4% and 79.4 ± 39.6% excess weight loss . In non-surgical patients, however, the mean weight was 108.5 ± 9.5 kg at baseline, 106.7 ± 15.5 kg at 4 months, 104.5 ± 12.5 kg at 8 months, and 103.5 ± 17.5 kg at 12 months . Accordingly, the BMI was 37.2 ± 12.5 kg/m2, 36.6 ± 11.3 kg/m2, 35.8 ± 12.8 kg/m2, and 35.3 ± 13.2 kg/m2 at baseline and at 4, 8, and 12 months of follow-up, respectively  . The body mass index (BMI) was 38.4 ± 4.8 kg/mAn ACR20 response rate was observed for 12/28 patients (43.8%) in the bariatric surgery group and for 11/33 patients (33.3%) in the non-surgery group at 4 months (p < 0.05); the ACR20 response was observed for 15/26 (56.3%) and 13/30 (42.4%) patients in the bariatric surgery and non-surgery groups, respectively, at 8 months (p < 0.01); the ACR20 response was observed for 20/26 (75.0%) and 14/28 (51.5%) patients in the bariatric surgery and non-surgery groups, respectively, at 12 months (p < 0.01). ACR50 responses were achieved by 28.1%, 34.4%, and 53.1% of RA patients who underwent bariatric surgery at 4, 8, and 12 months, respectively, which were significantly different from those in patients in the non-surgery group at each time point  (p < 0.05 or p < 0.01). ACR70 responses were achieved by 15.6%, 21.9%, and 31.3% for RA patients who underwent bariatric surgery at 4, 8, and 12 months, respectively, which were significantly different from those in patients in the non-surgery group  (p < 0.05 or p < 0.01)  (p < 0.05 or p < 0.01). The mean DAS28-CRP scores were 6.6 ± 3.7, 2.9 ± 2.1, 1.7 ± 1.4, and 1.2 ± 0.9 in the surgical patients at baseline and at 4, 8, and 12 months of follow-up, respectively, which were significantly different from the scores for non-surgical patients at each time point  (p < 0.05 or p < 0.01). Finally, the mean cDAI scores were 38.9 ± 24.5, 21.4 ± 18.3, 14.5 ± 8.9, and 9.5 ± 6.8 at baseline and at 4, 8, and 12 months of follow-up in surgical patients, respectively, which were also significantly different from the scores in the non-surgery group  (p < 0.05 or p < 0.01) at each time point , biological agents in 14/32 patients (43.8%), combination treatment including 2 or more immunosuppressive medications in 21/32 patients (65.6%), glucocorticoids in 4/32 patients (12.5%), and NSAIDs in 16/32 patients (50.0%) in the bariatric surgery group. The patients’ medication for RA in the non-surgical group included csDMARDs, biological agents, combination treatment, glucocorticoids, and NSAIDs in 31/33 (93.9%), 10/33 (30.3%), 19/33 (57.6%), 5/33 (15.2%), and 17/33 patients (51.5%), respectively. There were no significant differences between the 2 groups . Compared to baseline, after 12 months, there was a significant reduction in the use of leflunomide, biological agents, combination treatment, and NSAIDs in both groups (p < 0.05 or p < 0.01). However, medication use did not differ between the 2 groups at baseline or at the 12-month follow-up   patients, including anastomotic ulcer/leak (n = 3), intra-abdominal hernia (n = 1), and postoperative gastric functional emptying (n = 1). Late complications more than 1 month after surgery were observed in 3/32 (9.4%) patients, including an incisional hernia (n = 1), intestinal obstructions (n = 1) and serious wound infection (n = 1). In this study, no patients who underwent bariatric surgery had more than one postoperative complication.17. The benefits of bariatric surgery beyond the weight loss effect on disease and prognosis continue to be reported in other autoimmune or inflammatory diseases, including systemic lupus erythaematosus, inflammatory bowel disease, autoimmune thyroiditis and autoimmune liver diseases21. The association between obesity and RA might present a strategy for the prevention or control of RA activity23. Although it has been shown that substantial weight loss from bariatric surgery results in lower disease activity and improved response to treatment14, the effect of bariatric surgery on the treatment of RA was unconfirmed in a prior cohort study. Our study demonstrated that compared with non-surgical patients who were followed for up to 12 months, RA patients with obesity who underwent bariatric surgery had significant weight loss and a better response to treatment. RA-related medication usage, on the other hand, had no difference between the surgical and non-surgical patients at baseline or at the 12-month follow-up.Obesity, with a higher prevalence, is one of the described risk factors for the onset of RA, and it negatively affects disease activity and treatment outcomeset al.16 showed that both severe obesity and weight loss were associated with worsening disability, and weight loss was a risk factor for death in patients with RA. Mounting studies have shown that obesity augments RA disease activity and decreases the likelihood of achieving sustained remission despite intensive treatment25. Non-surgical weight loss interventions contributed to a weight loss of 4.5 kg and significantly improved physical function in 19 overweight RA patients26. Furthermore, a more substantial strategy for weight loss, such as surgery, also has an impact on the levels of various inflammatory markers induced by obesity30. Santos et al.31 enrolled 46 patients with obesity-related metabolic dysfunction and showed that gastric banding surgery resulted in a significant decrease in the inflammation process associated with adipose tissue loss. Interestingly, in a Swedish study followed up for 29 years, there was no association between bariatric surgery and the incidence of RA in obese patients32, which might demonstrate that bariatric surgery has no preventive effect on the occurrence of RA.However, studies from Baker 14 of bariatric surgery in 53 RA patients with moderate to high RA disease and a BMI of 47.8 ± 7.7 kg/m2. This study concluded that bariatric surgery resulted in a significant disease remission rate of 74% at a follow-up of 5.8 ± 3.2 years after surgery compared to a rate of 26% at baseline14. This study design was an uncontrolled cohort without a control group. It is therefore unclear whether the observed improvement in RA activity after bariatric surgery is related to RA-related medications or to surgery-specific effects. In the present study, both surgical and non-surgical patients had significantly decreased disease activity at 12 months compared to baseline, which was consistent with the abovementioned study. More importantly, after 12 months of follow-up, compared with the non-surgery group, the surgical patients also had a better response rate to RA-related medication in terms of ACRs, DAS28, and cDAI scores, providing powerful evidence to support the potential role of bariatric surgery in the treatment of RA.Few prior studies have specifically focused on the effects of more substantial weight loss on RA severity before and after bariatric surgery. To the authors’ knowledge, the only report is a retrospective cohort study33. These findings suggest that surgery-induced weight loss is associated with decreased SLE immunosuppression medication requirements33. The study has the same issue that, due to the lack of comparison group data, it is uncertain whether the medication reduction is attributable to bariatric surgery or SLE-related treatment. A study of bariatric surgery in RA patients showed a significantly decreased usage of RA-related medication after bariatric surgery (66% at 1 year after surgery compared to 98% at baseline)14. In our study, there was a significant reduction in the use of leflunomide, biological agents, combination treatment, and NSAIDs in both surgical and non-surgical patients after 12 months compared to the baseline (p < 0.05 or p < 0.01). However, there were no differences in medication use between the 2 groups either at baseline or after 12 months of follow-up. Our data suggested that medication tapering for RA patients who underwent bariatric surgery was not superior to that in non-surgical patients.The study of bariatric surgery’s efficacy on 31 patients with systemic lupus erythaematosus demonstrated that 42% of patients showed a reduction in the number of immunosuppressive medications and that 19.3% were off steroids completely at a mean follow-up of 3 years36. However, as the setup of the parameters was mainly focused on the change in RA disease activity before and after treatment, data on these metabolic factors were not collected in this study. The deficiency of these metabolic factors might confound the treatment efficacy of bariatric surgery on RA disease activity. Finally, other limitations, including a lower case number, nonrandomized design, and short-term follow-up, need to be perfected in future studies.There are some limitations to our study. First, the study was an open-label clinical study, and the treating doctors and patients were not blinded to the therapeutic strategy. Even so, the disease activity measurements were performed by different trained evaluators who were unaware of the specific therapeutic regimen to make the study more objective. Second, previous studies demonstrated that metabolic factors, including dietary intake and physical activity, were associated with the occurrence and disease activityTo the best of our knowledge, this is the first cohort study to evaluate the treatment efficacy of bariatric surgery on RA patients with obesity compared with non-surgical patients. Our data demonstrated that bariatric surgery appears to be feasible in RA patients with obesity. The activity-improving effects of bariatric surgery were evident 4 months after surgery and persisted for at least 12 months. In conclusion, the present results suggest that weight loss may have an important role in the nonpharmacologic management of RA patients. These findings warrant further study concerning the treatment of RA patients, especially those with obesity.Approval for this retrospective study was granted by the ethics review board of the First Affiliated Hospital of Anhui Medical University (AHEC2013-015), and all methods were performed in accordance with the relevant ethical guidelines and regulations. Prior written informed consent was obtained from all patients. This 12-month cohort study was conducted between May 2016 and August 2018. RA patients with obesity who were referred to our hospital for surgical treatment of obesity and pharmacotherapy for RA were included.37; (2) age of 18–65 years; (3) BMI ≥30 kg/m2 in 3 continuous years; (4) no abuse of alcohol or psychotropic drugs, and no serious behaviour disorder or mental retardation; (5) TJCs ≥5 and SJCs ≥3; and (6) ESR ≥20 mm/h or CRP ≥20 mg/L. Patients were excluded if they had (1) a history or current infection or any type of malignant cancer; (2) secondary obesity, such as Cushing syndrome, hypothyroidism, polycystic ovary syndrome, hypopituitarism, hypothalamic obesity, or prolactinoma; and (3) intolerance to surgery due to serious organ diseases. During the study, nonsteroidal anti-inflammatory drugs (NSAIDs) and low-dose glucocorticoids of ≤50 mg prednisone or equivalent/day were allowed when necessary.The eligible patients for this study had to meet the following criteria: (1) RA diagnosis determined by 2010 ACR/EULAR classification criteriaAll patients with RA were classified into either a bariatric surgery group or a non-surgery group after the comprehensive assessment of surgery indications according to the patient’s decision. Bariatric surgery was defined as Roux-en-Y gastric bypass (LRYGB) or laparoscopic sleeve gastrectomy (LSG). The RA-related treatment regimen was determined by rheumatologists. The disease activity measurements were performed by different trained evaluators who were unaware of the specific therapeutic regimen.2 lost)14 at the 4-, 8-, and 12-month follow-up, and complications and changes in immunosuppressive medication at the 12-month follow-up.Data collected included baseline demographic characteristics, clinical parameters, operative data, RA activity index scores, and postoperative and follow-up outcomes. The demographic characteristics of the recruited patients included age, sex, height, weight, BMI, comorbidities, and the disease duration of RA. The clinical parameters included rheumatoid factor (RF), anti-cyclic citrullinated peptide antibody (Anti-CCP), ESR, and CRP. Operative data included surgery type and time, estimated blood loss, and intraoperative complications. The RA activity index included the patient’s global assessment of overall well-being (PtGA), the physician’s global assessment (PyGA) at baseline, the 28-joint count disease activity score (DAS28), and the clinical disease activity index (cDAI) at baseline and at the 4-, 8-, and 12-month follow-up. The postoperative and follow-up outcomes assessed were the percentage of patients who achieved the American College of Rheumatology criteria , weight loss and excess weight loss (percentage of baseline weight above BMI 25 kg/mt test (or the Mann-Whitney U test) for measurement variables and the χ2 test (or Fisher’s exact test) for categorical variables. Covariance analysis was used to compare the difference in disease activity over time in surgical and non-surgical patients. Patients with missing end points were considered to be nonresponders. Values of p < 0.05 were considered statistically significant. Statistical analysis and plotting were performed using SPSS 23  and GraphPad Prism 7 .Data are presented as the mean and standard deviation (SD) for measurement variables and frequency percentages for categorical variables. Parameters were analysed using the unpaired Student’s The study was reviewed and approved by the Medical Science Ethics Committee of First Affiliated Hospital of Anhui Medical University.All study participants, or their legal guardian, provided informed written consent prior to study enrollment.


Psidium cattleyanum is a widely used plant for the management of various ailments such as gastrointestinal, respiratory, and cardiac disorders, but it lacks proof on a scientific basis, and therefore, this is the major emphasis of the current research work. Crude extract of Psidium cattleyanum (Pc.Cr) was preliminary analyzed for the presence of different classes of bioactive molecules. The aqueous and dichloromethane fractions of Pc.Cr were subjected to in vitro and in vivo studies. It was applied at variable concentrations (0.1–10 mg/ml) to isolated rabbit jejunum to investigate spasmolytic effect. Concentration dependent curves of calcium were constructed to check the calcium channel antagonistic activity. For the evaluation of tracheorelaxant activity, isolated tracheal tissue was treated with High-K+ (80 mM) and carbachol (CCh) and then challenged cumulatively with Pc.Cr. To study the antidiarrheal effect of the plant extract, castor oil-induced diarrhea model was adopted. For evaluation of the hypotensive effect of Pc.Cr, it was given intravenously to preanesthetized normotensive rats, and the response was recorded using pressure transducer. Platelet rich plasma was used for the assessment of the antiplatelet activity when challenged with purinergic and adrenergic agonists. Concentration-dependent inhibition of spontaneous and High-K+ mediated contractions in isolated jejunum was observed by the application of Pc.Cr. Contractions induced in isolated tracheal tissue by High-K+ and CCh were inhibited by application of Pc.Cr to these tissues. Similarly, application of Pc.Cr to High-K+ and phenylephrine (PE) treated aortic strips resulted in vasodilation. Platelet aggregation inhibition was shown by Pc.Cr against adenosine diphosphate (ADP) only. The antidiarrheal effect was observed as a reduction in the total number of feces in Pc.Cr-treated mice when given castor oil. Dose-dependent hypotension was seen in normotensive rats when treated with Pc.Cr intravenously. This study showed the spasmolytic, tracheorelaxant, vasodilator, platelet aggregation inhibitory, antidiarrheal, and hypotensive activities of P. cattleyanum which may be due to the blockage of calcium channels, but the involvement of any other pathway cannot be ignored.Traditionally, in the Southern Asian countries,  Psidium cattleyanum (P. cattleyanum) is an ornamental aromatic tree and known as Jeju guava, Cattley guava, strawberry guava, and Chinese guava. It belongs to family Myrtaceae, the guava family. There are about 3,800 species and 133 genera in this family, and about 92 species fall into genus Psidium. Many fruits of this family are medicinally important and used in traditional medicine. Among these 92 species, Psidium cattleyanum is commercially important due to its utility as a fruit and food [P. cattleyanum [P. cattleyanum has better antioxidant, antibacterial, and anti-inflammatory activities than P. guajava. It is a good source of vitamin C and total phenolic contents [P. cattleyanum had been reported to contain glycitin, glycitein, sissotrin, and ononin [P. cattleyanum contains sesquiterpenes and monoterpenes. Main sesquiterpene was beta-caryophyllene followed by alpha-humulene and alpha-thujene 1, myrcene, alpha-pinene, 1,8-cineole, epi-alpha-muurolol, alpha-cadinol, and epi-alpha-cadinol [and food . It has and food . It possand food –7, antioand food , inflammtleyanum , 12. Itscontents , 15. Morcontents . Aqueousd ononin . Differed ononin . Oil of P. cattleyanum and other members of the Myrtaceae family and the limited availability of scientific literature, this study was conducted to give pharmacological basis to some of its therapeutic uses.Because of the wide traditional uses of P. cattleyanum were randomly collected from “Bio Park” of Bahauddin Zakariya University, Multan, Pakistan, in October and shed-dried after taxonomical identification. Identification voucher number was R. R. Stewart 504. This plant material was converted into a coarse powder. One-kilogram powder was macerated in 70% methanol (aqueous methanol) with occasional shaking. After a period of 8 days, the macerated material was filtered. The filtrate was dried by using a rotary evaporator . The obtained crude extract of P. cattleyanum (Pc.Cr) was stored at −40°C.Aerial parts of For the purpose of fractionation, aqueous solution of plant extract was prepared, and then dichloromethane was added to it in a volume similar to water. This solution was continuously shaken in a separating funnel for few minutes and left there overnight. This solution split into two separate layers, i.e., aqueous and dichloromethane layers. These layers were separated and get dried, aqueous layer as Pc.Aq (aqueous fraction) and dichloromethane as Pc.DCM (dichloromethane fraction).2, MgSO4, KH2PO4, NaHCO3, MgCl2, glucose, NaCl, and NaH2PO4. All the chemicals used were of high purity and analytical standard.Drugs used in his study are verapamil hydrochloride, acetylcholine (Ach), carbachol (CCh), and sodium citrate. Chemicals used are KCl, CaCl2CO3) was added, mixed, and incubated for 0.5 h. The optical density was measured at 760 nm utilizing UV-visible spectrophotometer. The total phenolic contents were expressed as mg gallic acid equivalents (GAE)/g of the extract [Total phenolic content of Pc.Cr was determined by the standard Folin–Ciocalteu spectrophotometric method. Briefly, 0.5 ml of extract was added to 0.1 ml of Folin–Ciocalteu reagent (0.5 N), and the contents of the flask were mixed thoroughly. Later, 2.5 ml of sodium carbonate  was added sequentially. The test solution was vigorously shaken. Absorbance was recorded at 415 nm after 30 minutes of incubation. A standard calibration plot was generated at 415 nm using known concentrations of quercetin. From calibration plot, flavonoid concentration was calculated and expressed as mg quercetin equivalent (QU)/g of the extract [For total flavonoid content, standard aluminium chloride spectrophotometric method was used. In brief, 1 ml of  extract .P. cattleyanum extract (Pc.Cr) were determined using gas chromatography and a mass spectrometer . The temperature was set to 40°C, followed by a 2 min hold. Then the temperature was increased at the rate of 5°C/min to a maximum of 200°C and then put on hold for 2 min. After 2 minutes, temperature was again increased up to 300°C at the rate of 5°C/min and held for another 2 min again. For the determination of chemical composition of plant extract, its mass spectra were compared with the mass spectra from the National Institute of Standard and Technology and Wiley Spectral Libraries. The mass spectra of compounds were also compared with those of similar compounds in the Adams Library and the Wiley GC/MS Library [The chemical constituents of  Library .Balb/C mice having weight range of 20–40 g, Sprague Dawley rats (150–300 g), and locally bred rabbits, both males and females, were used for the experiments. These animals were kept under standardized conditions of diet, humidity, temperature and 12-hour light and dark cycle with free access to water. Protocols for the use of animals in this study were framed under the guidelines of Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council (1996). Departmental ethical committee approved these protocols via no. 09/PEC/2015.n = 10 rabbits) were used. Rabbits were kept on starvation for 24 hours before the start of experiment but with access to water. On the day of experiment, the rabbits were sacrificed, and different tissues like jejunum, trachea, aorta, and atrium were isolated and kept in physiological salt solution [For ex vivo studies on isolated tissues, locally bred rabbits (solution –24.(1) Study on Rabbit Jejunum. Antispasmodic effect of P. cattleyanum was studied by using rabbit jejunum. 2-3 cm long pieces of isolated jejunum were made after cleaning the adhering tissues like mesenteries. Each segment was left in Tyrode's solution-filled organ bath. The solution was kept on continuous perfusion with carbogen and maintained at pH 7.4 and 37°C. A weight of 1g was applied as preload, and tissue response was recorded by MLT0015 isotonic transducer which was connected with PowerLab. To equilibrate the tissue, Tyrode's solution was repeatedly changed after every 30 minutes Acetylcholine (0.3 µM) was repeatedly applied to the tissue to stabilize it every 3 minutes. After stabilization of jejunum, antispasmodic response of respective plant extract was investigated by application in a cumulative fashion. Spasmolytic effect of the extract was calculated using Calcium plays a vital role in the contraction of smooth muscles, and this calcium comes from the extracellular fluid via calcium channels or comes from intracellular stores. Any drug which blocks these calcium channels is considered as calcium channel blocker and causes the relaxation of muscles. To investigate the involvement channel blockade in the spasmolytic potential of Pc.Cr, we construct calcium curves as described earlier by –27.(2) Tracheorelaxant Study on Isolated Rabbit Trachea. Isolated trachea of rabbits was cut into small pieces having a size of about 3-4 mm in width, with 2-3 cartilages. These tracheal pieces were opened by cutting the cartilage in the opposite direction of smooth muscle layer. Tissue organ bath was cleaned and filled with Krebs solution, and prepared tracheal tissue was put in it. Solution in organ bath was kept on continuous perfusion with carbogen. 1 gram preload tension was applied to the suspended tissue. This tissue was kept free to equilibrate for 30–45 minutes, but the solution was replaced repeatedly. The tracheorelaxant response of test drug was observed on precontracted isolated tracheal strip with High-K+ and CCh via PowerLab connected isometric transducers [nsducers , 29.3) Vasorelaxant Study on Rabbit Aorta. Aorta was isolated from rabbits and cleaned off the mesenteries. Cleaned aorta was divided into 2-3 cm small rings/strips. This aortic ring was suspended in Krebs solution-filled organ bath. It was ensured that supply of carbogen should not be interrupted throughout the experiment and temperature should be kept at 37°C. Suspended tissue was kept free for 30 minutes to equilibrate, and 2.0 gram preload tension was applied to it. In order to stabilize the tissue, it was repeatedly treated with either phenylephrine (1 µM) or High-K+. After stabilization, tissue was cumulatively treated with plant extract to observe its vasodilator effect. Results were recorded by MLT 0201 transducer linked with PowerLab [(PowerLab .(4) Study on Rabbit Atrium. To study the cardiac inhibitory effect of the plant extract, right atrium of the rabbits was isolated and used. A 10 mL organ bath was cleaned and filled with Krebs solution, and isolated atrium was put in it. The solution in the bath was kept on continuous perfusion with carbogen, and temperature maintained at 37°C throughout the experiment. A preload tension of 1.0 gram was applied to the atrium. Atrial contractions were recorded by PowerLab linked isometric transducer. The left atrium was allowed to equilibrate for 30 minutes with repeated changing of Krebs solution. During this time no drug was applied to the left atrium. After equilibration, the tissue was treated with acetylcholine (1 µM) or isoprenaline (1 µM) to check its responsiveness. Plant extract was applied to this tissue cumulatively to observe the response of the tissue, and results were calculated as percentage of the baseline response [response , 32.n = 5). Collected blood was centrifuged at 1,500 rpm for a period of 15 minutes, and supernatant was collected as platelet rich plasma (PRP). Remnant was recentrifuged at 4,000 rpm speed for 20 minutes, and supernatant separated and collected as platelet poor plasma (PPP). Chrono-log 490-2D aggregometer was utilized for this study. This aggregometer works on light transmission aggregometry principle. Machine was turned on and kept free to maintain the temperature. 0.23 mL of PRP and 10 µL of plant extract were taken in micro cuvette and placed in PRP chamber. A spacer was attached at the base of the cuvette. Then, 10 µL of ADP (5 µM) or epinephrine (40 µM) was added to this cuvette. 450 µL of PPP was added to another micro cuvette and kept as a reference in PPP chamber [Inhibition of aggregation of platelets by the application of Pc.Cr was studied by using a previously described method , 33, 34. chamber . EthicalCastor oil induced diarrhea model was used to study the antidiarrheal effect of Pc.Cr as reported previously , 24. Thin = 5) were anesthetized with intraperitoneal administration of ketamine and diazepam at a dose range of 50–80 mg/kg and 5 mg/kg, respectively. After the development of anesthesia, the animal was placed on a dissection board in an upward position. Isothermic warming cushions were utilized to maintain the temperature at 37°C. A small incision was made for cannulation of trachea, jugular vein, and carotid artery. Polyethylene tube (PE-20) was used for cannulate trachea, while jugular vein and carotid artery were cannulated with PE-50. Cannula of carotid artery was filled with heparin solution (60IU/Ml) and attached to disposable pressure transducer (MLT0699) which was attached to PowerLab for data recording. After cannulation, 0.1 mL heparin was administered to rat through jugular vein. Plant extract and standard drugs were administered in a volume of 0.1 mL which was followed by a flush of 0.1 mL normal saline. Epinephrine (1 µg/kg) and acetylcholine (1 µg/kg) were given to check the response of the animal to hypertensive and hypotensive drugs. After equilibration, 0.1 mL Pc.Cr was administered through jugular vein, and the response to different doses was recorded [SD rats  was measured with standard aluminium chloride spectrophotometric method. It was shown that Pc.Cr contained a total flavonoid content of 33.2 mg QU/g.The total flavonoid content of P. cattleyanum extract and fractions was measured with the Folin–Ciocalteu reagent assay. It was shown that Pc.Cr contained .On the other hand, the total phenolic content of the The identified compounds of Pc.Cr, along with their retention times and area percentages, are shown in + induced contractile responses were inhibited by the application of Pc.Cr in a concentration-dependent way to isolated jejunums. Respective values of EC50 were 0.76 ± 0.11 (0.46–1.29 mg/mL) and 0.25 ± 0.14 (0.11–0.44 mg/mL). Verapamil, a standard antagonist of calcium channel, showed similar behavioral against jejunum with EC50 value of 0.20 ± 0.06 (0.14–0.27 µmol) and 0.08 ± 0.06 (0.06–0.08 µmol)  and 0.16 ± 0.15 (0.08–0.27 mg/mL). Application of Pc.Aq resulted in partial inhibition of spontaneously contracting jejunum, and complete inhibition of potassium induced contractions was seen. EC50 value was 2.86 ± 0.15 (2.21–6.41 mg/mL) (Spontaneous and High-K08 µmol) . These rs proved . Similar1 mg/mL) .+ and CCh were inhibited by the application of Pc.Cr, Pc.DCM, and Pc.Aq in a concentration dependent way. These effects were similar to verapamil, but the effect of Pc.DCM was more potent than Pc.Cr and Pc.Aq. Respective EC50 values against High-K+ and CCh generated Pc.Cr contractions of 0.80 (0.62–1.07 mg/mL) and 4.99 ± 0.20 (3.29–7.83 mg/mL). For Pc.DCM, EC50 values were 0.15 ± 0.12 (0.08–0.21 mg/mL) and 0.56 ± 0.08 (0.40–0.80), respectively, but for Pc.Aq, EC50 values were 3.55 ± 0.12 (2.36–5.33 mg/mL) and 6.90 ± 0.20 (4.12–12.84 mg/mL), respectively  and 1.92 ± 0.10 (1.03–1.72 mg/mL). Similarly, vasorelaxant by Pc.DCM was observed at EC50 values of 0.11 ± 0.06 (0.05–0.13 mg/mL) and 0.52 ± 0.07 (0.36–0.76 mg/mL). Vasorelaxant effect of verapamil was observed with respective EC50 values of 0.20 ± 0.06 (0.14–0.29 µmol) and 0.32 ± 0.17 (0.23–0.45 µmol). Pc.Aq also showed inhibitory effects at higher concentrations with EC50 values of 0.97 ± 0.08 (0.72–1.30 mg/mL) and 4.25 ± 1.16 (3.05–6.0 mg/mL)  . Vasorel50 value of 2.20 ± 0.20 (1.17–4.36 mg/mL) was observed, but no effect on rate of contraction was observed (Concentration dependent inhibition of force of contractions by applying Pc.Cr to isolated atrium with ECobserved .Inhibition of aggregation in platelets was shown by Pc.Cr in the presence of agonists like ADP with minimum inhibitory concentration of 0.27 ± 0.09 (0.22–0.34 mg/mL), but no inhibition was observed in the presence of epinephrine (Epi) up to dose of 5 mg/ml .P ≤ 0.01) as evident by decrease in total feces in a time period of six hours. The dose of 50 mg/kg showed no significant protection. Protection was observed by decrease in total numbers of feces in a time period of six hours like loperamide (10 mg/kg) (P ≤ 0.001). No antidiarrheal effect was observed by the administration of saline (Castor oil treated mice were protected significantly by giving Pc.Cr (p.o.) in a dose range of 100 mg/kg to 500 mg/kg . Administration of Pc.Cr 1.0 mg/kg resulted in reduction of SBP to 95.67 ± 2.16 mmHg vs normotensive SBP 140.67 ± 11.61 mmHg, DBP to 71.68 ± 4.68 mmHg vs normotensive DBP 121.05 ± 21.45 mmHg, and MAP to 127.48 ± 21.18 mmHg. Similarly, Pc.Cr 3.00 mg/kg inhibited SBP to 79.86 ± 3.0 mmHg, DBP to 68.74 ± 8.71 mmHg, and MAP to 68.55 ± 6.90 mmHg compared to normotensive rats. However, Pc.Cr 10.00 mg/kg reduced SBP to 39.23 ± 2.41 mmHg, DBP to 30.71 ± 0.84 mmHg, and MAP to 34.19 ± 0.29 mmHg compared with control group .No sign of toxicity was observed in any animal for a period of 24 hours.P. cattleyanum and other members of its family in the treatment of such ailments, it was planned to explore the pharmacological insights of these traditional uses.Smooth muscle relaxant drugs are used usually in the treatment of cardiovascular, respiratory, and gastrointestinal disorders . Conside+) applied to the tissue, contraction of smooth muscle takes place due to the opening of calcium ion channels. Increased intracellular calcium activates the contractile machinery of the smooth muscle cells. Calcium channel blockers are the drugs which block the opening of these channels and cause relaxation. Concentration dependent curves of calcium were constructed for the confirmation of calcium channel blocker potential of plant extract. It was observed that pretreatment of tissue with Pc.Cr caused the rightward shift of calcium curves with inhibition of maximum contractile response, thus confirming the presence of calcium antagonist effect of plant extract [Spasmolytic effect of plant extract was seen when applied to isolated jejunum. These spasmolytic effects were similar to those of verapamil. Smooth muscle contraction is based on intracellular free calcium. Increase in intracellular calcium may induce contraction in smooth muscles , 39. Cal extract , 28, 42.+ and carbachol mediated contractions in isolated tracheal strips. Tracheorelaxant effect of Pc.DCM was more potent as compared to Pc.Aq. CCh has different mechanism of contraction from that of High-K+. It causes the stimulation of muscarinic receptors of M3 type. Muscarinic receptor stimulation causes increase in intracellular concentration of calcium through phospholipase C second messenger system, and as a result contraction takes place [Pc.Cr and its fractions also showed inhibition of High-Kes place , 43. It + mediated vasoconstrictions were inhibited by the application of Pc.Cr and its fractions. However, like calcium channel antagonists, inhibition against High-K+ was more than that against PE, again giving a clue for the presence of calcium channel blocker property in P. cattleyanum. As blood pressure is the product of cardiac output and peripheral vascular resistance (BP = CO × PVR), Pc.Cr was applied to isolated rabbit atrium to see its influence on cardiac output. Partial reduction in force of contraction with no change in rate of contraction was seen. Therefore, it can be said that P. cattleyanum selectively blocks the calcium channels of the smooth muscles but has little influence on calcium channels of cardiac muscles.PE and High-KPsidium cattleyanum contains trans-caryophyllene as indicated by GC-MS analysis which is reported to possess spasmolytic and calcium channel blocker activity [trans-Caryophyllene may be the one of those macromolecules which cause the relaxation of smooth muscles.Dose dependent diarrheal protection was also seen. Reduction of blood pressure in normotensive rats treated with Pc.Cr was observed. Due to vasodilator and cardiac inhibitory properties of calcium channel antagonists, they are used in the treatment of hypertension and other ailments of cardiac system –46. Flavactivity . trans-C2Y1 and P2Y12 glycoprotein receptors exist on the surface of platelet and play a vital role in aggregation. ADP causes the stimulation of these receptors and results in the aggregation of platelets. It can be said that plant extract possesses ADP antagonistic potential which caused the inhibition of platelet aggregation [Platelet aggregation was blocked because Pc.Cr epinephrine (Epi) induced aggregation was not inhibited. Pregation , 35. Calregation , 49. It regation –53. Our P. cattleyanum were observed in this study, thus giving a justification for using it in treating ailments of gastrointestinal, respiratory, and cardiovascular systems. These results of P. cattleyanum may be due to the blockage of voltage gated calcium channels, and presence of trans-caryophyllene and flavonoids may be the reason for these effects. However, the involvement of any other mechanism cannot be ignored. Platelet aggregation inhibition by Pc.Cr may be due to antagonism of P2Y1 and P2Y12 receptors. This is a preliminary research, and exploration of the exact mechanism of action and identification of active compounds from Psidium cattleyanum require further investigation.Spasmolytic, tracheorelaxant, vasodilator, atrial inhibitory, platelet aggregation inhibitory, diarrheal protective, and hypotensive activities of