Retinoic acid receptor-related orphan receptors (RORs) are frequently abnormally expressed in several human malignancies, including gastric cancer (GC). RORs are involved in the development and progression of GC through Wnt signaling pathway receptors and other common receptors. However, the prognostic roles of individual RORs in patients with GC remain elusive. We accessed the prognostic roles of three RORs  through “The Kaplan–Meier plotter” (KM plotter) database in patients with GC. For all patients with GC who were followed for 20 years, the low mRNA expression of all three RORs showed a significant correlation with better outcomes. We further accessed the prognostic value of individual RORs in different clinical pathological features including Lauren classification, clinical stages, pathological grades, HER2 status, and different treatments methods. The RORs demonstrated critical prognostic roles in GC. Expressions of RORs were higher in GC tissues when compared with normal gastric tissues. Moreover, knockdown of RORs significantly inhibited cell proliferation and migration, suggesting an oncogenic role of RORs in human GC. These findings suggest potential roles of RORs as biomarkers for GC prognosis and as oncogenes in GC. GC is ranked as second highest in mortality among malignant tumors, which indicates an obvious threat to human health . The occRetinoic acid receptor-related orphan receptors (RORs) belong to the nuclear receptor super family and consist of three subtypes: RORα , RORβ , and RORγ . RORs play an important role in a series of physiological and pathological processes. These include reproductive development, physiological rhythm regulation, metabolic disorders, inflammation, immune system regulation, and cancer ,8,9. RecThe online Kaplan–Meier (KM) plotter is capable of assessing the effect of any gene or gene combination on survival in breast, ovarian, lung, and GC using patient samples measured by gene chips or RNA-seq. So far, a number of genes have been identified and validated by the KM plotter in these four cancer types ,15,16,1722.1http://kmplot.com/analysis/). In this online database, gene expression data, relapse free, and OS information are downloaded from GEO (Affymetrix microarrays only), EGA ,and TCGA. The clinical data from 876 patients with GC including gender, perforation history, stage, Lauren classification, HER2 status, pathological grades, and different treatment methods were collected. After entering the three ROR sub-members types into the database , data were compared using a Kaplan–Meier survival plot. The hazard ratio (HR) with 95% confidence intervals and log-rank P value were also calculated. P values of < 0.05 were considered statistically significant. HR is defined as the ratio of the hazard rates corresponding to the conditions described by two levels of an explanatory variable in the survival analysis.The correlation between mRNA expression of RORs and overall survival (OS) was analyzed using the KM plotter database (2.22. The cells were transfected with siRNA by Lipofectamine 2000 once confluency reached approximately 70%.Human GC cell line AGS was obtained from the Type Culture Collection of the Chinese Academy of Sciences. The cells were cultured in dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum and incubated at 37℃ with 5% CO2.3The proliferation of AGS cells transfected with siRNA was confirmed using CCK-8. SiRNA transfected cells were moved into a 96-well plate at 5,000 cells per well. Ten microliters of CCK-8 solutions was added to the well, and the cells were cultured for 1.5 h. Finally, OD values were measured on a microplate reader. Each experiment was performed in triplicate.2.4Transwell assay was performed to evaluate cell migration ability with the transwell chamber . SiRNA transfected cells were seeded into the upper chamber. After incubation for 48 h, cells that migrated to the lower chamber were stained with crystal violet for 10 min. Migrated cells were calculated from triplicate determinations.2.5GC tissues were stained by immunohistochemistry. Antibodies against the RORs were purchased from Abcam. All samples were fixed in 4% polyformaldehyde and then embedded in paraffin wax. Sections were cut at a thickness of 4 µm. Antigen retrieval was done by heating in a microwave for 12 min. Slides were blocked with goat blocking serum for 40 min followed by incubation with the antibody to h RORs antibody at room temperature for 1 h. The results were observed by a microscope, and all image processing was completed with ImageJ software.2.6After the transfection for 48 h, total protein was extracted from cells for Western bolt assay. The primary antibodies were purchased from Abcam . The relative expressions of RORα, RORβ, and RORγ were calculated by target protein/GAPDH using QUANTITY ONE software.33.1www.kmplot.com. The valid Affymetrix IDs of RORα, RORβ, and RORγ were, respectively, 210479_s_at, 206443_at, and 206419_at. Survival curves were drafted for all patients with GC (n = 876), patients with intestinal-type GC (n = 320), patients with diffuse-type GC (n = 241), and patients with mixed-type GC (n = 33).We examined the prognostic value of mRNA expression of RORs via www.kmplot.com. Low mRNA expression of RORα demonstrated an association with a better prognosis  (P = 0.013] (P = 0.00045] (P = 0.061)  .www.kmplot.com. As shown in P < 0.0001] (P < 0.0001] (P = 0.01] (P = 0.36)  .P < 0.0001] (P < 0.0001] (P = 0.18] (P = 0.13)] ] .P = 0.0063; stage 2: HR = 2.71, 95% CI: (1.3–5.67), P = 0.0085; stage 3: HR = 1.65, 95% CI: (1.17–2.32), P = 0.0036; stage 4: HR = 1.61, 95% CI: (1.1–2.37), P = 0.014]. Low mRNA expression of RORα was associated with a better prognosis in clinical stages 2 and 4 in patients with GC . Low mRNA expression of RORγ was associated with a better prognosis in clinical stages 3 and 4 in patients with GC . As shown in P = 0.00064; HER2 positive: HR = 1.36 95% CI: (1.04–1.79), P = 0.024] and RORβ  was associated with a better prognosis in both HER2-negative and HER2-positive patients with GC. In comparison, low mRNA expression of RORγ was only associated with a better prognosis in HER2-negative patients . As shown in P < 0.0001] and in 5 FU-based adjuvant patients . Low mRNA expression of RORβ was only associated with a better OS in patients who received surgery alone . Finally, low mRNA expression of RORγ was only associated with a better OS in 5 FU-based adjuvant patients with GC .According to these results, low mRNA expression of RORα, RORβ, and RORγ were all associated with a better prognosis in patients with GC. Although there was not a significant difference in prognosis in the mixed-type GC subgroup, this could be due to the small cohort of only 33 cases. Next, we analyzed the correlation between ROR family members and GC prognosis regarding different clinical pathological features. This included comparing clinical stages , HER2 st3.2According to our results, high ROR expression presented as a poor prognosis in GC, so we sought to confirm whether RORs were overexpressed in GC tissues. The differentiated expression of RORs in 60 gastric normal tissues and 60 GC tissues of humans was detected using immunohistochemistry. As shown in 3.3ROR expression was inhibited by specific siRNAs  to build knockdown cells for elucidating their biological functions in AGS cells . We also4Nuclear receptors are a class of ligand dependent receptors that belong to the transcription factor superfamily, which is similar to steroid hormone receptors. This family is widely distributed in the human body and plays an important role in cell development, cell differentiation, physiological rhythm, metabolism, and immune regulation. Nuclear receptors have three subfamilies consisting of steroid hormone receptors, nonsteroid hormone receptors, and orphan nuclear receptors . RORs arThe RORα gene, located on chromosome 15q22.2, contains 15 exons and spans approximately 730 kb of genomic regions. It has many important physiological functions in organisms. Defects of RORα in mice can lead to a series of abnormalities. These abnormalities can include impaired bone formation and muscle atrophy, ataxia and cerebellar atrophy, decreased size of the spleen and thymus, circadian rhythm disorders, and impaired lipid metabolism ,21. FuncRORβ was originally identified as a member of the orphan nuclear receptor family by Carlberg in 1994. This gene, located on chromosome 9q21.13, contains 10 exons and spans 188 kb of genomic regions . RORβ plThe coding gene of RORγ is located on human chromosome 1 and consists of 10 exons. RORγ is mainly expressed in tissues of the immune system, such as the thymus. However, it is also expressed in the liver, skeletal muscle, adipose tissue, and kidney. It plays an important role in the secretion of IL-17 and other proinflammatory factors in Th17 cells . By inhiin vivo. These findings suggest that all three ROR subfamilies play an important role in the development of GC and have potential as anticancer supplements. This provides new ideas and approaches for the treatment of GC.In conclusion, expression levels of RORα, RORβ, and RORγ were all correlated with the prognosis of GC. Low mRNA expression of RORs indicated a better OS based on the existing pathological samples. RORs were upregulated in GC tissues and their knockdown inhibited the proliferation and migration of GC cells. Therefore, it is necessary to expand the sample size to study the prognostic value of RORs and to study the specific pathways of RORs and their roles


Higher levels of omega–3 EPA and docosahexaenoic acid prevented the reduction in the MVC and limited joint flexibility after ECCs. Conclusions: The present study reveals that higher levels of EPA are important to promote muscle strength and preserve the strength loss after exercise.Background: Although the ingestion of total omega–3 fatty acids (omega–3) is positively related with muscular strength in older persons, little is known about the effect of omega–3 plasma levels on muscular function before and after exercise in young men. Moreover, omega–3 supplementation has a positive role in exercise-induced acute muscle damage. This study investigated the relationship between plasma omega–3 in the blood and promotion and preservation of muscle strength after eccentric contractions (ECCs) in young men. Methods: Thirty-two healthy young men participated in this study. We assessed plasma omega–3 level and the maximal voluntary contraction (MVC). Twenty-six out of them exercised 60 ECCs at 100% MVC. We measured the MVC torque, flexibility before and immediately after exercise, 1–5 days post exercise. Results: The levels of eicosapentaenoic acid (EPA) and EPA/arachidonic acid were positively associated with muscle strength ( Omega–3 was focused on because the Greenland Eskimos had a lower heart disease rate and consumed more fatty acids [Omega–3 fatty acids (omega–3) are the polyunsaturated fatty acid family of omega–3. Omega–3 fatty acids are included in fish oil and contain mainly eicosapentaenoic acid , or lengthening  contractions. Especially ECC produces larger muscle force than other contractions, and causes greater muscle damage in untrained individuals ,3. It isSome studies have studied the relationship between omega–3 intake and muscular function in older adults and young athletes ,6,7. RosIt is widely known that omega–3 effectively improves fatigue recovery and exercise performance ,9. In reAccordingly, we examined the relationship between plasma omega–3 levels at baseline and muscle function, and muscle damage following ECCs without the ingestion of supplementation in young men. Collectively, the preliminary findings indicate that plasma omega–3 level is associated with muscle strength before and after exercise.Thirty-two healthy untrained men were included for this study . All subjects participated in our study. Twenty-six out of them agreed to participate in a study on the magnitude of muscle damage following ECCs. We requested to all subjects that they do not to attend other clinical interventions. No subject had done continuous exercise training within one year. All subjects were explained the whole study protocol before intervention and signed the informed consent form. The study was approved by the ethics committee for human experiments of Teikyo Heisei University (ID: R01–040) and followed the Declaration of Helsinki.Subjects visited the laboratory on one (6 subjects) or four (26 subjects) occasions throughout the study period. On the first visit, the blood samples were drawn from all subjects, and muscle strength of elbow flexion was assessed on the same day. After measurement of muscle strength, 26 subjects performed ECCs. All outcomes were obtained before and after ECCs, and on day 1, day 2, and day 5 after ECC at 9:00–11:00 a.m. in a temperature-controlled room maintained at 23 °C. According to previous studies ,14,15,16The subjects fasted for 8 h before a nurse took blood from the forearm. Then, the obtained blood was centrifuged for 10 min at 3k rpm at 4 °C . The levThe MVC torque was calculated 3 times for 3-s contractions at 90° with a 1-min interval. Participants were encouraged to perform a maximal contraction during the tests. The mean value of the highest 1-s for each contraction was used for the MVC torque analysis. The detailed method of analysis is shown in our previous study .The participants sat on an arm curl bench. The weight of dumbbell was converted from MVC. The subjects performed 6 sets of a total of 10 ECCs of elbow flexors with a 1.5-min interval. The dumbbell was given to the participant with the elbow flexed at 90°, then made lower until it reached the extended position at 30°/s. A well-trained investigator returned the dumbbell so that the subject could restart the ECC.This study evaluated the MVC and range of motion (ROM) as muscle damage markers. The MVC is similar to the method of muscle strength measurement described above. The investigator measured the extended and flexed arm to calculate the elbow ROM as shown in a previous study .p < 0.05 to be significant.Statistical analysis was conducted with SPSS software version 26.0 . Values were expressed as means ± standard deviation. The physiological characteristics of subjects and muscle strength were generated for participants by quartiles of EPA, DHA, and EPA/AA in order to investigate whether blood EPA and DHA level affect muscle function based on previous studies . A 1-wayp < 0.05). Furthermore, Quartile 4 of DHA was higher than Quartile 2 (p < 0.05). Moreover, as for BMI, Quartile 4 of EPA was higher than Quartile 1 (p < 0.05), and Quartile 4 of DHA was higher than Quartile 1 and 2 (p < 0.05). In addition, we have analyzed anthropometric data by Tertiles 1, 2, and 3 and the results are similar to Quartile 4. The range of concentration were as follows; EPA: Tertiles 1, 9.4–15.7 μg/mL, Tertiles 2, 17.3–25.6 μg/mL, and Tertiles 3, 26.0–94.5 μg/mL, DHA: Tertiles 1, 35.4–58.4 μg/mL, Tertiles 2, 58.4–78.2 μg/mL, and Tertiles 3, 82.9–119.2 μg/mL, EPA/AA: Tertiles 0.06–0.09, Tertiles 2, 0.09–0.1, and Tertiles 3, 0.13–0.5. For body mass, Tertile 3 of EPA (71.2 ± 6.7 kg), DHA (70.6 ± 6.6 kg), and EPA/AA (68.1 ± 6.1 kg) were significantly higher than that of Tertile 1 . For BMI, Tertile 3 of EPA (24.2 ± 2.2) and DHA (23.9 ± 2.2) were significantly higher than that of Tertile 1 .The subject physical characteristics and range of concentration in the plasma EPA, DHA, EPA per AA by the Quartile can be seen in p < 0.05). In addition, Quartile 4 and 3 of EPA/AA were higher than Quartile 1 (p < 0.05). However, there was no association between DHA and muscle strength (p < 0.05).As for muscle strength, Quartile 4 of EPA was significantly higher than that of Quartile 1 (strength B. This wp < 0.05). As for MVC, Tertile 3 of EPA was higher than Tertile 1 and 2 at post-exercise (p < 0.05). In addition, Tertile 3 of EPA was higher than Tertile 1 at 2 d and 5 d post exercise (p < 0.05). Tertile 3 of DHA was significantly higher than that of Tertile 1 and 2 at post- and 1 d post-exercise (p < 0.05). In addition, there was a group × time effect for ROM in EPA, DHA, and EPA/AA (p < 0.05). As for ROM, Tertile 3 of EPA was significantly higher than that of Tertile 1 and 2 at post- and 1 d post-exercise (p < 0.05). Moreover, Tertile 3 of EPA was higher than Tertile 1 at 2 d post-exercise (p < 0.05). Similarly, Tertile 3 of DHA was significantly higher than that of Tertile 1 and 2 immediately after exercise and 1 d, 5 d post-exercise (p < 0.05). Moreover, Tertile 3 of EPA/AA was significantly higher than that of Tertile 1 immediately after exercise (p < 0.05).Regarding the association between omega–3 and ECC-induced muscle damage, statistical analysis showed a group × time interaction effect for the MVC in EPA and DHA  the concentrations of EPA and EPA per AA were correlated with muscular function in healthy young males; (2) higher levels of EPA, DHA, EPA per AA have preventive roles for preserving muscle strength and joint flexibility after strenuous exercise in healthy young men. Therefore, higher plasma omega–3 is positively associated with muscle strength in young men, and that preserves muscle damage following exercise. To the best of our knowledge, this is the first study to show their relationship.This study showed that higher EPA, DHA, and EPA per AA resulted in higher body weight and BMI. A previous study reported that higher consumption of omega–3 showed higher body mass, higher lean mass, and lower body fat . In addiOmega–3 supplementation enhances the rate of muscle protein synthesis in older adults , and impThe present study also reveals that higher EPA and DHA levels are important to reduce muscle damage. Our previous study showed that 8–week omega–3 supplementation caused inhibition in decreased muscle strength, reduced ROM, muscle soreness . In addiThis study has several limitations. The subjects were limited to young men, and the sample size was not enough to reach a conclusion on our hypothesis. Further studies are needed to compare elderly participants with young ones in a large sample. Moreover, the only muscle damage markers evaluated muscle strength and ROM in this study. It is necessary to study the EPA and DHA levels on another symptom of muscular damage such as plasma CK, Mb, and inflammatory response.We conclude that higher plasma omega–3 is positively related with muscular function in young males. Furthermore, higher blood levels of EPA and DHA preserve muscle and joint function following exercise. Therefore, although the present study shows preliminary data, we propose a daily intake of omega–3 might be important to maintain to promote and preserve the muscle strength after exercise.


Recent studies regarding the relationships between plasma amyloid-β (Aβ) levels and cognitive performance had inconsistent results. In this study, we aimed to characterize the relationship between cognitive decline and plasma Aβ levels in a large-sample cognitively normal population.40, Aβ42, and Aβ42/40).This population-based, prospective cohort study included 1,240 participants with normal cognition. The Mini-Mental State Examination (MMSE) was used to assess cognitive function at baseline and 2 years later. Restricted cubic splines, multivariate logistic regression, and multivariate linear regression models were used to evaluate the type of relationship between cognitive decline during the 2-year follow-up period and plasma Aβ levels : 0.60, 95% confidence interval (CI): 0.45–0.81, p < 0.001] or high Aβ40  levels. The association between Aβ40 and cognitive decline did not depend on sex, education level, or APOE ε4 status. There was an interaction found between age (≤ 65 and > 65 years) and Aβ40 (p for interaction = 0.021). In individuals older than 65 years, there was a positive linear relationship between plasma Aβ40 and cognitive decline . For participants ≤ 65 years old, the lower Aβ40 and higher Aβ40 groups had a lower risk of cognitive decline than the medium Aβ40 group . None of relationship between plasma Aβ42, Aβ42/40 and cognitive decline was found during a 2-year follow-up.Participants with moderate Aβ40 and cognitive decline was not linear, but an inverted-U shape in a cognitively normal population. The underlying mechanism requires further investigation.The relationship between plasma Aβ Amyloid-β (Aβ) pathology has been confirmed as a pathological change in the early phase of Alzheimer ‘s disease (AD) . Abnorma42/40 or Aβ42 increased the risk of cognitive decline and the progression of dementia  scans or measured in cerebrospinal fluid (CSF), has been extensively studied and has proven clinical applications for AD . Howeverdementia . Other spairment .In order to rapidly identify patients with cognitive decline earlier, and therefore delay the occurrence of AD, the present study aimed to determine the change in cognitive status, its relationship with plasma Aβ, and the type of association, during a 2-year follow-up in a cognitively normal population. This study also investigated whether this association was altered by sex, age, education level, or Apolipoprotein E (APOE) genotype, and estimated the relationship between plasma Aβ and cognitive disorder and cardio-cerebrovascular diseases during a 2-year follow-up.Participants were enrolled from a village in the suburbs of Xi’an city, located in northwestern China, between October, 2014 and November, 2015. This study was a community population cohort study in which each interviewee received a face-to-face questionnaire. Participants were asked about detailed history by neurologists, and received a nervous system examination, a systemic physical examination, blood biochemical examination aimed at determining whether there were any related neurological diseases. All patients with cerebral disease had received the CT or MRI scans. For patients who had a diagnosis of mild cognitive impairment (MCI) or dementia due to AD, we provided free MRI scan to confirm the diagnosis of AD in a local designated hospital. Inclusion criteria was that participants were (1) older than 40 years and (2) a current resident of the village and had lived there for more than 3 years. Exclusion criteria was that (1) the Mini-Mental State Examination (MMSE) was not completed; (2) there was evidence of MCI or dementia, or (3) other neurological conditions that may influence cognitive function ; (4) plasma Aβ was not measured; and (5) other baseline information was missing. This study was approved by the First Affiliated Hospital of Xi’an Jiaotong University. Written informed consent was obtained from all participants.The MMSE was conducted as global cognitive screening at enrollment. The MMSE, other neurological, physical and imaging examinations were assessed at baseline and repeated 2 years later. Cutoff values were ≤ 17 for subjects with illiteracy, ≤20 for primary school educated subjects, and ≤ 24 for those educated at the junior high school level or above . After i40 and Aβ42 tests were measured using enzyme-linked immunosorbent assay kits   at OD 450 nm. Apolipoprotein E (APOE) was genotyped by polymerase chain reaction (PCR), followed by sequencing, as previously described  . Concentescribed .The primary outcome was cognitive decline, which was defined as the difference of MMSE between the first visit and the second visit > 0. Cognitive disorder was defined as MMSE score ≤ 17 for illiterate, ≤ 20 for primary school educated, and ≤ 24 for junior high school educated or above . New carLack of physical exercise was defined as exercising fewer than three times a week, fewer than 30 min a time. Smoking was defined as a current smoker. Diabetes mellitus was defined as a self-reported medical diagnosis, diabetic medication use, a fasting glucose of ≥ 7.0 mmol/l, a random plasma glucose concentration ≥ 11.1 mmol/l, or glycated hemoglobin ≥ 6.5%. Hypertension was defined as a self-reported medical diagnosis, antihypertensive medication use, a systolic blood pressure of ≥ 140 mmHg, or a diastolic blood pressure of ≥ 90 mmHg.2 or Fisher’s exact test for categorical variables, and Student’s t-test or Mann–Whitney U-test for continuous variables. Differences of basic characteristics between different Aβ groups were compared using Pearson’s χ2 or Fisher’s exact test for categorical variables, and ANOVA test or Kruskal-Wallis H test for continuous variables.Continuous variables are presented as mean ± SD, and categorical variables are presented as proportions. We determined the association between baseline information and cognitive decline using Pearson’s χt-test.Restricted cubic splines with a logistic regression model were applied to test the relationship between cognitive decline and Aβ levels, and to evaluate the type of correlation of cognitive decline with Aβ levels. Four knots defined at the 5th, 35th, 65th, and 95th percentiles of Aβ levels were prespecified . The ref40 and cognitive decline by sex, age (≤ 65 and > 65 years), education (≥ high school and < high school), and APOE ε4 status in multivariable-adjusted logistic regression models. SPSS ver. 24.0 and R ver. 3.5.3 were used for statistical analysis. A p < 0.05 was considered significant.To assess the association between Aβ levels  and various outcomes, unadjusted, age- and sex-adjusted, and multivariable-adjusted logistic regression models were used. A multivariable linear regression model was used to evaluate the relationship between Aβ and 2-year MMSE score change. In multivariable models, adjustments were made for age, sex, diabetes, hypertension, smoking, lack of physical activity, APOE ε4 status, and education. In subgroup analyses, we examined the relationship between AβThe study population screening process is shown in p = 0.005). MMSE score was higher (27 ± 3.2) in the group with cognitive decline than that in the population without cognitive decline.The associations between continuous Aβ40 showed a non-linear relationship  with cognitive decline. Aβ42 and Aβ42/40 didn’t show any relationship with cognitive decline .The associations between Aβ levels and cognitive decline using restricted cubic splines are shown in 40 and cognitive decline, and the inverted U shape found in the restricted cubic spline, we transformed continuous Aβ40 into two kinds of categorical variables: three tertiles defined by the 33rd percentiles and 66th percentiles, and 3-class classified variables defined by the 25th and 75th percentiles. In multivariate logistic regression analyses evaluating the relationship between cognitive decline and categorical Aβ40, 3-classified Aβ40 had a better goodness of fit  than Aβ40 defined by the 33rd and 66th percentiles . Therefore, 3-classified Aβ40, defined by the 25th and 75th percentiles, was used in the present study.Because of the non-linear relationship between plasma Aβ40 groups are shown in 40 groups were significant , medium (45 pg/mL ≤ Aβ40 < 58.4 pg/mL), and high Aβ40 (≥58.4 pg/mL) are listed in Two-year changes in MMSE score in the three Aβ2 groups . The cha40 and various outcomes during follow-up using multivariable logistic regression models are listed in 40 between 45 and 58.4 pg/mL, participants in the low Aβ40 group and high Aβ40 group had lower risks of cognitive decline . After adjustments for age and sex, the results were the same as before. In the fully adjusted model, participants in the low Aβ40 (< 45 pg/mL) and high Aβ40 groups (≥58.4 pg/mL) still had a lower risk of cognitive decline, compared with the medium Aβ40 group . In addition, participants in the low Aβ40 group had a lower risk of a new cognitive disorder  than participants in the medium Aβ40 group, while participants with high Aβ40 levels did not show this association. There were no significant associations found in other outcomes. The relationships of categorical Aβ40 defined by the 33th percentiles and 66th percentiles, and cognitive decline are listed in The relationship between plasma Aβ40 and 2-year change in MMSE score using a multivariate linear regression model is shown in 40 and MMSE score change was still apparent. Compared with participants with medium Aβ40, those with low Aβ40 and high Aβ40 had a lower risk of MMSE score change .The relationship between plasma Aβ40 and cognitive decline are listed in 40 and cognitive decline did not depend on sex, education level, or APOE ε4 status. There was an interaction found between age (≤ 65 and > 65 years) and Aβ40 (p for interaction = 0.021). For participants ≤ 65 years old, the lower Aβ40 and higher Aβ40 groups had a lower risk of cognitive decline than the medium Aβ40 group . In participants aged > 65 years, lower Aβ40 levels correlated with cognitive decline  compared with the medium Aβ40 group. Continuous Aβ40 level was associated with the increased risk of cognitive decline .Subgroup analyses of the relationship between plasma Aβ40 and cognitive decline during a 2-year follow-up in individuals with normal cognition. People with medium plasma Aβ40 concentrations (45–58.4 pg/mL) had the highest risk of cognitive decline than persons with plasma Aβ40 < 45 pg/mL and ≥ 58.4 pg/mL.In this large, prospective, population-based cohort study, we found an inverted U-shaped relationship between plasma Aβ42 and plasma Aβ42/40 ratio  or the incidence of dementia for a long follow-up time in old and high-risk dementia populations  and younger population in our study. It is why we only find the correlation of cognitive decline with plasma Aβ40, not plasma Aβ42.Previous studies have shown evidence of an association between plasma Aβ levels and the progression of AD, although most studies focused on plasma Aβsma Aβ40 . Chen etsma Aβ40 . This isulations . In our l volume . There wl volume . Therefo40 and cognitive decline remain unclear. There may be both of neurodegenerative and cerebrovascular pathologies promoting cognitive impairment in the study participants , while there seems to be a positive linear relationship between plasma Aβ40 and cognitive decline in individuals older than 65 years. This result agrees with other studies that suggested that amyloid-cognition association varied by age (40) were not suitable for assessing the relationship between plasma Aβ40 and cognitive decline.Of note, we found that the inverted U-shaped relationship between plasma Aβd by age . Jansen d by age . In olded by age . For you40 with CSF Aβ40 and brain Aβ40, and the mechanism of transportation and clearance of Aβ40, were not studied. It would be useful to explore the dynamic transportation mechanism of Aβ40 between the brain and peripheral blood in future studies, in order to verify the present results.There are still limitations that need to be considered. First, only the total MMSE score was evaluated for every participant in this study; sub-items of MMSE and other cognitive scores should be included to confirm cognitive decline. Second, ELISA is not the most sensitive technique for assessing plasma Aβ levels. Recent studies have employed single-molecule array or hybrid mass spectrometry techniques which may be more accurate approaches than ELISA . Neverth40 and cognitive decline is not linear, but an inverted-U shape, in a cognitively normal population. Medium concentration of plasma Aβ40 is associated with a greater risk of cognitive decline, compared with low and high levels of plasma Aβ40, primarily in younger persons (≤65 years old). The underlying mechanism needs to be further elucidated.This population-based, prospective cohort study showed that the relationship between plasma AβThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The studies involving human participants were reviewed and approved by the ethics committee for medical research at the First Affiliated Hospital of Xi’an Jiaotong University. The patients/participants provided their written informed consent to participate in this study.FG and QQ: conception and design of the study. SS, CC, LD, LG, SW, JW, and QQ acquisition of the data. FG, SS, and CC: analysis of data. FG, JW, KH, MD, and QQ interpretation of data. FG: wrote the first manuscript draft. SS, CC, LD, LG, SW, JW, KH, MD, JW, and QQ: revised the manuscript for intellectual content. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Morinda officinalis How. To clarify the pharmacokinetics and tissue distribution of these three compounds, we employed liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to determine the contents of the three components in rat plasma and tissues. Separation was achieved on an Acquity UPLC HSS T3 column . Formic acid aqueous solution  and acetonitrile  were used as the mobile phase system with a programmed elution of 0∼5 min with 70% A and then 5∼7 min with 60% A. All analytes were measured with optimized multiple reaction monitoring (MRM) in negative ion mode. Geniposide and 1,8-dihydroxyanthraquinone were used as the internal standards (IS). The linear ranges were 1.2∼190, 1.3∼510, and 0.047∼37.5 μg/mL, respectively. Compared with the Morinda officinalis without wood (MO) group, the Cmax and AUC0-t parameters of rubiadin and rubiadin 1-methyl ether elevated remarkably for the salt-processed Morinda officinalis (SMO) groups, which indicates that steaming by salt could increase the bioavailability of rubiadin and rubiadin 1-methyl ether. The Tmax for monotropein is shorter (0.5 h) in SMO groups than that in MO group, which means that monotropein was quickly absorbed in the SMO extract. Moreover, the contents of three compounds in the small intestine were the highest.Salt processing, which involves steaming with salt water, directs herbs into the kidney channel. After being salt processed, kidney invigorating effects occur. However, the underlying mechanism of this method remains elusive. The compounds monotropein, rubiadin, and rubiadin 1-methyl ether are the major effective components of  Morinda officinalis is a commonly used traditional Chinese medicines (TCMs) [s (TCMs) , which hs (TCMs) –6. Its ms (TCMs) –9.Chinese medicinal herb processing is a unique pharmaceutical technique, which can turn Chinese medicinal materials into decoction pieces using processing methods like stir-frying, steaming, and frying with sand or wheat bran . DecoctiMorinda officinalis in herbal markets are M. officinalis without wood (MO) and salt-processed products (SMO). It was revealed that MO extract exerts tonifying kidney and supporting yang effects by regulating the functions of the hypothalamus-pituitary-adrenal axis [The commercially available products of nal axis , 16. Thenal axis , 18.M. officinalis [M. officinalis.Monotropein, which possess anti-inflammatory, analgesia, and antiosteoporotic activities, is the main iridoid glycoside isolated from icinalis , 20. Anticinalis –24. TherM. officinalis. Also, the effect of processing methods on the pharmacokinetics, bioavailability, and tissue distribution characteristics of iridoids and anthraquinones has barely been reported.There have been reports on the pharmacokinetics or tissue distribution of iridoids and oligosaccharides using HPC-DAD and LC-MS –28. HoweIn this study, we developed a selective and sensitive UPLC-MS/MS method for simultaneous determination of monotropein, rubiadin, and rubiadin 1-methyl ether in rat plasma and tissues. The analysis method was successfully applied to the pharmacokinetics study of MO and SMO extracts.M. officinalis were collected from Gaoliang County, Guangdong Province, and identified by Prof. Feng Li of the Liaoning University of Traditional Chinese Medicine. For reference substance, rubiadin (J0111AS), rubiadin 1-methyl ether (J0307AS), monotropein (00605AS) were purchased from Meilun Biological Technology Co. Ltd. . The internal standards (ISs) geniposide (1203A023) and 1,8-dihydroxyanthraquinone (100398–200701) were provided by the National Institute for Food and Drug Control .Merck KGaA  supplied LC-MS-grade formic acid and acetonitrile (HPLC-grade). The ultrapure water was generated with a Milli-Q water purification system . Other reagents were of analytical grade from Tianjin Kermol Chemical Reagent Co., Ltd.  and Waters Xevo TQD Mass Spectrometer .Sprague-Dawley (SD) rats  were purchased from Changsheng Biotechnology Co. Ltd. (License No. SCXK (Liao) 2015-0001, Benxi, Liaoning Province). The experimental animals were kept at 25 ± 2°C and 60% ± 10% humidity with a 12 h light/dark cycle. Water and chow were provided ad libitum. All animal pharmacological experiments followed the ethical regulations of the Liaoning University of Traditional Chinese Medicine.μm, Waters) with a temperature of 40°C. The mobile phase was acetonitrile containing 0.1% formic acid (A) and 0.1% formic acid aqueous solution (B). The elution gradient was 0.00–0.50 min with 2% B; 0.51–5.00 min with 2%–30% B; and 5.01–7.00 min with 40% B. The flow rate was 0.2 mL/min.For LC-MS/MS analysis, data acquisition and instrument control were performed using MassLynx 4.1 software . The analysis column was an Acquity UPLC HSS T3 column  equipped with an electrospray ionization source (ESI) was used in negative ion mode. The desolvation gas was nitrogen with a flow rate of 500 L/h at a temperature of 250°C. All detected compounds were measured in multiple reaction monitoring (MRM) mode. m/z 389.01 ⟶ 191.01 for monotropein (collision energy 15 eV); m/z 253.05 ⟶ 195.21 for rubiadin (collision energy 40 eV); m/z 267.07 ⟶ 252.04 for rubiadin 1-methyl ether (collision energy 18 eV); m/z 387.03 ⟶ 225.03 for IS geniposide (collision energy 8 eV); and m/z 238.94 ⟶ 166.99 for IS 1,8-dihydroxyanthraquinone (collision energy 35 eV).The precursor and product ion pairs for MRM were Monotropein, rubiadin, and rubiadin 1-methyl ether were dissolved in methanol to prepare a stock solution with a concentration of 0.5 mg/mL. The stock solution was diluted with methanol to get the appropriate concentrations for the working standard solutions. The IS stock solution (1 mg/mL) and IS working solution (200 ng/mL) were also prepared in methanol, as described above. All prepared solutions were stored at 4°C before use.μg/mL for monotropein, 396∼1.54 μg/mL for rubiadin, and 596∼1.16 μg/mL for rubiadin 1-methyl ether. The QC concentrations for monotropein were 0.14 μg/mL, 2 μg/mL, and 28.1 μg/mL; for rubiadin, they were 1.6 μg/mL, 50.6 μg/mL, and 712.5 μg/mL; and for rubiadin 1-methyl ether, they were 3.9 μg/mL, 54.6 μg/mL, and 765 μg/mL. The QC samples used for the recovery, matrix effect, intra- and interday accuracy, precision, and stability studies were prepared in the same way as that of the calibration standards. The solutions were stored for one week at −20°C.The preparation of calibration standards was done as follows: The appropriate working solution was spiked into blank plasma or tissues to obtain concentrations of 75∼0.047 M. officinalis without wood and salt-processed M. officinalis by following processing methods.Morindae officinalis Radix for 0.5 h, remove the woody cores while hot, cut into sections, and dry.  MO: steam the clean Morindae officinalis Radix with salt water for 4.0 h, until it is steamed thoroughly, remove the woody cores while hot, cut into sections and dry.  SMO: steam the clean We obtained the products of −1.Then, MO and SMO were refluxed with 80% ethanol three times for 1 h each to make a combined filtrate. Then, the extracts were concentrated to 4 g·mLThe contents of the three constituents in the MO and SMO extracts were detected by the same UPLC-MS/MS method, including the same column, mobile phase system, and column temperature as used for the pharmacokinetics and tissue distribution study. The contents of monotropein, rubiadin, and rubiadin 1-methyl ether in the MO and SMO extracts were 6.44, 0.088, and 0.174 and 3.95, 0.091, and 0.178 mg/g, respectively. The concentrated extract was redissolved in distilled water.μL of IS working solution and 400 μL of methanol were added to 100 μL plasma. The solution was vortexed for 10 min and then centrifuged at 12,000 rpm for 5 min. The supernatant was shifted to another centrifugation tube and dried with nitrogen at 37°C. The residue was redissolved in 100 μL acetonitrile and centrifuged for 5 min at 12,000 rpm. Five microliters of supernatant was drawn and analyzed by UPLC-MS/MS.10 μL of tissue homogenate, the tissue samples were prepared in the same way as the plasma samples.The whole tissue was cut into pieces and homogenized with ice-cold 0.9% (m/v) sodium chloride solution . Using 50 Six individual sources of blank plasma were used to measure the specificity of this analysis method. Meanwhile, we also analyzed the lower limit of quantification (LLOQ) of samples, the IS in blank plasma samples, and plasma samples within 2 h of being subjected to the MO extract (40 g/kg).X2) was applied to determine the regression equations. The lowest concentration in the calibration curve was determined as the LLOQ with signal-to-noise of ≥10.The peak-area ratio of the compounds to the IS was plotted to obtain the linearity. The weighted least-squares linear regression (weighting factor 1/n = 5) after one day and then for three days in a row, we obtained the intra- and interday precision, along with the accuracy data. The permitted range was within 15%.Through evaluating three levels of QC samples  freeze-thaw stability, (2) at room temperature for 4 h, and (3) put into the autosampler for 8 h, and (4) at −20°C for 30 days. The permitted range was within 15%.Three different QC sample concentrations were used for the recovery experiment. The absolute extraction recovery was measured as the ratio of the QC sample concentration extracted from the blank plasma/tissue to those in the QC samples.The peak areas of the biosamples extracted in blank plasma/tissue versus those dissolved in methanol solution were measured to obtain the matrix effect.The SD rats  were housed at 22 ± 2°C and fasted for 12 h with drinking water available ad libitum. MO and SMO ethanol extract were orally administered at a dose of 40 g/kg . Blood samples (0.5 mL) were collected from the venous plexus of the eye socket at 0.083, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 4.0, 6.0, 12.0, and 24 h under anesthesia. Then, the blood samples were centrifuged at 4500 rpm for 10 min, and then the plasma was immediately transferred to new tubes and stored at −80°C until analysis. The pharmacokinetic parameters were calculated using DAS software  based on noncompartmental method. All data were recorded as mean ± SD.n = 6), at 0.25, 1, 3, and 6 h, respectively. Heart, liver, spleen, stomach, kidney, brain, and small intestine tissues were extracted, washed with saline, blotted with filter paper, weighed, and then stored at −80°C.MO and SMO ethanol extract (40 g/kg) was orally administered to SD rats  and acetonitrile (containing 0.1% formic acid), the responses of the three components and two ISs were considerably better. To reach a better and more rapid separation effect, we optimized the gradient elution program.We carried out both positive and negative modes in mass spectrometry. Three components and the ISs all had high responses under the negative mode. The MS/MS transitions and parameters are given in Protein precipitation (PP) was used for the pretreatment of biological samples. We applied methanol and acetonitrile for PP. The samples had better peak shapes and recovery after being treated by acetonitrile, so we chose acetonitrile for further PP in this study.The MRM chromatograms of three components and the ISs are shown in r2 > 0.9907) in the linear ranges. The regression equations, correlation coefficients, linear ranges, and LLOQs of the three components in the plasma and tissues are listed in The internal standard method was applied for the establishment of calibration curves, which showed good linearity , we successfully determined the concentrations of monotropein, rubiadin, and rubiadin 1-methyl ether in rat plasma using the established method. Cmax in 0.5 h, whereas the Tmax of monotropein in the MO groups was 1.0 h, which indicates that monotropein was quickly adsorbed into the blood after oral administration. The Tmax for rubiadin was 1.5 h, longer than that for rubiadin 1-methyl ether, whereas the Cmax for rubiadin 1-methyl ether was the highest, especially in the SMO groups. In the plasma concentration-time curves of monotropein, an obvious double-peak phenomenon was found, which is related to enterohepatic recirculation. The pharmacokinetics properties shown in the present assay could be helpful for further studies on the pharmacokinetics of MO extract and further applications in different processing methods.The concentration of monotropein in SMO reached The concentrations of monotropein, rubiadin, and rubiadin 1-methyl ether in the liver, kidney, lung, and small intestine were determined at 0.25, 1, 3, and 6 h after administration. The results are shown in M. officinalis because of better bioavailability.In this study, we established an efficient and accurate UPLC-MS/MS method for the determination of three components from MO and SMO in plasma and tissues after oral administration in rats. Meanwhile, this is the first simultaneous determination of monotropein, rubiadin, and rubiadin-1-methyl ether in rat plasma and tissues. The study examined the pharmacokinetics and tissue distribution. The information found might partially illustrate their metabolic mechanisms in vivo, as well as provide a scientific basis for the strengthening of kidney-yang using the salt-processed principle of


Lamiaceae family is one of the most diverse and common plant families in terms of ethnomedicine due to their potential therapeutic effects. The aim of this study is to investigate the correlation between the chemical composition and the antibacterial effect of five essential oils from this family against five reference bacterial strains responsible of nosocomial diseases and foodborne illnesses.Rosmarinus officinalis, Thymus capitatus, Origanum majorana and Salvia officinalis were analyzed by GC/FID and GC-MS. Essential oils were evaluated for their antibacterial activities by disc diffusion and microbroth dilution methods against five reference bacterial strains: Pseudomonas aeruginosa, Escherichia coli, Salmonella enterica, Bacillus subtilis and Staphylococcus aureus. The inhibition zone diameter values and the twenty major compounds of the selected essential oils were subjected to PCA and HCA analysis.The commercial essential oils of Tunisian 1,8-cineole, α-thujone, α-terpineol and α-pinene. The PCA and the HCA of the selected essential oil components and the inhibition zone diameter (IZD) values identified four species groups and subgroups. Each essential oils group constituted a chemotype responsible for their bacterial inhibition ability. Thymus capitatus essential oil showed the strongest antibacterial activity with MBC ranging from 0.73 to 2.94 mg mL− 1.Analysis by GC/FID and GC/MS allowed the identification of ninety-one components representing 96.0 to 98.2% of the total oils. The different component contents varied according to the species. The main components were carvacrol, Rosmarinus officinalis, Thymus capitatus, Origanum majorana and Salvia officinalis essential oils have shown promising antibacterial activities against reference bacterial strains responsible for nosocomial diseases and foodborne illnesses. T. capitatus were used for many purposes such as unguents for embalming by Egyptians . Ho. HoO. maR. officinalis EOs were in accordance with previous studies reporting that 1,8-cineole (37.6 and 47.2%), camphor (7.1 and 13.3%) and α-pinene (7.0 and 19.4%) were the major components of R. officinalis EO [R. officinalis EO from Corsica and Sardinia [Results obtained for nalis EO , 44whileSardinia .T. capitatus EO antibacterial activity was even higher than that of the standard antibiotic Gentamicine®. The IZD of T. capitatus ranged from (15.0 ± 1.0 to 30.0 ± 1.0 mm) and MIC ranged from (0.73 to 2.94 mg/mL). In fact, T. capitatus EO was characterized by the most important mean percentage of the monoterpenoid phenol carvacrol (56.1 ± 3.0%), and important contents of hydrocarbon monoterpenes p-cymene (7.4 ± 0.8%) and ϒ-terpinene (10.8 ± 3.2%). Previous study proved that the thyme essential oil activity was mainly due to the presence of phenolic compounds and monoterpene hydrocarbons [The essential oils antibacterial activity showed considerable variation among the different Lamiaceae species and bacterial strains. ocarbons . It was ocarbons . IncreasActivity amongst the phenols and alcohols is at least partly due to the hydroxyl group, which has intrinsic antimicrobial activity and contributes to the relatively greater solubility of these components in biological membranes. Furthermore, the ability of components to release or accept protons has been postulated as an important factor in antimicrobial activity , 47, 48.p-Cymene, the third major element according to percentage, does not show antibacterial efficacy when used alone. Moreover, other studies [Another study attributed the activity of carvacrol to its hydroxyl group. In fact, they confirmed the importance of this group in term of activity in the phenolic structure when they compared carvacrol to its methyl ether  it was d studies , 51 haveO. majorana oil with MIC values ranging from 22.5 to 45.0 mg/mL. This EO contained a very lower content of carvacrol and higher mean percentage of p-cymene and ϒ-terpinene. O. majorana oil was also characterized by the highest amount of monoterpene hydrocarbons, and alcohol monoterpene α-terpineol. The combination of these results permitted as to deduce that the main antibacterial activity against the studied strains might be attributed to the richness of the essential oils in the phenol carvacrol.The activity was decreased with T. capitatus EO of P. aeruginosa, which is known to be a very resistant bacterium even to synthetic drugs, with MICs ranging from 0.73 to 2.94 mg/mL. This result confirm those found with EL Jalel et al. (2018) [It is interesting to note the susceptibility to the . (2018) .S. officinalis, R. officinalis1 and R. officinalis2 were placed in the same group in both antibacterial ACP and HCA analyses, but within chemical ACP and HCA analysis, they were classified in two different subgroups. S. officinalis was characterized by the most important amount in monoterpene ketones  while R. officinalis were characterized by the highest amounts of monoterpenes oxides (47.2 ± 0.5 and 37.6 ± 0.5%).1,8-cineole were the principle components responsible for the antibacterial activity against B. subtilis, E. coli and S. aureus [S. officinalis against E. coli, P. aeruginosa, B. subtilis and S. aureus to the presence of high concentrations of thujone, 1,8-cineole and camphor [These results suggested that the major components from these two chemical classes could be responsible for producing a similar inhibition. Our results were confirmed by Hammer et al. who demonstrated that camphor and . aureus . In the  camphor .R. officinalis EOs was noted to be active against all the bacterial strains but in different degrees. In fact, R. officinalis1 was more potent with MICs values ranging from 11.38 mg/mL to 22.75 mg/mL when R. officinalis2 MICs ranged from 45.00 mg/mL to 91.00 mg/mL. These results could be explained by the difference in the mean percentages of some components in these EOs. Actually, R. officinalis1 EO was richer in oxygenated monoterpene active compounds such as 1,8- cineole (47.2 ± 0.5% Vs. 37.6 ± 0.5%) and camphor (13.3 ± 2.9% Vs. 7.1 ± 1.3%). However, R. officinalis2, was characterized by higher amounts of hydrogenated monoterpene compounds, like α- pinene (19.4 ± 5.4% Vs. 7.0 ± 5.4%) and camphene (6.5 ± 1.6% Vs. 2.7 ± 1.4%). These finding confirm the results of Griffin et al. (1999) and Van de Vel et al. (2017). These authors suggested that the water solubility and the hydrogen bonding capacity are the main factors influencing MIC values of terpenoids against P. aeruginosa, S. aureus, and E. coli [1,8- cineole and camphor are distinguished by a higher water solubility and hydrogen bonding capacity than α- pinene and camphene. Thereby, we can confirm that the qualitative and quantitative differences between R. officinalis EOs chemical composition could affect their antibacterial activity [The  E. coli , 53, sinactivity .R. officinalis had a bacteriostatic effect on S. aureus [O. majorana had a bactericidal effect on E. coli and was bacteriostatic on S. aureus and B. subtilis [The MBC/MIC ratio ranged from 1 to 4 for all the EOs, indicating a bactericidal activity for these lamiaceae EOs against the tested bacterial strains. Other Moroccan studies showed that . aureus , whereassubtilis . The varsubtilis , 57 as wsubtilis .T. capitatus was the richest species in carvacrol, S. officinalis was the richest species in α-thujone, and R. officinalis1 was the richest species in 1,8-cineole. The EOs antibacterial activity varied significantly within species and within strains. In general, EOs have shown promising antibacterial activities we noted a pronounced activity of T. capitatus against P. aeruginosa. These results may represent useful and valuable leads for the development of new disinfectant products.The PCA and the HCA of the chemical composition of the studied Lamiaceae EOs separated them in four groups and sub-groups with different chemotypes.


Hypercalcemia of malignancy (HCM) is a serious metabolic complication, and the highest rates are in multiple myeloma (MM). The cause of hypercalcemia in newly diagnosed multiple myeloma (NDMM) remains unknown. We sought to evaluate the prognostic impact and mechanism of hypercalcemia in patients with symptomatic NDMM.We studied all consecutive MM patients who were initially diagnosed and followed up at Beijing Jishuitan Hospital between February 2013 and December 2019; 357 patients were included in the retrospective analysis.p < 0.05) based on univariate analysis, and it remained an independent poor prognostic factor  in a multivariate model that included age and R‐ISS stage.A total of 16.8% of MM patients presented with hypercalcemia at the time of MM diagnosis. The presence of hypercalcemia was associated with higher serum levels of β2 microglobulin, creatinine, phosphorus, uric acid, procollagen I N‐terminal peptide, β‐carboxy‐terminal cross‐linking telopeptide of type I collagen and osteocalcin, lower serum levels of hemoglobin, parathyroid hormone (PTH), and advanced ISS and R‐ISS stages. Multivariate analysis showed that serum PTH, hemoglobin, creatinine, and uric acid levels were the main factors affecting hypercalcemia. The presence of hypercalcemia was associated with significantly inferior survival (40 months vs 57 months, This study shows that hypercalcemia is associated with poor survival and is caused by manifold factors with humoral effects and local bone destruction. This study has shown that hypercalcemia is associated with poor survival and is caused by manifold factors with humoral effects and local bone destruction. The median follow‐up time was 24 months (5‐86 months), with follow‐up ending in April 2020. Hypercalcemia was defined as a serum calcium level >2.75 µmol/L and anemia as a hemoglobin level <10.0 g/dL. Renal failure was defined as creatine ≥177 µmol/L. Bone lesions were defined as one or more osteolytic lesions identified by skeletal radiography, computed tomography (CT), 18F‐fluorodeoxyglucose positron emission tomography with computed tomography (PET‐CT), or magnetic resonance imaging (MRI) according to the International Myeloma Working Group (IMWG) criteria.2.22, administered intravenously on days 1, 8, 15, and 22; dexamethasone 40 mg, administered intravenously on days 1 through 4, 8, 15, and 22; pegylated liposomal doxorubicin 25 mg/m2, administered intravenously on day 1. VTD: bortezomib 1.3 mg/m2, administered intravenously on days 1, 8, 15, and 22; dexamethasone 40 mg, administered intravenously on days 1 through 4, 8, 15, and 22; thalidomide 100 mg po qn; VCD: bortezomib 1.3 mg/m2, intravenously on days 1, 8, 15, and 22; CTX 300 mg/m2, intravenously on days 1, 8, and 15; dexamethasone 40 mg, on days 1‐4, 8, 15, and 22. TAD: thalidomide 100 mg po qn; pegylated liposomal doxorubicin 25 mg/m2 on day 1; dexamethasone 40 mg on days 1‐4, 8, 15, and 22). Patients who achieved PR underwent stem cell mobilization with cyclophosphamide and granulocyte colony‐stimulating factor, followed by high‐dose melphalan and ASCT. Consolidation therapy typically consists of two to four cycles of a repeated induction regimen, which aims to improve the degree of response after transplant. Lenalidomide maintenance was considered to be post‐ASCT for all patients.Patients up to 65 years of age and in good clinical condition received four cycles of bortezomib‐ or thalidomide‐based induction regimens, such as VAD, VTD, VCD, or TAD . The inter‐/intra‐assay coefficients of variation in plasma βCTX was between 0.010 ng/ml and 6.00 ng/ml. The assay range of plasma P1NP was 5 ng/ml to 1200 ng/ml, that of plasma osteocalcin was 0.500 ng/ml to 300 ng/ml, that of plasma vitamin D total was 3.00 ng/ml to 70.0 ng/ml, and that of plasma PTH was 1.20 ng/ml to 5000 ng/ml.2.4p values <0.05 were considered significant. Comparisons of categorical variables among different groups were carried out using the chi‐square test or Fisher's exact test when appropriate. Pearson correlation analysis was performed to assess linear correlation between two parameters. OS was measured from the date of treatment initiation until the date of death or the date of the last follow‐up. Time to event curves were plotted with the Kaplan–Meier method, and comparisons among groups were conducted using the log‐rank test. For multivariate analysis, factors associated with time to event were introduced into a Cox proportional hazards model. SPSS 18.0 software  was used for the statistical analysis. A p‐value of <0.05 was considered statistically significant.Differences between mean values were evaluated by both Student's t and Mann–Whitney tests, and 33.12 microglobulin, hemoglobin, creatinine, uric acid, phosphorus, ISS stage, and R‐ISS stage  (Tables The baseline characteristics of the patients with NDMM are described in Table ) Tables .3.2p = 0.035, data not shown).Serum levels of β‐CTX, tPINP, and osteocalcin (OC) were significantly elevated but PTH levels significantly decreased in patients with hypercalcemia Table . Serum a2D3, PTH, creatinine, and uric acid, laboratory parameters  were found to be the most important factors of hypercalcemia ia Table .p = 0.001, respectively  accounted for 35%, moderate hypercalcemia  for 46.7%, and severe hypercalcemia  for 18.3%. The degree of hypercalcemia was closely associated with renal failure, and the proportion of mild, moderate, and severe hypercalcemia patients with renal failure was 23.8%, 60.7%, and 81.8%, 3.3p = 0.019). We examined the prognostic impact of hypercalcemia in a multivariate model that also included established prognostic factors , and hypercalcemia remained an independent poor prognostic factor  .Early death occurred in 18.33% and 8.41% of patients with and without hypercalcemia, respectively (42D), which modulate calcium movement at the level of the bone, kidneys, and intestines.2D‐induced disorder are the two main reasons for HCM. In patients with localized, as well as generalized, myeloma bone disease, bone destruction, together with a lack of bone formation, results in excess bone resorption and causes excessive release of calcium, leading to hypercalcemia.The bone remodeling process, intestinal absorption, and renal tubule resorption are the three main metabolic pathways that contribute to maintaining the serum calcium concentration in a narrow physiological range. Hyper‐ or hypocalcemia occurs due to imbalances in these regulatory pathways. HCM is the most common cause of hypercalcemia, which is a potentially life‐threatening but relatively common clinical problem.2D3 and PTH levels were significantly decreased in patients with hypercalcemia compared with patients without hypercalcemia. Among the 357 patients with NDMM, 14.6% had renal failure, and 59.6% with renal failure had hypercalcemia. Serum levels of phosphorus, creatinine, and uric acid were significantly increased in patients with hypercalcemia compared with patients without hypercalcemia. PTH, hemoglobin, creatinine, and uric acid were the factors most closely related to hypercalcemia in multivariate analysis. Hypercalcemia may develop in patients with osteoporosis and treated with recombinant PTH (1‐84), and serum calcium correlates with urinary calcium excretion, serum ALP, and β‐CTX in these patients.Unfortunately, to date, there are no reports on the association between hypercalcemia and the amount or degree of bone damage in patients with MM. In our study, patients with bone destruction accounted for 91.2% of all patients; the vast majority of patients had more than 3‐5 lesions, though only 16.8% of patients had hypercalcemia. Thus, hypercalcemia mainly caused by localized bone disease could not well explain our clinical results. The presence of hypercalcemia was associated with higher serum levels of β‐CTX, which reflects bone resorption, or tPINP and OC, which reflect bone formation. Nonetheless, β‐CTX and OC were not significant factors in multivariate analysis. This result indicates that although local bone lesions are related to hypercalcemia, they are not the most important factors for hypercalcemia. We think that humoral hypercalcemia is still the main cause of hypercalcemia in MM. On the other hand, both 25‐(OH)p < 0.001, data not shown).In particular, kidney impairment accounted for 81.8% of patients with severe hypercalcemia , further indicating the important role of the kidneys in hypercalcemia. Due to the excessive deposition of immunoglobulin in patients with MM, renal tubular function is often impaired, which leads to increased calcium reabsorption by renal tubules. Subsequently, the ability of the kidney to effectively remove excessive calcium from the circulation is hampered, resulting in elevated serum calcium, which may lead to severe hypercalcemia and renal failure. In addition, hemoglobin had a faint effect on hypercalcemia, as shown in Table Overall, we think that although bone destruction is a characteristic change in MM, serum calcium is still regulated by internal secretion. Although the reasons for hypercalcemia are manifold, among them, humoral hypercalcemia may be highly important. Unfortunately, we only retrospectively analyzed bone turnover metabolism and biochemical markers and did not detect PTHrP. Despite the lack of PTHrP analysis, previous studies have confirmed that PTHrP produced by plasma cells in MM regulates their survival and pro‐osteoclast activity, promoting bone disease progression.2 microglobulin, ISS stage, and R‐ISS stage. Our results are consistent with those of many previous studies showing that hypercalcemia is a factor for a poor prognosis.The presence of hypercalcemia was strongly associated with features of advanced disease, such as βIn our patient population, hypercalcemia was associated with a significantly higher early mortality rate than no hypercalcemia, with rates of 18.33% vs 8.41%. This result is consistent with that reported by Zagouri et al.In our study, which was based on a single‐center database of consecutive, unselected patients, thalidomide‐containing or bortezomib‐based antimyeloma therapy was used in patients presenting with hypercalcemia. The symptoms and serum calcium level of all patients with hypercalcemia improved quickly within the first week after bisphosphonate application and chemotherapy. However, the use of these drugs did not alter the poor prognosis of patients with hypercalcemia.5Our study based on patients with symptomatic NDMM at a single center showed that hypercalcemia is associated with poor survival and is caused by manifold factors with humoral effects and local bone destruction.The authors have no conflicts of interest to disclose.L. B designed the study; Y.‐T.W. collected the data; L. B analyzed the data and wrote the manuscript. All authors contributed to the interpretation of the data, prepared the manuscript, and approved the final version.


Gardenia ternifolia is utilized in traditional medicine of Ethiopia for malaria treatment and possessing in vitro antimalarial activity. However, no in vivo study was conducted to substantiate the claim. The aim of this study was to judge the antimalarial activity of Gardenia ternifolia extract in vivo in Plasmodium berghei-infected mice. Plasmodium berghei was inoculated to healthy mice, and hydromethanolic crude extract and chloroform fraction of G. ternifolia leaves at 100 mg/kg/day, 200 mg/kg/day, and 400 mg/kg/day were administered. Percent parasitemia inhibition, percent change in bodyweight, hemoglobin level, and mean survival time were determined. Data were analyzed using one-way ANOVA followed by post hoc Tukey HSD test with IBM SPSS software version 20.0 statistical package and P < 0.05 considered as statistically significant. P < 0.001) compared to the negative control. Maximum effects on chemosuppressive, curative, prevention of weight loss, and reduction in hemoglobin were observed at higher doses of the hydromethanolic crude extract and chloroform fraction.  The chemosuppressive test of hydromethanolic crude extract at 100 mg/kg/day, 200 mg/kg/day, and 400 mg/kg/day ranged from 27.09% to 67.72%, and chloroform fraction had 35.21%–78.19% parasitemia suppression, respectively. For curative test on day 5, hydromethanolic crude extract at 100 mg/kg/day, 200 mg/kg/day, and 400 mg/kg/day ranged from 25.58% to 48.76%, chloroform fraction at 100 mg/kg/day, 200 mg/kg/day, and 400 mg/kg/day and chloroquine base at 10 mg/kg showed 46.36%–74.42% and 92.87% percent parasitemia inhibition, respectively, and also the results to both tests were highly significant (G. ternifolia leaves have shown promising antimalarial activity. The findings support the traditional claim of G. ternifolia leaves for malaria treatment; however, species variation could also limit such a straightforward extrapolation of the findings of this study in humans. From this study, hydromethanolic crude extract and chloroform fraction of  Plasmodium falciparum to the cheap and safe chloroquine (CQ) and to sulfadoxine-pyrimethamine (SP) caused a major drawback in effective malaria control in sub-Saharan Africa [Gardenia ternifolia is an evergreen small tree that grows to six meters tall [G. ternifolia (Rubiaceae) has been utilized by traditional healers to palliate malaria and its related fevers [Globally, in 2019, there were an estimated 229 million malaria cases of which an estimated 94% 215 million) cases occurred in Africa [n Africa . Gardeni million d fevers , 7. Fresd fevers . The macG. ternifolia showed IC50 values of 1.06 and 0.94 μg/ml against chloroquine-resistant Indochina (W2) and chloroquine sensitive to Sierra-Leone (D6) strains of Plasmodium falciparum [The crude acetone extract of lciparum .Gardenia ternifolia leaves in vitro and ethnobotanical studies showed antimalarial activity; no report is offered in the literature whether the leaves possess in vivo activity. This study was therefore planned to evaluate the efficacy as well as acute oral toxicity of G. ternifolia leaves used as malaria treatment in the Ethiopian folk medicine.G. ternifolia were collected on October 13, 2016, from Gondar Zuria district, Degola Chinchaye kebele, about 45 km south of the town, northwest Ethiopia. The collected material was wrapped and covered with plastic sheets during transportation. The specimen of the plant was authenticated as Gardenia ternifolia by a botanist (Getnet Chekole), in the Department of Biology, the University of Gondar, and was deposited in the herbarium with a voucher specimen (No. KA1784) for future reference.Fresh leaves of G. ternifolia were dried under shade, grounded to a course powder using hand compression, and stored in glass jars at room temperature until extraction. 400 g of G. ternifolia leaves powder was macerated in 2400 ml in 80% hydromethanol and shook frequently. After 72 hours of maceration, extraction was performed using thick layers of 40 mesh gauze and filtered with Whatman paper No. 1 twice, and the 2nd extraction was repeated after 72 hours of after maceration; the 3rd extraction was repeated 72 hours later (in a complete of nine days). The extracts were combined and concentrated in a dry oven at 40°C [The leaves of  at 40°C .The hydromethanolic crude extract was subjected to fractionation using solvents with differing polarity (chloroform and water). 40 g of hydromethanolic crude extract powder was suspended in a separatory funnel using 400 ml water and similar volume of chloroform was added and shaken to mix and left for 12 hours. The chloroform portion was powered in a clean beaker and concentrated in a dry oven at 40°C and stored in a refrigerator at 4°C until use , 12.G. ternifolia leaves using standard chemicals and procedures which were utilized in previous researches [The qualitative phytochemical screening tests were performed on 80% hydromethanolic crude extract of searches –21.Swiss albino mice, weighing within 20−28 g, 6–8 weeks old, and either sex, were purchased from the Ethiopian Public Health Institute (EPHI), Addis Ababa. Mice were kept in cages and housed in a standard animal house under a natural 12/12 h light-dark cycle at room temperature with free access to food and water. Before the experiment, they were acclimatized to the test environment for five days. The care and handling were consistent with the international guidelines for the employment and maintenance of experimental animals , 23.P. berghei, was obtained from EPHI in the Traditional Medicine Research Department, Addis Ababa, and transported by infecting the donor mice and maintaining the parasite until the actual procedure of the study.Chloroquine-sensitive malaria, G. ternifolia leaves at 2000 mg/kg was administered by the oral route. Strict observation was carried out for half an hour and periodically for the first 24 hours, for consecutive fourteen days. Observation was majorly focused on physical and behavioral changes of the mouse. But preliminary toxicity observation of the mouse did not show any sign of toxicity for four days. The remaining 4 mice were dosed by similar doses and followed for 14 days [Five female mice were kept for five days before dosing to acclimatize. One mouse was randomly taken to work out preliminary toxicity observation on free access to water but fasted for four hours and weighed and hydromethanolic crude extract of  14 days , 24.P. berghei infected mice (parasitemia of roughly 30%) were sacrificed by decapitation; then, blood was collected through cardiac puncture with a sterile disposable needle and syringe. The blood was diluted with sterile sodium chloride 0.9% in water in such a way that 0.2 ml of blood contained about 107 infected red blood cells (RBCs) and mice were infected by 0.2 ml blood suspension intraperitoneally expected to produce steadily rising consistent infection of the specified intensity in mice [Donor  in mice . Then, m in mice , 26 admiG. ternifolia leaves was distributed consistent with the strategy described by curative methods [7 infected erythrocytes were inoculated in mice intraperitoneally and randomly divided into their respective groups, weighed, and maintained on a pellet diet. Seventy-two hours later, they were dosed with hydromethanolic crude extract at 100 mg/kg/day, 200 mg/kg/day, and 400 mg/kg/day and chloroform fraction at 100 mg/kg/day, 200 mg/kg/day, and 400 mg/kg/day, chloroquine base at 10 mg/kg/day and solvent 10 ml/kg/day, which was prepared from 3 ml of 96% ethanol + 7 ml of Tween 80 + 90 ml of water once daily for five days. A Giemsa-stained thin blood film was prepared from the tail of each mouse daily for five days  to determine the parasitemia level. The mean survival time for each group was resolute arithmetically by calculating the mean survival time (days) of mice starting from the date of infection over a period of 30 days (D0-D29) [An evaluation of the curative potential of hydromethanolic crude extract and chloroform fraction of  methods . On Day (D0-D29) .On day 4 (96 hours), after infection, a drop of blood was collected from the mice by the vein section of the tail and transferred onto the edge of a microscopic slide and drawn evenly across the second slide to create a thin blood film and allowed to dry at room temperature, fixed with methanol, and stained with 10% Giemsa stain for quarter an hour. Slides were viewed using light microscopy with oil immersion (1000 x magnification). Percentage parasitemia was determined by counting the number of parasitized red blood cells (PRBCs) and non-infected red blood cell in random fields of the microscope. Five fields were counted and the parasitemia was calculated as the percentage of the total RBCs containing PRBCs .Percent parasitemia was calculated according to the following formula :(1)% parAverage percentage of parasitemia suppression was calculated as follows:G. ternifolia leaves prevented bodyweight loss. The weights were taken on day 0 (after an infection was initiated) and day 4 [The bodyweights of the mice were measured to observe whether the test extract of nd day 4 , 26:(3)bG. ternifolia leaves prevented anemia. Hemoglobin was evaluated by using hemoglobin machine and hemoglobin cuvette. From vein section of tail of mice, blood sample was collected by a trained medical laboratory technologist with optimum pressure applied on the tail and filled microcuvette (Hb 301) approximately 10 μl. Before inserting the microcuvette into the machine, excess blood was removed. The machine was run; hemoglobin level in g/dl was displayed automatically and recorded. Finally, mean hemoglobin level was determined [The hemoglobin level of the mice was measured to evaluate the effectiveness of hydromethanolic crude extract and chloroform fraction termined :(4)mean Any death was recorded within 28 days of the study period to evaluate the hydromethanolic crude extract and chloroform fraction effect for improvement in survival days. The mice were maintained with free access to food and water , 26:(5)mM ± SEM). Comparison of parasitemia and statistical significance resolved by one-way ANOVA descriptive statistics. Post hoc tests for multiple comparisons of Tukey HSD and paired t-test were employed to test significance for the difference between initial and final results within the identical and in between groups using IBM SPSS for window (Version 20.0) statistical package. All data were analyzed at a 95% confidence interval and P < 0.05 was considered statistically significant.The results of the study were expressed as the mean ± standard error of the mean (The yield and percent yield of hydromethanolic crude extract were found to be 42.6 g (10.65%) and stored in a refrigerator at 4°C until use.G. ternifolia leaves for the probable presence of different secondary metabolites is summarized in The preliminary phytochemical screening of 80% hydromethanolic crude extract of G. ternifolia leaves did not cause mortality of mice within 24 hours up to 2000 mg/kg bodyweight. Gross physical and behavioral observation of the experimental mice also revealed no visible signs of acute toxicity like lacrimation, hair erection, and reduction in their motor and feeding activities. They were physically active for 14 days after plant extract administration.The acute toxicity study indicated that the hydromethanolic crude extract of G. ternifolia leaves in P. berghei infected mice resulted in reduced parasite load as compared to their respective negative control group. Positive control groups treated with chloroquine base 10 mg/kg/day cleared the parasite on day 4 under identical conditions. Hydromethanolic crude extract at 100 mg/kg/day, 200 mg/kg/day, and 400 mg/kg/day showed 27.09, 44.72, and 67.72% parasitemia inhibition, respectively, and chloroform fraction at 100 mg/kg/day, 200 mg/kg/day, and 400 mg/kg/day showed 35.21, 63.95, and 78.19% parasitemia inhibition, respectively, and the results were highly significant (P < 0.001) compared to the negative control group at day 4  change in bodyweight, 200 mg/kg/day −7.85 ± 1.65% (P=0.118) change in bodyweight, 400 mg/kg/day −2.90 ± 1.82% (P < 0.001) change in bodyweight and chloroform fraction at 100 mg/kg/day −9.14 ± 0.88% (P=0.324) change in bodyweight, 200 mg/kg/day −5.95 ± 0.72% (P=0.017) change in bodyweight, 400 mg/kg/day −2.85 ± 1.37% (P < 0.001) of bodyweight and chloroquine base at 10 mg/kg/day gain 3.87% (P < 0.001) of bodyweight and mice treated with solvent 10 ml/kg/day loss 14.68% of bodyweight (see ight see .G. ternifolia leaves showed average hemoglobin levels at 100 mg/kg/day 7.57 ± 0.24 g/dl (P=0.668), 200 mg/kg/day 9.87 ± 0.40 g/dl (P < 0.001), 400 mg/kg/day 12.95 ± 0.54 g/dl (P < 0.001), chloroform fraction at 100 mg/kg/day 8.98 ± 0.32 g/dl (P < 0.001), 200 mg/kg/day 11.82 ± 0.13 g/dl (P < 0.001), 400 mg/kg/day 13.97 ± 0.40 g/dl (P < 0.001), chloroquine base at 10 mg/kg/day 14.15 ± 0.32 g/dl (P < 0.001) and non-infected mice 14.37 ± 0.24 g/dl (P < 0.001) compared to the negative control (6.55 ± 0.68 g/dl) (see The hydromethanolic crude extract of /dl) see .G. ternifolia leaves at 100 mg/kg/day, 200 mg/kg/day, and 400 mg/kg/day showed 6.5 ± 0.22 (P=1.000), 7.330.33 (P=0.999), 11.17 ± 0.60 (P=0.440), chloroform fraction at 100 mg/kg/day, 200 mg/kg/day, 400 mg/kg/day 7.33 ± 0.33 (P=0.999), 10.17 ± 0.70 (P=0.689), 14.17 ± 0.31(P=0.042) and chloroquine base at 10 mg/kg/day showed 24.00 ± 1.83 (P < 0.001) days of mean survival time in four-day suppressive test, respectively, and chloroform fraction at 400 mg/kg/day showed significant difference (P < 0.05) in survival days compared to negative controls and infected mice treated with chloroquine base at 10 mg/kg/day showed highly significant (P < 0.001) in survival days compared to negative controls (5.50 ± 0.43) (see Hydromethanolic crude extract of .43) see .G. ternifolia leaves at 100 mg/kg/day, 200 mg/kg/day, 400 mg/kg/day and chloroform fraction of G. ternifolia leaves at 100 mg/kg/day, 200 mg/kg/day, 400 mg/kg/day and chloroquine base 10 mg/kg/day in P. berghei infected mice at day 5 showed 25.58, 36.98, 48.76, 46.36, 62.87, 74.42, and 92.87 percent parasitemia inhibition, respectively , 8.33 ± 0.33 days, 10.33 ± 0.33 days, 9.00 ± 0.26 days, 11.83 ± 0.48 days, 14.50 ± 0.62 days and 30.00 ± 0.00 days of mean survival time in curative test, respectively, and were highly significant differences (p < 0.001) compared to negative control which was 5.33 ± 0.21 days  has been employed by traditional healers to palliate malaria and its related fevers [Plasmodium falciparum [G. ternifolia leaves were evaluated. The hydromethanolic crude extract yield of G. ternifolia leaves was 10.65% w/w which is not up to the G. ternifolia root bark (19% w/w) [G. ternifolia) contains considerable secondary bioactive constituents like alkaloids, anthraquinones, flavonoids, phenols, saponins, sterols, tannins, and terpenoids [G. ternifolia leaves have higher mean percent parasitemia inhibition, reduction in weight, prevention of anemia, and a rise in mean survival time in days in a dose-dependent manner. Alkaloids are attributed to their ability to intercalate with DNA and terminate division of cells [G. ternifolia leaves did not cause any overt morbidity and mortality in the experimental mice at 2,000 mg/kg oral administration. This showed that a median lethal dose of 50% (LD50) is larger than 2,000 mg/kg, which attests that plant products are frequently considered to be safe and have fewer adverse effects than synthetic ones [G. ternifolia leaves were investigated by evaluating the chemosuppression during early infection and curative tests. Then, determination of percent parasitemia inhibition and improved survival days are widely used parameters for evaluating of antiplasmodial activities [G. ternifolia leaves produced dose-dependent mean percent parasitemia inhibition and prolonged the survival days in P. berghei infected mice, the results recorded at the doses administered. Parasitemia level P. berghei infected mice treated with hydromethanolic crude extract and chloroform fraction of G. ternifolia leaves was due to a suppressive effect on the multiplication and erythrocyte infectivity of P. berghei parasites in mice. Chloroform fraction demonstrated dose-dependent higher activity in blood parasitemia inhibition in P. berghei than hydromethanolic crude extract. The relatively higher activity of the chloroform fraction might flow from/to secondary metabolites which can be partitioned during fractionation [G. ternifolia leaves prolonged the survival time of P. berghei infected mice dose dependently. This could be due to the presence of secondary metabolites that prevent the pathologic effect of the parasite in the infected mice [Currently, malaria management causes an unlimited threat due to resistance of plasmodium species to available drugs , 3. Therd fevers , 7. It id fevers , from thlciparum . The in 19% w/w) . The medrpenoids –21. The of cells . Flavonoof cells . Phenoliof cells . Tanninsof cells . The terof cells . Hydroettic ones , 23. Howtivities , 26. Chltivities , 26. In ionation –16. The ionation . The hydted mice –16, 29.G. ternifolia leaves showed significant mean percent parasitemia inhibition compared to the negative control. In vivo antiplasmodial activity is additionally classified as moderate, good, and extremely good if extract displayed percent parasite inhibition is ≥50% at a dose of 500 mg/kg/day, 250 mg/kg/day, and 100 mg/kg/day in a 4-day suppressive test, respectively [G. ternifolia leaves at 400 mg/kg/day showed 67.72%, and chloroform fraction at 400 and 200 mg/kg/day showed 78.19% and 63.95% parasitemia inhibition in P. berghei infected mice, respectively, and exhibited moderate and good antiplasmodial activity. A stronger percentage of parasitemia inhibition was observed in chloroform fraction than the hydromethanolic crude extract at 100 mg/kg/day, 200 mg/kg/day, and 400 mg/kg/day. These results indicated that compounds which have antimalarial activity were soluble in the solvent accustomed extract and fractionate the leaves of G. ternifolia for antimalarial activity. Weight is eminence of plasmodium infection in mice, indicating that a decent antimalarial agent should prevent weight reduction in Plasmodium infected mice. The dose-dependent suppressive activities of hydromethanolic crude extract and chloroform fraction G. ternifolia leaves propose that hydromethanolic crude extract and chloroform fraction have not got any toxic effects in experimental mice at the doses evaluated. In the curative test, the mean percent parasitemia was increased in the negative control from the 3rd day to the 5th day. In the Plasmodium berghei infected mice treated with hydromethanolic crude extract and chloroform fraction of G. ternifolia leaves, the mean percent parasitemia increased from day 3 to day 4 and showed a decrease in percent parasitemia from the day 5 to day 7 in a dose-dependent manner compared to the negative control. The infected mice treated with chloroquine base showed a decrease in average percent parasitemia from 3rd day to the 7th day.Comparing the mean survival time of chloroform fraction and hydromethanolic crude extract, mice treated with chloroform fraction showed higher mean survival time; this may be due to the feed intake depressing effect of metabolites and interfere with the protein digestion by secondary metabolites that may be absent in chloroform fraction . In compectively . SupportG. ternifolia leaves produced significant difference in percent hemoglobin level compared to the negative control. The hydromethanolic crude extract and chloroform fraction of G. ternifolia leaves at doses of 100, 200, and 400 mg/kg/day showed increased mean survival time with significant difference compared to the negative control and produced highly significant difference (P < 0.001) at 400 mg/kg/day compared to negative control. The promising result from the curative test suggests that the crude extract incorporates a therapeutic efficacy against established malarial parasitic infections.In the curative test, hydromethanolic crude extract and chloroform fraction of G. ternifolia leaves have in vivo anti-malarial activities. Chloroform fraction showed relatively better antimalarial effect in all parameters determined than hydromethanolic crude extract of G. ternifolia leaves. The dearth of acute toxicity and antimalarial activity upholds the earlier in vitro findings as well as the folkloric use in Ethiopian traditional medicine. However, species variation would also limit such a straightforward extrapolation of the findings of this study to humans.The hydromethanolic crude extract and chloroform fraction of P. falciparum infection are justifiable. Gardenia ternifolia is multipurpose which is employed for the building of homes, firewood, and animal feed and for treatment of various diseases in both humans and stock that the concerned bodies are advised to require measures to conserve it.Our study confirmed the claim in Ethiopian traditional medicine that the plant has therapeutic values in malarial infections. So, it needs further in-depth investigation by using primate and other models against


Aging Cell, 19, e13146. https://doi.org/10.1111/acel.13146Tzu‐Ting Huang, Hironori J. Matsuyama, Yuki Tsukada, Aakanksha Singhvi, Ru‐Ting Syu, Yun Lu, Shai Shaham, Ikue Mori, Chun‐Liang Pan. In the original publication of the article by Huang, T.T. et al. (2020), the authors would like to correct the following:1. In section 4.6, the definition of onset:Original:y(t) to reach the value of ymaxe from t = 0, where ymax is the maximal value of y(t), and e is Napier's constant.Onset of activation is defined as the time required for Correction:y(t) to reach the value of ymax2 from t = 0, where ymax is the maximal value of y(t).Onset of activation is defined as the time required for 2. In the legend of Figure 3c:Original:sl(t).(C) AFD operating range  defined by yCorrection:ysl(t).(C) AFD operating range (the temperature range between the onset and the end of the relaxation phase) defined by 3. Figure 4h, N numbers of D1 group in the Operating Range:Original:Correction:4. In the legend of Figure 1, the statistical test:Original:Tukey HSD multiple comparison test.one‐way  or two‐way ANOVA  followed by Correction:t test.one‐way ANOVA followed by Tukey HSD multiple comparison test. Unpaired 5. In the legend of Figure S3, the statistical test:Original:p < 0.05, two‐way ANOVA test followed by Tukey HSD multiple comparison test.*Correction:p < 0.05, one‐way ANOVA test followed by Tukey HSD multiple comparison test.*The authors apologize for the inconvenience caused for the readers.