Berchemia plants were important materials for Chinese traditional medicines due to their special secondary metabolites. Unlike the root, stem and leaf tissues, Berchemia floribunda  Brongn. fruit was lacked of systematic metabolic investigation. Biochemical analysis found that the total flavonoid and total phenolic content of Berchemia fruit pulp showed a peak value at red ripe stage, and then decreased, but the total anthocyanin content sharply increased along with the coloration. By widely targeted metabolomic analysis, 644 metabolites were identified and categorized into 23 groups mainly including flavonoid, organic acids, amino acids, lipids, phenylpropanoid, nucleotides, alkaloids, carbohydrates, alcohols, anthocyanins & proanthocyanidins, vitamins, terpenes, polyphenols, phenolamides, quinones, indole derivatives, and sterides. Among them, 111 metabolites and 123 metabolites respectively showed up‐ and down‐regulation from break stage to full mature. KEGG enrichment analysis indicated that active secondary metabolism such as biosynthesis of phenylpropanoids, flavonoid, and alkaloids happened during Berchemia fruit ripening. More importantly, Cyanidin‐3‐O‐galactoside and other 3 cyanidins were found to be the predominant pigments in mature Berchemia fruit and increased cyanidins and pelargonidins but decreased anthocyanins might be contributed to the purple pigmentation of Berchemia fruit. Interestingly, 29 pharmaceutical compounds previously reported in other Berchemia tissues were also detected in ripening Berchemia fruit pulp: 8 flavonoid, 2 quinones & sucrose showed up‐regulated accumulation while 6 polyphenols, 5 flavonoid, 3 phenylpropanoid, 2 organic acids, 1 quinones and β‐sitosterol showed down‐regulated accumulation In conclusion, our first comprehensive metabolic fingerprint will promote the further study of B. floribunda fruit and its medical and food application. O‐galactoside and other 3 cyanidins was the predominant pigments in mature Berchemia fruit and increased cyanidins and pelargonidins but decreased anthocyanin might result in purple pigmentation of Berchemia fruit.The first comprehensive metabolic fingerprint of B. floribunda fruit was drew by a targeted metabolome. Totally 644 metabolites including 23 groups such as flavonoid, organic acids, amino acids, lipids, phenylpropanoid, nucleotides, and alkaloids, etc, were identified. Active secondary metabolism such as biosynthesis of phenylpropanoids, flavonoid, and alkaloids happened during Berchemia fruit ripening. Cyanidin‐3‐ Leioclada, Berchemia floribunda  Brongn. And Berchemia lineata (L.) DC.). In addition, polyphenols such as gallocatechin, catechin, epigallocatechin, epicatechin, protocatechuic acid and protocatechuic acid O‐glucoside were found to be abundant in these Berchemia plants. Phenylpropanoid such as ferulic acid, vanillic acid, phillygenin, quinones such as emodin, chrysophanic acid, and aurantio‐obtusi, organic acids , and β‐sitosterol were also isolated from these Berchemia plants. Although more than 30 metabolites were isolated and investigated in the root, stem, vine, leaf and whole plant of Berchemia plants, limited information is reported about the chemical constituents of Berchemia fruits.The largest group of secondary metabolites found in Fragaria × ananassa)  Brongn. fruits from break stage (start coloring) and full‐mature stage by a widely targeted metabolomic method using HPLC‐ESI‐triple quadrupole‐linear ion trap. We further screened out and annotated the significantly differently accumulated metabolites (DAM) in Berchemia fruits during the ripening process. Our comprehensive metabolic fingerprint was expected to guide the maturity grading of Berchemia floribunda  Brongn. fruits and their further applications in food and pharmaceutical industry.In recent years, liquid chromatography–mass spectrometry (LC–MS)‐based metabolomics has been facilitated by the construction of MS2 spectral tag (MS2T) library from the total scan ESI MS/MS data, and the development of widely targeted metabolomic method using MS/MS data gathered from authentic standards  Brongn.) fruits were harvested from the mountainside at an altitude of 2000 m located in Danba town, Ganzi Tibetan Autonomous Prefecture, Sichuan Province, China. The harvested fruits were immediately taken to the laboratory and graded according to maturity and coloring stages: break (B), red ripe (RP), and full‐mature (FM) stage. After the removal of seeds, the pulp of fruit was immediately frozen in liquid nitrogen and stored at −80°C until be used.The Gou‐er‐cha . Ultrapure water was prepared by distilled water through a Milli‐Q A10 system (Millipore). Ethanol, Folin‐Ciocalteau reagent, Na2.3g for 5 min, the supernatant was transfered into a 10 ml volumetric flask. The residue was then extracted twice with 3 ml 80% ethanol as described above. The combined ethanolic extract in volumetric flask was adjusted to 10 ml using 80% ethanol. The ethanolic extract was stored at amber colored air‐tight containers at 4°C.The ethanolic extract used for determination of total phenolics and flavonoid contents were prepared as follows: the frozen sample was ground into powder in liquid nitrogen; 0.1 g powder was added into 3 ml 80% ethanol in 10 ml tuber and then extracted under a ultrasonication for 30 min (with a ice bath to cool); after a centrifugation at 5,000 2CO3 was added and fully mixed. The solution was adjusted to 25 ml using 80% ethanol and kept under dark for 30 min. The optical density of the blue‐colored samples was measured at 760 nm. The total phenolic contents were calculated according to the standard curve and expressed as mg gallic acid equivalent (GAE)/g fresh weight. The assay was subjected to three repeats.The total phenolic content (TPC) was determined by the Folin–Ciocalteu method . Before LC‐MS analysis, each sample was filtrated .The frozen pulp was crushed using a mixer mill  with a zirconia bead for 1.5 min at 30 Hz. Sample powder of 100 mg was weighted and extracted overnight at 4°C with 1.0 ml 70% aqueous methanol, vortexed for three times during the period to increase the extraction efficiency. After be centrifuged at 10,000 2.4.218, 100 mm × 2.1 mm i.d., 1.8 µm, Waters). Mobile phase was composed of phase A (ultrapure water containing 0.04% acetic acid) and phase B (acetonitrile containing 0.04% acetic acid). The elution program was performed as follows : , , , , . The flow rate was 0.40 ml/min, and the column temperature was kept at 40°C. The effluent was alternatively connected to the ESI‐triple quadrupole‐linear ion trap (Q‐TRAP)‐MS.After the filtering, 2 μl sample was injected and analyzed using an ultraperformance liquid chromatography  coupled with tandem ESI‐MS/MS . The UPLC conditions were performed according to a previous reported method  and the self‐built database MetWare database .The metabolites were quantified using multiple reaction monitoring (MRM) of triple quadrupole mass spectrometry. The ions corresponding to other molecular weight substances were excluded, and the precursor ions of the target substance were screened. Meanwhile, in the collision cell, the precursor ions were ionized to break and form fragment ions, and the characteristic fragment ions were selected by triple quadrupole filtration. This makes the quantitative results more accurate and repeatable |≧1 and p‐value < .05 (t tests) were screen out as DAMs. The annotation of all of the metabolites by KEGG database  were filtered out. The rest metabolites were used for Orthogonal Partial Least Squares‐Discriminant Analysis (OPLS‐DA) . Multiple comparisons were performed by One‐way ANOVA based on Duncan's multiple range tests, while paired‐samples 33.1Berchemia fruits showed a yellowish‐pink pigmentation at break stage and turned to be red at red ripe stage. It was very interesting to note that the fruit color further turned to be purple black at the full‐mature stage ; while 36 metabolites containing 8 organic acids and derivatives, 5 phenylpropanoids, 4 flavones, 4 others, 3 nucleotide and derivatives, 2 phenolamides, 2 polyphenols, 2 terpenes, 2 vitamins and derivatives, 1 isoflavone, 1 anthocyanin, 1 flavonoid and 1 alkaloid were undetected in two or three repeats of the full‐mature (FM) fruit pulp (named as UFM); One metabolite (DGMG (18:2) isomer 2) was undetected both in two repeats of the break fruit samples and two repeats of the full‐mature fruit samples (named as UBFM) ‐MS analysis was used to identify and quantify the metabolites in ) Figure . Thus, tBerchemia fruit was flavonoid which containing 95 flavones, 40 flavanols, 27 flavonoids, 20 flavanones, and 11 isoflavone. Moreover, 100 organic acids and derivatives, 78 amino acids and derivatives, 63 lipids, 58 phenylpropanoids, 49 nucleotide and derivates, 36 alkaloids, 19 carbohydrates, 18 alcohols, 16 anthocyanins, vitamins and derivatives, 14 terpenes, 13 polyphenols, 12 phenolamides, 7 quinones, 5 indole derivates, 5 sterides, and 3 proanthocyanidins were detected in Berchemia fruit pulp  < 0.05 were identified as the DAMs in Berchemia fruits from break to full‐mature stage  and significance difference analysis, the significantly differently accumulated metabolites (DAMs) were screened. The metabolites with variable importance in projection (VIP) value ≥ 1, fold change (FM vs. B) ≥ 2 or fold change (FM vs. B) ≤ 0.5, and e Figure . In detae Figure . The expe Figure .l‐tryptophan, methoxyindoleacetic acid, dimethylaniline, kynurenic acid, pantothenol, procyanidin B3, 3,4‐dihydroxy‐DL‐phenylalanine, procyanidin B2, xanthurenic acid, theobromine, l‐phenylalanine, epigallocatechin (EGC), cyanidin 3‐O‐malonylhexoside, l‐epicatechin, d‐(+)‐phenylalanine, 6‐methylmercaptopurine, l‐citrulline, and N‐feruloyl tryptamine.The top 20 significantly down‐regulated metabolites contained 6 amino acid and derivatives, 3 polyphenols, 2 organic acids and derivatives, 2 alkaloids, 2 proanthocyanidins, 1 alcohol, 1 anthocyanin, 1 nucleotide and derivates, 1 indole derivatives, and 1 others; these DAMs were respectively reduced glutathione, (+)‐gallocatechin (GC), O‐p‐coumaroyl shikimic acid O‐hexoside, LysoPE 14:0, 2‐Hydroxyisocaproic acid, (S)‐(‐)‐2‐hydroxyisocaproic acid, 2,6‐dimethyl‐7‐octene‐2,3,6‐triol, riboflavin, d‐erythro‐sphinganine, Di‐O‐methylquercetin, pelargonidin 3‐O‐beta‐d‐glucoside .The top 20 significantly up‐regulated metabolites contained 3 flavonols, 3 flavanones, 3 organic acid derivatives, 2 anthocyanins, 2 lipids, 1 amino acid, 1 flavonoid, 1 vitamin, 1 alcohol, 1 flavone, 1 phenolamides, and 1 Others. The highest up‐regulated metabolite was aromadedrin (dihydrokaempferol). The rest top up‐regulated DAMs were naringenin, phloretin, Gluconic acid, Pelargonidin, Eriocitrin, N‐Feruloyl spermidine, Fustin, LysoPE 14:0 (2n isomer), 5‐3.4Berchemia fruit. On the other hand, most of the DAMs of anthocyanins and all the DAMs of carbohydrates, isoflavone and lipids showed a down‐regulation from B to FM stage.The number and percentage of up‐regulated and down‐regulated DAMs in identified compounds were analyzed Table . More thp‐value < .05) into 34 pathways and the top 20 pathways including ABC transporters, aminoacyl‐tRNA biosynthesis, biosynthesis of alkaloids derived from histidine and purine, biosynthesis of alkaloids derived from shikimate pathway, biosynthesis of phenylpropanoids, biosynthesis of plant hormones, biosynthesis of secondary metabolites, flavonoid biosynthesis, galactose metabolism, metabolic pathways, phenylalanine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, phenylpropanoid biosynthesis, phosphotransferase system (PTS), tryptophan metabolism, biosynthesis of alkaloids derived from ornithine, lysine and nicotinic acid, benzoate degradation via hydroxylation, reductive carboxylate cycle (CO2 fixation), lysine biosynthesis and tyrosine metabolism  were able to be annotated by KEGG compound IDs. Moreover, 76 compounds from the 111 up‐regulated DAMs and 85 compounds from the 123 down‐regulated DAMs were respectively annotated by KEGG compound IDs. The DAMs were significantly enriched  into 14 pathways including flavonoid biosynthesis, galactose metabolism, metabolic pathways, phosphotransferase system (PTS), starch and sucrose metabolism, amino sugar and nucleotide sugar metabolism, purine metabolism, biosynthesis of phenylpropanoids, pentose phosphate pathway, biosynthesis of alkaloids derived from histidine and purine, benzoate degradation via hydroxylation, ABC transporters, and benzoate degradation via CoA ligation  into 19 pathways including biosynthesis of phenylpropanoids, tryptophan metabolism, phenylpropanoid biosynthesis, biosynthesis of alkaloids derived from shikimate pathway, aminoacyl‐tRNA biosynthesis, metabolic pathways, phenylalanine, tyrosine and tryptophan biosynthesis, flavonoid biosynthesis, biosynthesis of alkaloids derived from histidine and purine, biosynthesis of plant hormones, biosynthesis of secondary metabolites, biosynthesis of alkaloids derived from ornithine, lysine and nicotinic acid, nicotinate and nicotinamide metabolism, phenylalanine metabolism, tyrosine metabolism, cysteine and methionine metabolism, ABC transporters, 2,4‐Dichlorobenzoate degradation, and glucosinolate biosynthesis  and 3 proanthocyanidins  were detected in Berchemia fruits (Table 8–109) was cyanidin 3‐O‐galactoside, which was increased by 3.17 folds from B to FM stage. The abundance of cyanidin O‐syringic acid was also 108–109, which showed a non significant increase during the maturation process. Contrast with this, the level of other types of cyanidins  and cyanidin 3,5‐diglucoside) were significantly decreased. Interestingly, the level of pelargonidin and pelargonidin 3‐O‐β‐d‐glucoside increased by 33.41 and 13.64 folds, respectively, but the abundance of all of the 4 peonidins , peonidin and peonidin 3,5‐diglucoside chloride) decreased. The above results indicated that cyanidin‐3‐O‐galactoside and other cyanidins were the predominant pigments in mature Berchemia fruit and increased accumulation of cyanidins and pelargonidins but decreased level of other anthocyanins might be the metabolic basis for purple pigmentation of Berchemia fruit.Few large‐scale investigation of the pigments in the de Zhou, . The yel3.6Berchemia plant were flavonoid containing 1 flavone, 8 flavonols, 2 flavanones and 2 flavonoid , quercetin 3‐α‐arabinofuranoside, and kaempferol 3‐O‐glucoside were declined from break stage to full‐mature stage. Moreover, 6 polyphenols, 3 phenylpropanoids, 3 quinones, 2 organic acids and derivatives, 1 steride, and sucrose, which were identified in Berchemia leaves, wood, stem or root, were also detected in Berchemia fruit pulp. The above results indicated that the metabolic changes of Berchemia fruit during maturation might result in changes of pharmaceutical effect which need a further investigation.The largest class of DAMs was flavonoid including 7 flavanones, 24 flavones, 9 flavonoid, 10 flavonols, and 1 isoflavone. In previous works, the biggest group of medicinal metabolites reported in ge Table . Contras4Berchemia (Rhamnaceae) species, no comprehensive evaluation of the metabolites in Berchemia floribunda fruit has been reported. In the present study, a large‐scaled detection of metabolites in Berchemia floribunda fruit pulp were conducted by a widely targeted metabolic analysis. 730 metabolites were detected and 644 metabolites were identified in B. floribunda fruit pulp. The highest total flavonoid and total phenolic content in B. floribunda fruit pulp were found at red ripe stage but the total anthocyanin content was highest at full‐mature stage. During the ripening process, 123 metabolites were up‐regulated and 111 metabolites were down‐regulated, and the DAMs were enriched into biosynthesis of phenylpropanoids, flavonoid, and alkaloids. The unripe B. floribunda fruit showed relative high contents of the previous reported pharmaceutical compounds when compared to those in full‐mature fruits. More importantly, increased accumulation of cyanidins and pelargonidins but decreased level of other anthocyanins might be the metabolic basis for purple pigmentation of full‐mature Berchemia fruit. Thus, the unripe fruit should be used in medicine, while the full‐mature fruits were suggested to be used in food products. In summary, this study outlines the first comprehensive metabolic fingerprint of B. floribunda fruits which might benefit the further study of medicinal components and edible pigments of B. floribunda fruits.Although dozens of metabolites have been reported in leaves, stems, root, wood, bark and whole plant of The authors declare that they have no conflicts of interest.This study does not involve any human or animal testing.


Depodding of moringa which is still being carried out manually by removing with hand or by hitting a bag containing the pods is time-consuming, labour intensive and not economical. The demand for quality oil-bearing moringa seeds that have a wide area of industrial applications necessitates innovative deppoding techniques that will improve its market value. To ameliorate these problems, moringa depoddding machine has been developed but studies on performance evaluation and optimal parameter setting are sparsely reported. This study therefore, evaluated the effects of the processing factors (moisture content (MC) and speed of rotation (SR)) levels on the performance (throughput capacity (TP), effective throughput capacity (ETP), labour requirement (LR), depodding coefficient (DC), coefficient of wholeness (CW), depodding efficiency (DE), depodded kernel (DK), undepodded kernel (UK), small broken kernel (SBK), and big broken kernel (BBK)) of the designed and fabricated moringa depodding machine using the response surface methodology and test between subjects-effects. The experimental design used was a two factor, three levels i-optimal randomized design. Mathematical models relating the process factors to performance were developed. The predicted optimum results obtained were validated using the observed values of the experiment. MC and SR were found to have a significant effect on the performance of the machine. The predicted optimum performance of the machine were 113.73 kg/hr, 109.45 kg/hr, 0.85 man-hour required/Kg, 96.15 %, 0.96, 93.93 %, 0.98, 0.02, 10.64 %, and 1.24 % for TP, ETP, LR, DC, CW, DE, DK, UK, SBK, and BBK respectively at MC and SR of 10.10 % wet basis and 564 rpm. The experimental values at these processing conditions were close to the predicted optimum results obtained with little deviations which were statistically insignificant. The selected models sufficiently predicted the performance of the developed machine. Agriculture; Industrial engineering; Mechanical engineering; Performance efficiency; Moringa; Depodding; Response surface analysis; Speed of rotation; Moisture content Tial uses .Figure 1The process of depodding is a size reduction activity of breaking the case containing the seeds . Depoddiet al. [et al. [et al. [et al. [moringa oleifera seeds and some agricultural products using Response Surface Methodology (RSM), a statistical analysis tool in the modelling and optimization of more than two variables, to investigate the interactions between variables on selected responses have also been reported  for cocet al.  Agbetoye [et al.  Orhorhor [et al. ) for mel [et al.  for pea;nd Aremu , Fadele nd Aremu , Fakayod [et al. ; Fakayod [et al. , Fakayod [et al. ) for mor [et al. , Sobowal [et al. , Olayanj [et al.  for oranet al. [Moringa Oleifera Depodding Machine using a Response Surface Approach.However, the performance evaluation and optimization of depodding process of moringa to the best knowledge of the authors have not been reported. It was on this basis, Ikubanni et al.  designed22.1The moringa pods used for this experiment was harvested from the moringa tree domiciled in the Teaching and Research farm of Landmark University Throu2.5et al. [et al. [The RSM tool (Design-Expert version 12.0.1.0) was utilized for the experimental design, analyses, and generation of model equations that depicts the various performance of the developed moringa depodding machine. The predicted results were compared with the experimental results obtained as suggested by Fakayode et al. . The eff [et al.  and Fala [et al. . SPSS wi33.13.1.1o bar inclination but fluctuate using other bar inclination for an impact type moringa shelling device.The increase in the moisture content slightly decreased the TP from133 to 40 kg/hr and ETP from 120 to 40 kg/hr of the depodding machine a and b. 3.1.2et al. [et al. [et al. [It was observed that the increase in the speed of rotation increased the TP and ETP of the depodding machine to 133 kg/h and 120 kg/h respectively a and b. et al.  reported [et al.  also rep3.1.3et al. [The interactive effect of the moisture content and speed of rotation increased the TP from 40 to 118 kg/hr and ETP from 40 to 115 kg/hr of the moringa depodding machine since the speed of rotation had a higher significant effect on the TP than the moisture content a, b. It et al.  who repo3.23.2.1et al. [et al. [et al. [As indicated in et al. , it was  [et al. , it was  [et al.  for the  [et al.  reported [et al.  observed3.2.2et al. [et al. [et al. [et al. [et al. [et al. [et al. [et al. [It was observed that the increase in the rotational speed, increased DC of the un-dehulled moringa seeds to 100% b. Increaet al. , in whic [et al.  describe [et al.  revealed [et al.  for the  [et al.  revealed [et al.  also rep [et al.  for the  [et al.  reported [et al.  delineat [et al.  reported3.2.3et al. [et al. [et al. [The interactive effect of the moisture content and the speed of rotation revealed that increased moisture content with speed of rotation increased the depodding coefficient from 95 to 96 % b. A simiet al.  for the  [et al.  for the  [et al.  for the 3.33.3.1et al. [et al. [Increased moisture content increased the UK to 0.4 b. This met al.  increase [et al.  reported3.3.2et al. [Increased speed of rotation decreased the UK to 0 b. This met al.  increase3.3.3The interactive effect of moisture content and speed of rotation on the UK showed that a simultaneous increase in speed and moisture content decreased the UK from 0.1 to 0.01 b.3.43.4.1o cylinder bar inclination reduced with increased moisture content to18% but increased afterward for moringa in a shelling device. Falade and Aremu [From nd Aremu  revealed3.4.2et al. [Increased speed of rotation increased the SBK to 12% and the BBK to 1.6% a, b. At et al. , in whic3.4.3et al. [The interactive effects of the moisture content and the speed of rotation showed a simultaneous increase in moisture content with speed increased the BBK and the SBK. Similar results were also reported by Figueiredo et al. , in whic3.52) are the significant model terms, for DC, DE, SBK, and BBK  are the significant terms and for CW  are the significant terms . The speed of rotation was found to have the greatest effect on the responses as compared to the moisture content within the experiment conducted. The effect of moisture content and speed of rotation were quadratic for TP, ETP, LR, DC, DE, UK, SBK, BBK, but was 2FI for CW. From the optimization study, the optimal values for the performance of the moringa depodding machine were recorded at the moisture content (10.10% wet basis) and speed of rotation (564 rpm). The predicted values for TP, ETP, LR, DC, CW, DE, UK, SBK, and BBK were 113.73 kg/h, 109.45 kg/h, 0.85 man-hour required/Kg, 96.15%, 0.96, 93.93%, 0.02, 10.64%, and 1.24% respectively. The predicted values were in a reasonable agreement with the experimented values with very little deviation for all responses considered. The empirical models derived for the TP, ETP, LR, DC, CW, DE, UK, SBK, and BBK was considered to sufficiently relate the observations.Clement Adekunle Komolafe & Clinton Emeka Okonkwo: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.Peter Pelumi Ikubanni: Conceived and designed the experiments; Wrote the paper.Faith Olusola Ajao, Adewumi Samuel Alake & Tajudeen M. Adeniyi Olayanju: Performed the experiments; Wrote the paper.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors declare no conflict of interest.No additional information is available for this paper.


It showed that metabolomics method offer deeply insight into the therapeutic mechanisms of natural product.Chronic glomerulonephritis (CGN) as the culprit of kidney failure can increase the mortality of critically ill patients and seriously threatens people’s health all over the world. This study using metabolomics strategy is to reveal the potential therapeutic mechanism-related targets to evaluate the effects of rhein (RH) on CGN rats. Changes of serum metabolites and pathways were analyzed by non-targeted metabolomic method based on liquid chromatography-mass spectrometry (LC-MS) combined with ingenuity pathway analysis. In addition, the levels of biochemical indicators were also detected. A total of 25 potential biomarkers were identified to express serum metabolic turbulence in CGN animal model, and then 16 biomarkers were regulated by RH trending to the normal states. From metabolite enrichment and pathway analysis, pharmacological activity of RH on CGN were mainly involved in six vital metabolic pathways including phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, arachidonic acid metabolism, tricarboxylic acid cycle (TCA cycle), alanine, aspartate, and glutamate metabolism, arginine and proline metabolism. It suggested CGN treatment with RH, which may be mediated  CGN as a kind of auto-immunologically mediated glomerular injuries in chronic kidney disease is characterized by circulating inflammatory cells infiltration, glomerular cells proliferation, and extracellular matrix (ECM) accumulation . The patvia PI3K/Akt and Nrf2/HO-1 pathways  and formic acid (FA) was purchased from Fisher Scientific Corporation ; UPLC grade methanol (MeOH) was available from Merck . Distilled water for the solutions and mobile phase preparation was gained from Wahaha Group Co., Ltd. . Standard substance leucine enkephalin with the purity more than 99.2% and RH with the purity more than 98.7% were respectively obtained from Lonza  and Shanghai Chemical Reagent Co. . The chromatogram of RH was detected using HPLC : control group (Control), model group (Model), prednisone acetate tablets positive group (PA), and RH group (RH). The rats in model, PA, and RH group were injected with 2.5 ml C-BSA solution at bilateral armpits and groin, and then injected again 2.0 ml in the same way at the second week. The tail vein of animals were injected with 0.5 ml C-BSA solution at the third week, then was injected once every other day for a continuous period of 3 weeks. The control group was disposed by saline solution at the same day and place in the same way (Specified Pathogen Free (SPF) SD healthy male rats weighing 200 ± 20g in 8 weeks old were raised in laboratory animal center where indoor temperature was set at 22 ± 2°C and relative humidity was controlled at 50 ± 3% with 12 h light-dark cycle for 7 days adaptation. The experimental agreement ratified by the Animal Care and Use Committee of same way .via intragastric administration one time daily. The control and model group were intragastrically administered with distilled water daily for 6 weeks.From the first day of animals modeling, rats in PA group was administered orally prednisone acetate at a dose of 8 mg/kg per day, and the RH group were received 9 mg/ml RH solution The 24-h urine of rats in each group from 8:00 pm to 8:00 am was collected before and after treatment, and was measured by the Coomassie G-250 method, which the calculation formula of urinary protein content for 24 h is as follows: total quantity of urine protein for 24 h = urine protein concentration × urine output volume for 24 h.Orbital venous blood sample were collected before and after 12 h RH treatment. One hundred μl blood samples were respectively mixed with 10 μl of CD3+, CD4+, CD8+ monoclonal antibodies and 2 ml hemolysin, and then incubated at room temperature for 15 min in the dark. The mixture was centrifugated at 1,000 r/min, 4°C for 5 min and then gained supernatant was discarded. The residual liquid was added 2 ml PBS to resuspend the pellet, centrifuged at 1,000 r/min for 5 min. The supernatant was discarded again and added 2 ml resuspended cells for peripheral blood lymphocyte subsets analysis.After the rats were anesthetized, 8 ml blood sample was taken from the abdominal aorta, and then was allowed to stand at room temperature for 30 min until the serum was stratified. Subsequently, the sample was centrifuged at 3,000 r/min for 10 min at 4°C. The upper serum was used to detect the content SCr, BUN, TNF-α, IL-6, GSH, SOD, and MDA according to the ELISA kit instructions.The 0.1 g kidney tissue from each group was added 1 ml of pre-chilled Trizol for homogenization. The suspension was transferred to 1.5 ml EP tubes resting at room temperature for 5 min, and added 0.2 ml of chloroform, shaken vigorously for 15 s, and stand at room temperature for 2 min. The mixture was centrifuged at 12,000 r/min for 15 min at 4°C. The gained water phase was transferred into another EP tube, added respectively equal volume of isopropanol, 75% ice ethanol, and DEPC water solution to measure the OD value and estimate the purity of RNA that the value from 1.8 to 2.0 is used for reverse transcription. Ten μl of the cDNA obtained from the reverse transcription reaction system were stored in the refrigerator at −80°C until use.Before LC-MS analysis, blood sample collected from abdominal aorta in each group were placed 12 h under 4°C condition. Then, 1.0 ml sample was shifted into 5 ml centrifuge tube and mixed by chromatographically pure methanol in a ratio of 1:4. The mixture was vortexed for 60 s and centrifuged at 12,000 rpm for 12 min. One ml of supernatant was perked through the 0.22 μm filter membrane and push into the instrument for testing within several hours.Ten μl of each rat serum sample were added to 3,000 μl of the working solution to obtain a QC sample for ratifying and optimizing the chromatographic and MS condition. Ten characteristic peaks were selected for method validation assisted with dynamic background subtraction. For the successive operation of six replicates from the same QC samples, the relative standard deviation % (RSD%) calculation result of Rt and peak areas were respectively 0.79 and 2.10%, and those results were respectively 0.96 and 2.43% for six parallel samples in the repeatability evaluation.The chromatographic analytical procedures were employed on a Waters ACQUITY UPLC system equipped with binary pump, online degasser, auto plate-sampler, thermostatically controlled column compartment, and the analyte was separated by a Waters Acquity UPLC BEH C18 column  combined with a VanGuard Pre-Column precolumn , which the column temperature was set at 30°C. The binary gradient mobile phase system contains mobile phase A (water—0.1% formic acid) and mobile phase B (acetonitrile—0.1% formic acid), and the optimized procedure is described as follows: 0 to 1 min, 2 to 10% B; 1 to 2 min, 10 to 30% B; 2 to 7 min, 30∼50% B; 7∼9 min, 50∼80% B; 9∼10 min, 80∼100% B; 10∼11 min, 100∼100% B; 11∼13 min, 100∼2% B. The flow rate was 0.3 ml/min and the injection volume was 4 μl. The autosampler temperature was set 4°C.+ (556.2771) and [M - H]- (554.2614).Analytes were straightly imported into a Waters Micromass QTOF microt Synapt High Definition Mass Spectrometer  with an electrospray ion source (EIS) containing positive and negative ion mode. The main parameters were set as follows: ion source temperature is 600°C; in positive mode, source voltages was set 5,500 V, de-clustered voltage (DP) is set 80 V and collision energy (CE) is set 40 eV; in negative mode, source voltages are set 4,500 V, DP is set 60 V, and CE is set 30 eV. The MS existing 10 ppm mass errors possess a scan range of m/z (mass/charge ratio) 80–1,500 Da in centroid mode. As a reference to ensure accuracy and reproducibility of instrument, leucine enkaphalin in 0.2 ng/ml was calculated in [M+H]https://www.metaboanalyst.ca/) is used to establish interaction and pathway analysis based on differential metabolite to highlight the efficacy of RH on CGN rats. Statistical analysis was counted in the student’s t-test.The primitive MS spectra data were imported into Mass Hunter software in order to change into common data format, and then Progenisis QI 2.0 were applied to carry out noise filtering, peak detection, removal of isotope masses, and alignment of retention time (Rt) and mass (m/z). For refraining from the influence of concentration difference among samples, every ion intensity of each spectrum were normalized, and build a data matrix embodying the information of Rt, m/z value, and the normalized peak area. Multivariate analysis such as unsupervised principal component analysis (PCA), supervised orthogonal partial least squares discriminant analysis (OPLS-DA), S-score plot and variance importance for projection (VIP)-plot was performed in SIMCA-P14.1 software . The differential metabolites were selected by the criterion that VIP value ≥1.00 in VIP plot and p <0.05 in independent-sample t-test, and were identified by m/z, Rt, fragmentation patterns coupled with online databases such as HMDB, KEGG, Chemspider as well as LIPIDMAPS. Then, they are verified by the corresponding commercially available reference standards. MetaboAnalyst 4.0 , cyclic GMP, taurocholic acid, LysoPC(17:0) were increased compared with CGN rats. Hierarchical clustering analysis was employed to describe the relationships and differences among metabolites and samples as shown in From Pathway Analysis Features in MetaboAnalyst 4.0, CGN pathological changes are related to 25 differential metabolites, involving 20 metabolic pathways. Among them, the metabolic pathways with p value <0.1 including phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, arachidonic acid metabolism, pyruvate metabolism, citrate cycle (TCA cycle), arginine biosynthesis, alanine, aspartate, and glutamate metabolism, glycerophospholipid metabolism, arginine and proline metabolism, glycolysis/gluconeogenesis. After RH treatment, the levels of 16 metabolites were regulated by RH, involving 21 metabolic pathways. Among them, the metabolic pathways with p value <0.1 including phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, arachidonic acid metabolism, citrate cycle (TCA cycle), alanine, aspartate, and glutamate metabolism, arginine and proline metabolism  dialysis . Ornithi3-Hydroxyanthranilic acid (3-HAA) is a tryptophan metabolite with anti-inflammatory activity, which the immunoregulatory molecular mechanism of 3-HAA on macrophages is inhibiting the production of inflammatory mediators and reducing NF-κB activity. The results show that 3-HAA has an immunomodulatory effect, which may be due to the inhibition of PI3K/Akt/mTOR and NF-κB activation, thereby reducing the production of proinflammatory mediators . HydroxyPhenylalamine (PA) as an essential amino acid in the human body is involved in the formation of various protein components, but cannot be synthesized in the human body. Under normal circumstances, about 50% of the PA consumed is used to synthesize proteins of various components, and the rest is converted to tyrosine under the action of phenylalanine hydroxylase, and then converted into dopamine, epinephrine, norepinephrine, and melanin. When PA hydroxylase is lacking, these metabolites reach abnormally high levels and accumulate in tissues, plasma, and cerebrospinal fluid, which are excreted in large quantities from the urine . TaurochIn this study, a LC/MS-based serum metabolomics method has been employed to explore the metabolic changes of CGN rats in response to RH treatment for fully insight into the anti-inflammatory activity of RH and its action mechanism in molecule level. Six several metabolism pathways including Hphenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, arachidonic acid metabolism, citrate cycle (TCA cycle), alanine, aspartate and glutamate metabolism, arginine and proline metabolism were regulated after RH treatment, which 16 metabolites were involved such as isocitric acid, ornithine, 5’-methylthioadenosine, 3-hydroxyanthranilic acid, as well as citric acid. The action mechanisms of RH against CGN were may be through the regulation of inflammatory response, oxidative stress, kidney protective function, and the immune system response to achieve.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The animal study was reviewed and approved by Animal Care and Use Committee of Harbin Medical University.M-YZ conceived and designed the experiments. WYu, X-LM, WYa, and M-YZ performed the experiment. WYu and X-LM analyzed the data. WYu wrote the paper. All authors contributed to the article and approved the submitted version.This study was supported by grants from the National Natural Scientific Foundation of China (NO. 81772045), the Foundation of the First Affiliated Hospital of Harbin Medical University (NO.2017L004), and the Scientific Research Foundation of Heilongjiang Provincial Health Department (NO.2017-049).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.


Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, are responsible for a majority of all healthcare-acquired infections (HAI). The bacteria cause nosocomial infections in immunocompromised patients. Extracts from Callistemon viminalis have been shown to have antibacterial, antifungal, and anti-inflammatory activities. Tormentic acid congener, a pentacyclic triterpene saponin, was isolated from C. viminalis leaves. This study aimed to investigate the antibacterial effects of tormentic acid congener and leaf extracts on biofilm formation by A. baumannii, S. aureus, S. pyogenes, and P. aeruginosa. The antibacterial effects were determined by the microbroth dilution method, and ciprofloxacin was used as the standard antibacterial drug. Biofilm formation and detachment assays were performed using crystal violet staining. Production of extracellular polymeric DNA and polysaccharides from biofilms was also determined. Tormentic acid congener showed time-dependent antibacterial activity against P. aeruginosa with a MIC of 100 µg/ml and caused significant protein leakage. Antibacterial activity was found when tormentic acid congener was tested against both S. aureus and P. aeruginosa. The MICs were found to be 25 µg/ml and 12.5 µg/ml for P. aeruginosa and S. aureus cells, respectively. S. pyogenes was found to be susceptible to tormentic acid congener and the hydroethanolic extract with an MIC of 100 µg/ml and 25 µg/ml, respectively. A. baumannii was found not to be susceptible to the compound or the extracts. The compound and the extracts caused a significant decrease in the biofilm extracellular polysaccharide content of S. pyogenes. The extracts and tormentic acid congener caused detachment of biofilms and decreased the release of extracellular DNA and capsular polysaccharides from biofilms of P. aeruginosa and S. aureus. Tormentic acid congener and extracts, thus, have significant antibacterial and antibiofilm activities on these selected ESKAPE bacteria and can act as source lead compounds for the development of antibacterial triterpenoids.ESKAPE pathogens, namely,  Staphylococcus aureus (MRSA), and Gram-negative pathogens, Pseudomonas aeruginosa and Acinetobacter baumannii as well as extended spectrum-lactamase producing (RSBL) or carbapenem-resistant Enterobacteriaceae (CRE) were used [P. aeruginosa [ESKAPE pathogens are responsible for two-thirds of all healthcare-associated infections . The Infere used . Approxiruginosa . Nosocomruginosa .K. pneumoniae and P. aeruginosa have been found to cause life-threatening hospital infections in critically ill individuals [K. pneumoniae belongs to the family Enterobacteriaceae [K. pneumoniae naturally inhabits the gastrointestinal tract microbiome of healthy humans and animals [S. aureus is a Gram-positive bacterium carried in the nostrils of approximately 30% of the people [S. aureus does not cause any harm; however, in hospitalised immunocompromised people, it may cause infections. These bacteraemia invasions affect people with underlying lung disease, including those on mechanical ventilators and endocarditis, and this can lead to heart failure or stroke [S. pyogenes is a Gram-positive, nonmotile, nonspore forming, catalase-negative cocci that occur in pairs or chains [S. pyogenes infections cause pharyngitis and are responsible for up to 33% of diagnosed cases of sore throat in children and up to 10% in adults [A. baumannii has the ability of surviving for long periods on hospital surfaces and equipment. This is aided by its ability to develop resistance to multiple antibiotics [ividuals . These tividuals . K. pneueriaceae , and it eriaceae . K. pneu animals . S. auree people . In mostr stroke . S. pyogr chains . S. pyogn adults . ESKAPE n adults . A. baumibiotics  leading ibiotics .Biofilm formation is a bacterial strategy to survive under adverse conditions . BacteriCallistemon genus are known to possess antifungal, antioxidant, antithrombin, anti-inflammatory, antidiabetic, antibacterial, and herbicidal activities [Callistemon viminalis, also known as bottlebrush, is an ornamental plant which belongs to Myrtaceae family. Extracts from C. viminalis have been reported to have various medicinal properties including antibacterial, antifungal, and antioxidant activities [C. viminalis have shown antibacterial activity against S. aureus and E. coli with an inhibitory zone diameter about 16–20 mm [C. viminalis leaves has demonstrated the presence of phenolic, triterpenoid, flavonoid, saponin, steroid, alkaloid, tannin, carbohydrate, amino acid, and protein compounds [C. viminalis have shown antimicrobial properties for some bacteria [P. aeruginosa [K. pneumoniae [Plants of the tivities . Callisttivities . Compoun16–20 mm . Phytochompounds . Terpenobacteria . Triterpbacteria . Triterpbacteria . Severalruginosa . Severaleumoniae .C. viminalis, Sarcopoterium spinosum [Potentilla chinensis [C. viminalis, on K. pneumoniae, P. aeruginosa, S. aureus, S. pyogenes, and A. baumannii. In addition, we investigated the effects of tormentic acid congener on biofilm production of S. aureus, S. pyogenes, P. aeruginosa, and A. baumannii.Tormentic acid can be isolated from several plants which include spinosum , and Pothinensis  among otCallistemon viminalis crude extracts. Dimethyl sulfoxide (DMSO) was used for dissolving the extracts, ciprofloxacin (the standard antibiotic drug), and tormentic acid congener, and 3--2,5-diphenyltetrazolium bromide (MTT) was used as an indicator of cell viability after carrying out assays. The bacterial species P. aeruginosa (NCTC 10662), S. pyogenes, K. pneumoniae (ATC700603), A. baumannii (CECT(R) 911), and S. aureus (NCTC 6571) were obtained from Merck . Before resuscitation, the cells were kept in a −80°C freezer as 1 ml stock strains in 50% glycerol. Luria agar base, Miller, was used to plate cells on agar plates, and all the cells were cultured in Luria broth base, Miller. The tormentic acid congener used was isolated from Callistemon viminalis and characterised by NMR and mass spectrometry. All assays were performed in the biological safety cabinet Bioflow-II Labotec, Model 650. The incubator shaker, Model number S1-300 , and the incubator SI-300 were used for all incubations carried out. For analysis of cell viability using absorbance, a Tecan Genios Pro microplate reader  was used to record the results from a 96-well microwell plate. GraphPad Prism6  was used to record and analyse the results. For centrifugation, the centrifuge, Rotafix-32 Hettich Zentrifugen Microcent 94–2 Eppendorf centrifuge 541 was used.The chemicals and solvents used in this study were all obtained from Sigma-Aldrich Chemicals Company . Dichloromethane, ethanol, methanol, and water were used for extraction of Callistemon viminalis were collected in Harare, the University of Zimbabwe, 17.7840°S, 31.0530°E. The plants had been previously authenticated by a taxonomist from the National Herbarium and Botanic Gardens in Harare, Zimbabwe. The leaves of C. viminalis were separated from the branches and predried using a Labcon orbital incubator  at 60°C. The dried leaves were ground using a pestle and mortar to produce approximately 4 g of a powdered sample.The leaves of 2O. The powdered leaves and solvents were mixed in the ration 1 : 10, respectively. The leaves were left soaked in solvents for 3 days after which filtration was performed to obtain the extract. Filtration was done twice, first using cotton wool and using a filter paper thereafter. After filtration, the excess solvent was evaporated using a Buchi RII rotavapor , and the crude extract was dried under a fan.The leaves were washed with distilled water then dried in an oven  at 60°C. The dried leaves were pounded in a traditional mortar and pestle and sieved to obtain a powder. Two solvents were prepared, one with 50% v/v DCM: MeOH and the other one with 50% v/v EtOH:HThe leaf extract was run on a column with 100% hexane initially as the eluting solvent. Batch gradient system column elution was employed starting with a solvent of low polarity to that with high polarity. The batch gradient system was a 20-step gradient elution with a gradual increase of polarity with 100% ethyl acetate. Methanol was then added to 100% EA up to 90% EA and 10% methanol. Fractions of 250 ml were collected and concentrated using a Buchi RII rotary evaporator . Thin-layer chromatography was used for the analysis of the collected fractions. The fractions with similar retardation factor values were pooled. The pooled fractions were left to evaporate to dryness for crystals to form. Single spots observed on the developed chromatograms under UV (354 and 365 nm) after staining with sulphuring acid were deemed pure and were subjected to NMR analyses.1H NMR spectra were recorded at 400 MHz and 13C NMR spectra at 100 MHz. The chemical shifts for 1H NMR and 13C NMR were referenced to TMS via residual solvent signals . Two-dimensional (2D) NMR experiments were run using standard pulse sequences. Molecular formulae were determined by electrospray ionization with a 7T hybrid ion trap and a TOF detector running in a positive or negative mode. Tormentic acid congener was identified as a pure compound.2O leaf extracts of C. viminalis were investigated by adapting the microbroth dilution procedure used by Vipra et al. [2O leaf extracts of C. viminalis were dissolved to give a final concentration of 1% DMSO and 100 µg/ml. The resultant mixtures were serially diluted in a 2-fold manner with media containing 1% DMSO up to a minimum concentration of 12.5 µg/ml. For all experiments, bacterial cells exponentially growing were standardised using 0.5 McFarland's standard solution to give a cell suspension with a concentration of 1 × 106 CFU/ml.Bacteria were grown in Luria broth base, Miller (Sigma-Aldrich), supplemented by casein acid hydrolysate 10 g/L, yeast extract 5 g/L, and sodium chloride 5 g/L. The antibacterial effects of the compound tormentic acid congener as well as those for the DCM:MeOH and EtOH:Ha et al. . Brieflyµl. A positive control containing ciprofloxacin (highest concentration of 10 µg/ml) was set up in each well-containing media and bacterial growth culture. Relevant sterility and negative controls were set up containing media only and media with extract, respectively. Preincubation and postincubation cell density measurements were determined. After postincubation measurements, visualisation of viable cell growth on the plate was investigated by adding 20 µl of 3--2, 5-diphenyltetrazolium bromide (MTT) solution (1 mg/ml) to each well. The 96-well microplate was then covered and placed in an incubator for 2 hours.Tormentic acid congener, extracts, cells, and Luria broth were diluted into the 96-well microplate wells . Each well contained a final volume of 200 P. aeruginosa were performed in LB culture medium after carrying out serial dilutions starting from 100 to 12.5 µg/ml. After standardisation of cells using the 0.5 McFarland solution, ciprofloxacin was used as a control standard and untreated cells were used as a positive control. Aliquots of diluted cell suspension and tormentic were added to a microplate wells plate in equal volumes to produce a total volume of 200 µl. The microplate was incubated, and then, the absorbance was measured under suitable conditions for varied time intervals . The results of the absorbance representing cell densities were used to plot time-kill graphs for 100 µg/ml, 50 µg/ml, 25 µg/ml, 12.5 µg/ml, and the control. MTT was then added to the 96-well microplate to determine cell viability. The bactericidal effect was obtained from observing the graph with a lethality percentage of 90% for 6 hours, which is equivalent to 99.9% of lethality for 24 hours. This method was used to further confirm the MIC of tormentic acid congener on P. aeruginosa.The time-kill assays for tormentic acid congener on P. aeruginosa in consideration of their individual effects against this bacterium. This assay was aimed at determining and observing the presence of antagonism, synergism, and zero interaction between tormentic acid congener and ciprofloxacin [P. aeruginosa as shown by time-kill assays [µl of the inoculum was added to each well in a sterile 96-well microplate. Equal volumes of 50 µl tormentic acid congener and 50 µl ciprofloxacin were added to the wells. The plate was incubated at 37°C, and absorbance of the cells was measured at varied time intervals . The results of the absorbance were used to plot time-kill graphs for 100 µg/ml, 50 µg/ml, 25 µg/ml, 12.5 µg/ml, and control. The fractional inhibitory index (FICI) was calculated with the concentrations in the first nonturbid well found in each row and column of the microplate after extrapolating line graphs [The checkerboard assay was used to determine the effects of combining ciprofloxacin and tormentic acid congener on floxacin . Since tl assays  and to cP. aeruginosa cell suspension, a volume of 200 µl was subcultured in 200 ml of Luria broth base in a 1-litre container, and the cells were incubated overnight at 37°C at 100 rpm. The subcultured cells were centrifuged at 3500 rpm for 4 minutes, and the supernatant was removed. The pellet of P. aeruginosa cell suspension was diluted with 0.9% normal saline to produce absorbance of 1.5 OD using a centrifuge (Rotafix-32 Hettich Zentrifugen Microcent 94–2 Eppendorf centrifuge 541). To triplicates of labelled tubes A, B, C, D, and E, 6 ml of diluted cells was added. To tube A, 150 µl of tormentic acid congener was added to make a final concentration of 100 µg/ml. To tube B, 2.4 µl of ciprofloxacin was added to make up a final concentration of 0.16 µg/ml. To the control tube C, 1200 µl of SDS was added, to tube D, 180 µl of DMSO was added, and untreated cells were added to tube E. The tubes were centrifuged at 3500 rpm in the centrifuge for 4 minutes, and the pellets were discarded; the supernatants were used for protein determination assay. An exact volume of 150 µl of the supernatants was separately added to a labelled sterile 96-well plate. A calibration curve of concentration 0–50 µg/ml of the BSA stock was made. To each well, 150 µl of Bradford's reagent was added, and the optical densities of the wells were read at 590 nm using a Tecan Genios Pro microplate reader .From precultured µg/ml and 10 µg/ml for ciprofloxacin. After the incubation, 500 µl of antibacterial test solutions was added to the wells and left for further incubation for 72 hours at 37°C in a humid nonshaking incubator. After incubation, the contents of each well were gently discarded, and the plate was gently washed three times with 0.5 M phosphate buffer saline at pH 7.20. After washing, the plate was inverted on a paper towel for 15 minutes to drain off excess liquid, and the biofilms were subsequently fixed by incubating the plate at 60°C for 1 hour in an oven. After incubation, the biofilms were quantified as follows: 2.5 ml of 0.1% crystal violet was added to each well, the plate was further incubated at room temperature for 20 minutes before the crystal violet was discarded, and the plate was gently washed with sterile water. The plate was left to dry for overnight; then, 2.5 ml of absolute alcohol 99.9% was added to the wells of the plate. From each well, 200 µl was transferred to a 96-well plate, and optical density readings of the plate were measured at 590 nm using a Tecan Genios Pro microplate reader .For this assay, precultured cells were then centrifuged at 3500 rpm for 15 minutes in a Rotafix-32 Hettich Zentrifugen. The supernatant of the centrifuged cell culture was discarded, and 20 ml of 0.5 M phosphate-buffered saline prepared from sodium dihydrogen phosphate as the acidic salt and disodium hydrogen phosphate as the basic salt at pH 7.20 was used to wash the cells. The cell suspension was centrifuged again at 3500 rpm for 15 minutes, the supernatant was discarded, and 5 ml of sterile media was added and hand shaken to the dissolve the cell pellet in the media. A 2 ml volume of standardised cells was added to each well of a 24-well plate . Thereafter, the cells were allowed to adhere to the wells by incubating the cells at 37°C for 2 hours. During the incubation period, stock concentrations of test solutions were prepared to give a final concentration of 100 S. pyogenes and A. baumannii cells were centrifuged at 3500 rpm for 15 minutes. The supernatant of the centrifuged cell culture was discarded, and 20 ml of 0.5 M phosphate-buffered saline prepared from sodium dihydrogen phosphate as the acidic salt and disodium hydrogen phosphate as the basic salt at pH 7.20 was used to wash the cells. The cell suspension was centrifuged again at 3500 rpm for 15 minutes, the supernatant was discarded, and 5 ml of sterile media was added and hand shaken so as the dissolve the cell pellet in the media. A 2 ml volume of standardised cells were added to each well of a 24-well plate . The cells were allowed to adhere to the wells of a 24-well microplate by incubating the cells in a shaking incubator at 37°C for 2 hours. After 2 hours of incubation, the microplate is incubated in a nonshaking incubator for 72 hours. After incubation, 1.2 ml of 100 µg/ml of antibacterial test solutions and 1.2 ml of Tween 20 detergent solution were added to the plates. The plate was incubated in a nonshaking incubator, at 37°C for 2 hours; thereafter, the biofilm was quantified.Precultured S. pyogenes and P. aeruginosa cells were standardised and added as 4 ml volumes to 5 sterilised 50 ml centrifuge tubes with 100 µl of 100 µg/ml of C. viminalis extracts and tormentic acid congener as well as 0.64 µg/ml of the positive control, ciprofloxacin. The cells were further incubated in a shaking incubator at 37°C for 4 hours. After the incubation period, cells were separated by centrifugation at 4000 rpm for 15 minutes. The procedure was repeated thrice with chilled phosphate-buffered saline of pH 7.2. After centrifugation, the cells were suspended in 450 µl of autoclaved distilled water, and an equal volume of 450 µl of saturated phenol was added and then heated in a waterbath at 65°C for 20 minutes. For each sample, 300 µl was taken in triplicate and added to sterile Eppendorf tubes. To each tube, 150 µl chloroform was added, and the solution was mixed intensely by vortexing. The cell suspension mixture was then centrifuged, and 50 µl of the supernatant was collected and distributed into their respective wells of a flat-bottomed microplate. A standard curve, using 50 µl mannose at concentrations from 100 µg/ml to 6.30 µg/ml as well as a control with no mannose, was prepared. In each well, 150 µl of concentrated sulphuric acid and 30 µl of 5% phenol were also added, and the microtitre plate was put in a static waterbath of 90°C for 5 minutes. After heating, the plate was allowed to cool at room temperature for a further 5 minutes and wiped dry; absorbance was read at 492 nm using a Stat Fax 2 100 microplate reader .The extracellular polysaccharide of the extra polymeric substance was quantified using the phenol-sulphuric acid method. For the capsular polysaccharide analysis, precultured 2O extracts on extracellular DNA production in static biofilm formation was studied using a method developed by Hawser and Douglas [P. aeruginosa cells were centrifuged at 3500 rpm for 15 minutes. The pellet was washed using 0.5 M phosphate-buffered saline pH 7.2. The tormentic acid congener, DCM:MeOH extract, and EtOH:H2O extract were dissolved in 1% DMSO and Luria broth to give a final concentration of 12.5 µg/ml. Of the resulting solution, 500 µl was taken and added to the wells of a 24-well microplate. Positive control was set up using ciprofloxacin at a concentration of 0.80 µg/ml. Negative control of media and cells was also run in parallel.The effect of tormentic acid congener, DCM:MeOH extract, and EtOH:H Douglas . Briefly1H-NMR  δ (ppm): 5.14 , 3.49 , 2.74 , 2.12 , 2.00–1.20 (CH2 and CH region), 1.04 , 0.93 , 0.93 , 0.92 , 0.83 , 0.77 , 0.75 , and 0.72 . 13C NMR  δ (ppm): 178.7 (C-28), 138.7 (C-13), 124.9 (C-12), 82.7 (C-3), 70.2 (C-20), 67.6 (C-2), 55.2 (C-5), 52.8 (C-18), 47.5 (C-17), 47.5 (C-1), 47.4 (C-9), 46.4 (C-14), 40.5 (C-10), 39.4 (C-8), 39.3 (C-4), 38.9 (C-21), 38.8 (C-19), 36.8 (C-22), 33.0 (C-7), 30.6 (C-16), 29.3 (C-23), 27.9 (C-15), 23.7 (C-27), 23.4 (C-11), 21.5 (C-30), 18.6 (C-6), 17.6 (C-24), 17.5 (C-26), 17.4 (C-24), 16.9 (C-29), and 16.9 (C-25).The following spectral characteristics were obtained: white powder; +) at m/z 488, which agrees with the molecular formula C30H48O5. The 1H-NMR spectrum showed the presence of six singlet methyls and two doublet methyls, which were characteristic of the ursene skeleton, and exhibited signals of an olefinic proton (δ 5.14). The spectrum also showed a singlet at δ 3.49 (m) and two oxygen-bearing methine protons suggestive of the 2α, 3β, 19α-trihydroxy structure. These results indicated that the compound was an ursane-type triterpene. Furthermore, 13C NMR data substantiated the presence of a pair of olefinic carbons (δ 138.7 (C-13) and δ 124.9 (C-12)), a carboxylic acid group (δ 178.7 (C-28)), and three hydroxylated carbons (δ 82.7 (C-3), 70.2 (C-20), and 67.6 (C-2)) on the ursene structure. The structure was determined to be 2α, 3β, 20 β-trihydroxyurs-12-en-28-oic acid [The principle active showed the molecular ion , there was complete inhibition of the bacterial activity of P. aeruginosa.Another important finding was that all extracts of nganyama . P. aeruC. viminalis were also tested for antibacterial activity against S. pyogenes and A. baumannii. The 50% v/v EtOH:H2O extract of C. viminalis and tormentic acid congener proved were shown to have the most potent antibacterial effects against S. pyogenes at 100 µg/ml. Extracts of C. viminalis and tormentic acid congener did not have much inhibitory effects against A. baumannii, indicating intrinsic resistance of this bacterial strain. Antimicrobial resistance of A. baumannii is mainly due to reduced permeability of the outer membrane caused by loss or low porin expression, increased expression of multidrug efflux pumps, and mutations altering targets or different cellular functions [β-naphthoflavone on A. baumannii were also tested and were shown to have an inhibitory effect of 68%. Flavonoids are formed as antimicrobial barriers in plants response to microbial infection [S. pyogenes might be because of poor penetration to the cell membrane of the bacteria due to presence of a thick layer of peptidoglycan in their cell walls which act as a barrier. Tiwari and colleagues showed that herbal compounds are generally antibacterial agents but show better antimicrobial activity when used in synergy with other antibiotics [Extracts of unctions . The antnfection . Flavononfection . The pooibiotics .P. aeruginosa in relation to time. A range of five concentrations (100–0 µg/ml) were used, together with ciprofloxacin as a control. The antibacterial effect of tormentic acid congener on P. aeruginosa was shown to be time-dependent. At the highest concentration of tormentic acid congener 100 µg/ml, there was a complete inhibition of the bacterial activity of P. aeruginosa  was observed to be abundant in the biofilm matrix as shown in a study by Flemming et al. . A study species . Due to 2O extracts had any effect on the formation of biofilms in P. aeruginosa and S. aureus, the capsular polysaccharide production was quantified after exposure to these samples. The EtOH:H2O extract showed the most significant reduction in polysaccharide formation when compared to the reductions caused by tormentic acid congener and DCM:MeOH extract  which help them to bind the biofilm together in a matrix while anchoring it to surfaces . For thin et al. , it was a indica . Our resC. viminalis extracts had antibacterial activity against P. aeruginosa, S. aureus, S. pyogenes, K. pneumoniae, and A. baumannii. Extracts of C. viminalis reduced polysaccharide production and extracellular DNA production in P. aeruginosa and S. aureus biofilms. Tormentic acid congener caused significant cell lysis in P. aeruginosa cells. Exposure to tormentic acid congener produced detachment of S. pyogenes biofilms well as it reduced the extracellular polysaccharide content of S. pyogenes biofilms. However, tormentic acid congener and both C. viminalis extracts did not show significant antibacterial activity and antibiofilm formation activity against A. baumannii. Tormentic acid congener and extracts of C. viminalis, thus, had significant antibacterial and antibiofilm activities on selected ESKAPE bacteria and can act as source lead compounds for the development of antibacterial triterpenoids.


A mixture model on half‐maximal effective concentration identified a potential nonresponder group, with simulations suggesting that, in future, the model could be incorporated into a Bayesian therapeutic drug monitoring “dashboard” to individualize dosing and improve treatment outcomes.Variation in response to biologic therapy for inflammatory diseases, such as psoriasis, is partly driven by variation in drug exposure. Real‐world psoriasis data were used to develop a pharmacokinetic/pharmacodynamic (PK/PD) model for the first‐line therapeutic antibody ustekinumab. The impact of differing dosing strategies on response was explored. Data were collected from a UK prospective multicenter observational cohort ). Ustekinumab PKs were described with a linear one‐compartment model. A maximum effect (E WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?WHAT QUESTION DID THIS STUDY ADDRESS?☑ There is significant variation in response to biologic therapy in immune‐mediated diseases, some of which is driven by differences in drug exposure. Ustekinumab is a monoclonal antibody targeting the p40 subunit common to IL‐12/23, and is widely used in the treatment of psoriasis and inflammatory bowel disease. Studies investigating therapeutic drug monitoring and dose individualization for ustekinumab are limited.WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?☑ Can pharmacokinetic/pharmacodynamic (PK/PD) modeling define dose adjustments that will improve outcomes in patients with psoriasis treated with ustekinumab?HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?☑ Our PK/PD model reinforces findings from phase III clinical trials, and we additionally characterize a mixed distribution of half‐maximal effective concentration that could identify responder and nonresponder subgroups. Model simulations suggest that dose escalation/interval reduction may benefit partial responders.☑ Incorporating these findings into a Bayesian therapeutic monitoring algorithm could facilitate individualized ustekinumab dosing, including identifying nonresponders for early switching. These findings may be generalizable to other disease settings.The advent of biologic therapies means that complete disease remission is now achievable in patients with immune‐mediated inflammatory diseases, including psoriasis, rheumatoid arthritis, and inflammatory bowel disease. Nevertheless, poor response or loss of response remains a significant problem for many,Broadly speaking, TDM strategies advocate population‐based target trough concentrations for dose adjustment, using a reactive rather than proactive approach.Ustekinumab, a highly effective biologic targeting the IL23‐Th17 canonical pathway, is a fully human immunoglobulin G1 kappa monoclonal antibody binding to the p40 subunit shared by IL‐12 and IL‐23. Initially developed for psoriasisUstekinumab dosing for psoriasis comprises a fixed dose (45 mg/90 mg) stratified by bodyweight  given subcutaneously at week 0, week 4, and then 12‐weekly. Real‐world data show that those with higher baseline body mass index are less likely to respond,Psoriasis represents an ideal disease model to investigate the utility of TDM,As a first step to exploring the clinical potential of a Bayesian therapeutic monitoring algorithm for ustekinumab, we applied PK/PD modelling to this real‐world data set to investigate the relationship between ustekinumab exposure and treatment response, and compared our findings to published PK and PD models derived from phase III clinical trials.This study was conducted in the spirit of the 1996 International Conference on Harmonisation in Good Clinical Practice (ICH‐GCP) 1996, and in accordance with the 2008 Declaration of Helsinki. Two studies provided samples and data within the Psoriasis Stratification to Optimise Relevant Therapy (PSORT) consortium, aiming to understand determinants of response to biologics: BSTOP , and its nested study PSORTD . Written informed consent was obtained from all subjects prior to enrollment.n = 60) observational study, established in 2011 following a 2009 pilot, aiming to identify markers of outcomes to systemic therapies in psoriasis. All UK adults fulfilling BSTOP inclusion criteriaBSTOP is a prospective multicenter . In this pragmatic study, samples were not collected from every patient at every time point, and most were taken without reference to treatment administration . The ustekinumab assay was an enzyme‐linked immunosorbent assayPsoriasis severity was measured using PASI.A sequential population PK then PD model was developed using nonlinear mixed‐effects modeling in NONMEM (version 7.3).a priori with an exponent of 0.75 on clearance and 1 on volume.The PK structural model was one‐compartment with first‐order subcutaneous absorption and first‐order elimination (see max) model inhibiting the Kin parameter . Upon finding a possible bimodal distribution on half‐maximal effective concentration (EC50), a mixture model using the NONMEM $MIXTURE regime was used.A turnover model was used to describe PASI evolution over time (see P = 0.05) and backward elimination (P = 0.01) based on the likelihood ratio test.Baseline demographics , alcohol and smoking status, anthropometric measures (body mass index and waist circumference), psoriasis characteristics , and comorbidities were tested using the Stepwise Covariate Model‐building methodModel fit was assessed using plots of observations vs. model predictions, standardized residuals, likelihood‐based diagnostics , and assessment of model simulation properties via the visual predictive check (VPC).Guidelines suggest that ustekinumab is stopped if PASI75 has not been achieved by 16 weeks.To determine an early target trough concentration predictive of an 80% probability of longer‐term response (as defined by PASI75 at 6 months), 1,000 patients were simulated based on parameter estimates in the mixture 1 subpopulation. This was performed separately for both 45 mg and 90 mg doses on the standard regimen. Individual variability and significant PK covariates were taken into account during simulation. The trough concentrations at week 4  were then derived for an 80% probability of PASI75 at 6 months.Individual predictions of ustekinumab trough concentration at week 4 were plotted against the observed PASI change from baseline by 4 weeks for all patients to determine whether early response and trough concentration were related to 6‐month outcome.. Ustekinumab drug levels and ADA measurements were derived from 797 serum samples, and 1,590 PASI measurements were available over the first year of treatment. The percentages of subjects with 1/2/3/4/5 serum samples were 58.9/22.8/15.9/2.0/0.4, respectively. Of 491 patients, there were 239 responders, 107 partial‐responders, 131 nonresponders, and 14 with unknown response. Of note, the average number of serum samples per patient was similar between responders and nonresponders .Within a prospective multicenter observational study (60 dermatology centers across the United Kingdom), we identified 491 patients with psoriasis on ustekinumab monotherapy fulfilling our inclusion criteriaTableThe PK model included all patients’ PASI measurements, whereas the PD model included 348 patients and 1,136 PASI measurements. PD results from all 491 patients can be found in Table Baseline characteristics of the cohort Table  were conTable −1, 0.44 L/day, and 10.2 L) were similar to those reported in ustekinumab phase III development .As expected, ustekinumab PKs were linear,Figure Between‐subject variability (BSV) was 44.7% and 36.5% for clearance and volume of distribution, respectively. Relative SE values ranged from 6.7% to 42.7%. A VPC , whereas increased creatinine and biologic‐naïve status had the opposite effect (P < 0.01). Of note, when waist circumference was excluded from the model, diabetes status became a significant covariate . In patients with measurable ADA (n = 16), clearance was higher than in those with nonmeasurable ADA (P > 0.05) and increased with increasing ADA level  was 0.14 μg/mL, with large BSV (148.3%), which was not associated with any of the covariates using either manual or automatic selection methods. The BSV had an asymmetric bimodal distribution , whereas most nonresponders appeared in mixture group 2 (high EC50). There was an even proportion of partial responders in each group, but further $MIXTURE stratification into three subpopulations did not improve model fit.Comparing the mixture model classification with the actual observed response rate at 6 months, mixture group 1 comprised 68.6% who achieved PASI75 (full‐responders), 24.0% who achieved PASI50‐75 , and 7.4% who achieved PASI < 50 (nonresponders). By contrast, mixture group 2 comprised only 5.4% full‐responders, 23.2% partial‐responders, and 71.4% nonresponders. Most full‐responders, therefore, appeared in mixture group 1 , to explore the impact of alternative dosing regimens on response outcomes  suggested that the probability of achieving PASI75 dropped from near 75% to below 50% when the dosing interval changed from 12‐weekly to 16‐weekly, with a substantial decrease also seen in the 90 mg group  was identified as the median 4‐week drug concentration if the dose was adjusted to project an 80% response rate at 6 months for the mixture 1 subpopulation . We therefore divided simulated patients into four groups based on a trough concentration cutoff of 1.4 μg/mL (as above), with a pragmatically selected 4‐week PASI25 . The subsequent 6‐month response profiles for each group imply that 4‐week trough concentration could be combined with initial PASI trajectory to begin to stratify probable responder phenotype.In the subset of observed patients with PASI ≥ 10, individualized predictions of 4‐week ustekinumab trough concentration and 4‐week change from baseline PASI were evaluated with respect to three 6‐month responder categories: full (PASI75), partial (PASI50‐75), and nonresponder (PASI < 50). Responders tended to have higher trough concentrations and large initial PASI reduction from baseline, whereas nonresponders demonstrated low trough concentration and small initial PASI reduction  and patients failing to achieve PASI50 (nonresponders). Within the high EC50 group, there are likely to be a subgroup of true nonresponders for whom no amount of ustekinumab will be effective. However, our exploratory simulations suggest that dose escalation may improve probability of response toward 100% in patients already achieving at least PASI50‐75 , whereas this approach does not seem to substantially improve probability of response in nonresponders  was consistent for both (single and mixture) PD models  at week 4 indicates a higher likelihood that partial response would have been achieved without a dose increase than does a poor 4‐week response; our simulations show that a dose increase may “convert” such cases to full response. A poor early response with high 4‐week trough levels would indicate a group with a low probability of response, and may indicate a primary nonresponder group where therapy should be switched; certainly it would seem prudent to reassess PASI response in short order . Currently, serum ustekinumab concentrations are only sporadically measured in routine clinical practice, and clinical response is not evaluated as early as 4 weeks. Our recommendations point to a need for a change in practice, and should be evaluated in a prospective dose‐intervention study.In the future, similar models could be implemented into a Bayesian dashboard system, so that real‐time predictions of response could be used to inform dosing and treatment switching decisions.This work was funded by a Medical Research Council (MRC) Stratified Medicine award (MR/L011808/1). The Psoriasis Association (RG2/10), the NIHR Biomedical Research Centre at King's College London/Guy's and St. Thomas' NHS Foundation Trust, the NIHR Manchester Biomedical Research Centre, and the NIHR Newcastle Biomedical Research Centre. T.T. is supported by an MRC Clinical Research Training Fellowship (MR/R001839/1). N.D. is supported by Health Data Research UK (MR/S003126/1). N.J.R. is supported by the Newcastle MRC/EPSRC Molecular Pathology Node and the Newcastle NIHR Medtech and In vitro diagnostics Co‐operative. C.E.M.G. and N.J.R. are NIHR Senior Investigators. J.F.S. is supported by an MRC Fellowship (MR/M008665/1).http://www.psort.org.uk. All other authors declared no competing interests for this work.C.E.M.G. has received honoraria and/or research grant support (University of Manchester) from AbbVie, Almirall, Bristol Meyers Squibb, Celgene, GSK, Janssen, LEO Foundation, Lilly, Novartis, Pfizer, Sandoz, Sun Pharma, and UCB Pharma. N.J.R. has received honoraria, travel support, and/or research grants (Newcastle University) from AbbVie, Almirall, Celgene, Genentech, Janssen, Novartis, Pfizer, Sanofi Genzyme Regeneron, and UCB Pharma Ltd. J.B. has received honoraria, travel support, and/or research grants (King's College) from AbbVie, Pfizer, Novartis, Janssen, Roche, Regeneron, Lilly, UCB, Sun Pharma, Boehringer Ingelheim, and GSK. R.B.W. has received honoraria and/or research grants from AbbVie, Almirall, Amgen, Boehringer Ingelheim, Celgene, Janssen, Leo, Lilly, Novartis, Pfizer, Sanofi, Xenoport, and UCB. A.D.B. has received honoraria from AbbVie, Amgen, Boehringer Ingelheim, Celgene, Janssen, Leo, Lilly, Novartis, and Pfizer. T.R. has received honoraria for lectures from Pfizer, AbbVie, and Regeneron, and a research grant from Genmab. D.S. has received departmental research funding from AstraZeneca. C.S. has received departmental research funding from AbbVie, GSK, Pfizer, Novartis, Regeneron, and Roche. N.W. acts as statistician on a trial funded by AstraZeneca. The PSORT consortium has a number of industry partners; see S.P., T.T., N.D., J.F.S., and C.H.S. wrote the manuscript. C.H.S., J.F.S., and S.P. designed the research. All authors performed the research. S.P., T.T., N.D., J.F.S., and C.H.S. analyzed the data.Figures S1–S7.Table S1.Code S1, Code S2, Code S3.Click here for additional data file.


To evaluate the bleaching efficacy of at-home carbamide peroxide (CP) gels in two concentrations, containing or not a desensitizing agent. Forty incisors were divided into four groups (n=10), according to gel concentrations (10% or 22%), and presence or not of 3% potassium nitrate in the gel. A thin layer of gel was applied to the buccal surface of each tooth for 2h/day for 4 weeks. Bleaching efficacy was measured using a spectrophotometer, and ∆E*ab, ∆E00 and ∆WID were calculated. Measurements were performed at baseline, 7, 14, 21, 28, and 35 days following the first gel application. Data were analyzed by two-way RM-ANOVA and Tukey’s test (α=0.05). Regarding gel concentration or potassium nitrate inclusion, both gels resulted in color change above the perceptibility thresholds, which were similar between gels. Regarding time, significant differences were observed between color change values at 7 days and other time periods. ∆WID ranged from 3.8 to 9.6. Significant moderate to strong positive correlation was observed among the parameters. Nor the CP concentration, neither the inclusion of potassium nitrate in the gel, had influence on bleaching efficacy. All gels were effective and showed good results from the first weeks’ application. Key words:Tooth bleaching, carbamide peroxide, desensitizer, potassium nitrate, color. The demand for esthetic treatments is increasing among patients, and dental bleaching is a frequent esthetic procedure performed in clinical practice. At-home bleaching technique is effective, providing patient satisfaction with minimal long-term side effects . HydrogeHydrogen peroxide has low molecular weight, and ability to diffuse through the enamel and dentin and potentially reach the pulp, which may explain the frequent patient reports of tooth sensitivity during treatment . This shThus, this study aimed to evaluate the bleaching efficacy during the course of, and short-term after an at-home bleaching protocol using carbamide peroxide in two concentrations (10% and 22%), with or without the presence of a desensitizing agent (3% potassium nitrate) in the gels. The null hypotheses of the study were: 1) carbamide peroxide concentration would not influence the efficacy of at-home bleaching, 2) presence of potassium nitrate in the gel would not influence the efficacy of at-home bleaching.Forty bovine incisors free of enamel defects or evident discoloration were firstly disinfected in chloramine solution (1%) for 24h. Then the teeth were stored refrigerated (4°C) immersed in distilled water, which was changed weekly, until the beginning of the experiments. Prior to bleaching procedures, the teeth were cleaned with pumice and randomly divided into four groups (n = 10), according to the carbamide peroxide concentration (10% or 22%), and presence or not of 3% potassium nitrate (desensitizing agent) in the bleaching gel. The experimental gels were produced by a pharmaceutical laboratory and stored refrigerated during the course of the experiments.To standardize the area where the color readings were carried out, a 6 x 6 mm squared shallow groove was created with a spherical diamond bur  on the center of the buccal surface of each tooth. The baseline color readings were conducted immediately before the first bleaching application.Before gel application, the teeth were gently dried with absorbent paper to remove excess moisture. An even 2 mm thick layer of bleaching gel was applied to the buccal enamel surface. The teeth were kept in 100% humidity at 37°C for 2 hours in contact with the bleaching gel. This procedure was repeated daily for 4 weeks. After each application, the teeth were washed thoroughly under tap water for complete removal of the gel, followed by storage immersed in distilled water at 37°C. After the last application of the bleaching gel, the specimens were treated with topic fluoride gel.The bleaching efficacy of the four gels was evaluated by using an objective color measurement with a spectrophotometer . All color readings were performed within the previously defined area on the center of the buccal surface of the teeth. A research laboratory was chosen for the procedures, having standard temperature, humidity, and illumination conditions. Color readings were conducted at baseline (prior to bleaching), and then repeated at 7, 14, 21, 28, and 35 days after the first bleaching application.For the spectrophotometric evaluation, the specimens were gently dried with absorbent paper, and placed on a flat surface with a standard white background. The spectrophotometer was calibrated before the readings, and was always positioned at a 90° angle to the surface. The CIEL* a* b* tridimensional color space was used, where L* indicates luminosity axis (L*=0 is black and L*=100 is white), a* represents the greenness (-a*) and redness (+a*) axis, and b* represents the blueness (-b*) and yellowness +b* axis. Color stability was assessed by calculating the color difference between each time interval and baseline. Both the equations ∆E*ab Eq. 1) and the ∆E00 (Eq. 2) were used in this study :  in CIEDE2000. RT is a rotation function that accounts for the interaction between chroma and hue differences in the blue region. The weighting functions SL, SC, and SH adjust the total color difference for variation in the location of the color difference pair at the L*, a*, and b* coordinates, and the parametric factors kL, kC, and kH are correction terms for experimental conditions. In the present study, kL, kC, and kH were set to 1.The 50:50% acceptability thresholds for CIEL* a* b* (∆E*ab) and CIEDE2000 (∆E00) were 2.7 and 1.8, respectively .The CIEL* a* b*color space-based whitening index (WID) was calculated for each assessment time according to the formula : Fig. 33.The ∆WID was calculated by the difference between the indices at baseline and each time interval (∆WID=|∆WID2-∆WID1|). The whiteness difference threshold for acceptable bleaching effect was 2.60 WID units .The effectiveness of tooth whitening was determined based on the interpretation of visual thresholds, adopting a classification system from 1 to 5, derived from research results on PT (perceptibility threshold) and AT (acceptability threshold) . In thisThe color difference data were analyzed by three-way RM-ANOVA and Tukey’s test. The correlations among ∆E*ab, ∆E00 and ∆WID were determined by Pearson’s correlation coefficient. All analyses were performed with a 5% significance level.The means and standard deviations for ∆E*ab, ∆E00, ∆WID are shown in Tables 1 to 3, respectively.p = 0.04) and time (p <0.001), and for the double interactions of concentration of gel*potassium nitrate inclusion (p = 0.007) and concentration of gel*time (p = 0.04). Gel concentration (p = 0.91) and the other double and triple interactions were not statistically significant (p > 0.05).For ∆E*ab, statistically significant differences were observed for inclusion of potassium nitrate  were similar to those for 22% (7.3 ± 2.5). For the inclusion of potassium nitrate, pooled mean ∆E*ab data showed that gels with potassium nitrate (6.7 ± 2.4) resulted in statistically lower ∆E*ab values than those without potassium nitrate (7.8 ± 2.6). Regarding time, pooled mean ∆E*ab values were: 7 days (5.9 ± 2.7), 14 days (7.4 ± 2.3), 21 days (7.7 ± 2.1), 28 days (7.9 ± 2.5), and 35 days (7.4 ± 2.5). Significant difference was observed between ∆E*ab values at 7 days and other evaluation time periods, which presented statistically similar ∆E*ab.p < 0.001) and for the double interaction of concentration of gel*potassium nitrate inclusion (p = 0.001). Gel concentration (p = 0.81), potassium nitrate inclusion (p = 0.19) and the other double and triple interactions were not statistically significant (p > 0.05).For ∆E00, statistically significant differences were observed for time  were similar to those for 22% (4.3 ± 1.4). For the inclusion of potassium nitrate, pooled mean ∆E00 data showed that gels with potassium nitrate (4.2 ± 1.5) resulted in statistically similar values than those without potassium nitrate (4.6 ± 1.4). Regarding time, pooled mean ∆E00 values were: 7 days (3.5± 1.6), 14 days (4.4 ± 1.4), 21 days (4.6 ± 1.2), 28 days (4.7 ± 1.5), and 35 days (4.6 ± 1.3). Significant difference was observed between ∆E00 values at 7 days and other evaluation time periods, which were statistically similar.All groups had ∆E*ab and ∆E00 greater than the 50:50% acceptability thresholds at all evaluated time points. Regardless of gel concentration and presence of potassium nitrate, at-home bleaching was effective in all groups from the first week of application, and best results were attained after 14 days, and maintained in the subsequent times , Table 2p > 0.05). ∆WID ranged from 3.8 to 9.6, and was above the whiteness difference threshold for accepTable bleaching effect (2.60 units) in all groups.For ∆WID all individual factors, double and triple interactions were not statistically significant (p < 0.01). Significant moderate positive correlations were observed between ∆E*ab and ∆WID values  and between ∆E00 and ∆WID values , , Fig. .in vitro and clinical investigations, showing that gels with low peroxide concentrations are as effective as gels with high concentrations  to evaluate bleaching efficacy, all based on objective and standardized color readings, using a spectrophotometer to determine CIEL* a* b* coordinates . Visual ∆E*ab is one of the most used parameters for dental color evaluation . However∆E values do not show the direction of the color change, so it is usually not possible to know for sure if the bleaching procedure was effective or not, with increased ∆L*, without analyzing the individual coordinates. In bleaching studies, in addition to color change, it is important to evaluate a specific parameter for determining the whiteness (level of white). Many whiteness indexes have been described, but recently a new customized CIEL* a* b*-based whiteness index for dentistry (WID) was proposed . It has In the present study, bleaching effect was detected as early as the first week, and the results remained sTable from 14 to 35 days. In agreement with these findings, most clinical at-home bleaching protocols recommend a treatment period of 2 to 4 weeks, avoiding prolonged treatment periods, since saturation is attained and no major damage to the tooth structure occurs. In order to thoroughly evaluate bleaching efficacy, it is important to consider the changes in delta values for each individual color coordinate, along with the color difference values (∆E*ab or ∆E00). In the present study, ∆L* reveled a positive variation in all groups and times, indicating that the teeth became lighter , with ∆L* ranging from 1.3  to 6.2 . All groups also presented a coherent variation in ∆b*towards negative values, ranging from -1.3  to -6.6 , indicating less yellowish and more bluish colors.It can be concluded that the carbamide peroxide concentration and inclusion of potassium nitrate in the gel did not influence at-home bleaching efficacy. All gels evaluated in this study were effective and showed good bleaching results from the first weeks’ application.