Gnidia glauca and Plumbago zeylanica. Aqueous extracts of flower, leaf, and stem of G. glauca and leaves of P. zeylanica were prepared which could effectively reduce Cu2+ ions to CuNPs within 5 h at 100°C which were further characterized using UV-visible spectroscopy, field emission scanning electron microscopy, high-resolution transmission electron microscopy, energy dispersive spectroscopy, dynamic light scattering, X-ray diffraction, and Fourier-transform infrared spectroscopy. Further, the CuNPs were checked for antidiabetic activity using porcine pancreatic α-amylase and α-glucosidase inhibition followed by evaluation of mechanism using circular dichroism spectroscopy. CuNPs were found to be predominantly spherical in nature with a diameter ranging from 1 to 5 nm. The phenolics and flavonoids in the extracts might play a critical role in the synthesis and stabilization process. Significant change in the peak at ∼1095 cm−1 corresponding to C-O-C bond in ether was observed. CuNPs could inhibit porcine pancreatic α-amylase up to 30% to 50%, while they exhibited a more significant inhibition of α-glucosidase from 70% to 88%. The mechanism of enzyme inhibition was attributed due to the conformational change owing to drastic alteration of secondary structure by CuNPs. This is the first study of its kind that provides a strong scientific rationale that phytogenic CuNPs synthesized using G. glauca and P. zeylanica can be considered to develop candidate antidiabetic nanomedicine.Rapid, eco-friendly, and cost-effective one-pot synthesis of copper nanoparticles is reported here using medicinal plants like  Dioscorea bulbifera, Dioscorea oppositifolia, Gloriosa superba, Barleria prionitis, and Litchi chinensis are used for synthesis of gold, silver, platinum, and palladium nanoparticles with antimicrobial, antibiofilm, and anticancer activities [Nature has an infinite collection of medicinal plants which serve as repository of bioactive principles that are considered as complementary and alternative medicine. Combinatorial chemistry, nanotechnology, and cutting edge research on nutraceuticals have helped to expand the horizons beyond contemporary therapeutics , 2. Intetivities –12. Howetivities , 14. Alttivities . Hence, Gnidia glauca is also known to be of utmost medicinal importance as it is used for treatment of cancers, burns, wounds, abdominal pain, snake bites, and sore throat. Similarly, leaves are applied to treat back ache, joint aches, contusions, and swellings [Plumbago zeylanica, has exhibited carminative, anthelmintic, anti-inflammatory, antiplasmodial, antimicrobial, antifungal, antihyperglycemic, hypolipidaemic, and antiatherosclerotic activities [P. zeylanica which are caustic, vesicant, and aphrodisiac [G. glauca and P. zeylanica can be used for synthesis of metal nanoparticles since they are treasure house of both reducing as well as capping agents. However, there are no reports of synthesis and therapeutic applications of CuNPs from G. glauca or P. zeylanica. Hereby, there is a huge scope to design novel routes for synthesis of therapeutically active CuNPs using both the plants.wellings , 17. Anotivities . Furthertivities . It is rtivities . Alkaloirodisiac . Thus, fG. glauca and P. zeylanica followed by characterization and evaluation of antidiabetic and antioxidant activity.Development of antidiabetic nanomedicine is one of the thrust areas of nanotechnology as it is estimated that, by 2030, diabetes mellitus-afflicted population will shoot up to 366 million. Type II diabetes mellitus (T2DM) is the most prevalent ailment both globally as well as in the Indian subcontinent. Hereby, there is a continuous need to develop and screen novel antidiabetic agents that can target elevated postprandial hyperglycemia . Herein,α-Glucosidase and 4-nitrophenyl α-D-glucopyranoside were obtained from Sigma Aldrich, USA. DNSA  was obtained from SRL Pvt. Ltd. . Copper sulphate, dipotassium hydrogen phosphate (K2HPO4), potassium dihydrogen phosphate (KH2PO4), methanol, sodium potassium tartarate, and sodium hydroxide (NaOH) were procured from Qualigens, Mumbai, India. Porcine pancreatic α-amylase and sodium chloride (NaCl) were obtained from HiMedia Laboratories, Mumbai, India. Acarbose was obtained from Bayer Pharmaceuticals Pvt. Ltd. . All the chemicals and reagents procured were of AR grade.G. glauca flowers, leaves, and stems and P. zeylanica leaves were collected from the Western Ghats of Maharashtra and shade-dried for 2-3 days at room temperature. The dried plant materials were reduced to fine powder using an electric blender. G. glauca flower extract (GGFE), leaf extract (GGLE), and stem extract (GGSE) were prepared by adding 5 g of the powdered plant material in 100 mL distilled water in a 250 mL Erlenmeyer flask, followed by boiling at 100°C for 5 min. Similarly, P. zeylanica leaf extract (PZLE) was prepared. After filtering the extract through a Whatman No.1 filter paper, the filtrate was collected and stored at 4°C for further use [ther use .4·5H2O solution and incubated in darkness at 100°C. UV-visible spectra were recorded at regular intervals on a spectrophotometer  operated at a resolution of 1 nm; also, visible colour change was monitored to confirm the reduction of Cu2+ ions to CuNPs. Bioreduced CuNPs were characterized by employing a field emission scanning electron microscope (FESEM), high-resolution transmission electron microscope (HRTEM), energy dispersive spectroscopy (EDS), dynamic light scattering (DLS), X-ray diffraction (XRD), and Fourier-transform infrared spectroscopy (FTIR) as per our earlier reports [Synthesis of CuNPs was initiated by addition of 5 ml of GGFE, GGLE, GGSE, and PZLE separately to 95 ml of 1 mM aqueous CuSO reports .α-amylase inhibitory activity was checked using the chromogenic 3,5-dinitrosalicylic acid (DNSA) method as per our earlier report [µg/mL CuNPs and porcine pancreatic α-amylase (50 µg mL−1) were mixed and incubated for 10 min at 37°C. 1% starch was used as substrate which was added thereafter. DNSA assay was used to estimate the reducing sugar by recording the absorbance at 540 nm. Inhibitory activity was calculated by using the following formula:In order to study the antidiabetic activity of the bioreduced CuNPs, r report . In shorα-glucosidase activity in presence of CuNPs was checked by mixing 100 µL of α-glucosidase (0.1 unit/mL) with 200 µL of CuNPs (100 µg/mL) followed by incubation for 1 h at 37°C [p-nitrophenyl-α-D-glucopyranoside in 100 mM phosphate buffer of pH 6.8 was added to initiate the enzyme activity which was incubated for 10 min at 37°C and eventually stopped by addition of 2 mL Na2CO3 (0.1 M). Absorbance of the p-nitrophenol released from pNPG was recorded at 420 nm and percentage of glucosidase inhibition was evaluated using the following formula:Inhibition of α-amylase and α-glucosidase separately at 37°C followed to which CD spectra were recorded on a Jasco J-1500 spectropolarimeter at a scan speed of 40 nm/min with a response time of 1 s and a slit width of 1 nm, as reported earlier. The measurements were recorded in a range from 190 to 300 nm in a Quartz cell of 2 mm path length using a reaction mixture comprising 0.1 unit/mL of enzyme and CuNPs in phosphate-buffered saline [CuNPs were incubated with porcine pancreatic d saline .Synthesis of CuNPs was confirmed by the visible colour change from pale blue to yellow and finally to dark brown. The intensity of the spectra increased up to 5 h followed by which no significant increase was noticed which indicated the completion of the bioreduction process in 5 h. A similar pattern in the enhancement of the spectral intensity was observed in all the cases where CuNPs were synthesized using GGFE, GGLE, GGSE, and PZLE at 100°C . This syMorphological features of the bioreduced CuNPs were evaluated using HRTEM analysis as FESEM failed to show high-resolution images since the CuNPs were very small in size . CuNPs w−1 which is attributed to the hydroxyl group in alcoholic and phenolic compounds. However, no significant change was observed in the peaks after synthesis of CuNPs. The other significant and predominant peaks which were noticed before synthesis were not much altered after synthesis. The peaks at ∼1215 and ∼1624, 1365–1370, and ∼1740 cm−1 can be attributed to the unassigned amide mode, CH3 bend, and stretching of C=O bond, respectively. Remarkable change was observed in the peak at ∼1095 cm−1, which corresponds to C-O-C bond in ether. In case of GGFE, this peak was diminished significantly, while in rest of the extracts, there was notable change in the peak, after synthesis. This suggests that C-O-C bond is utilized during conversion of the Cu2+ to CuNPs. Moreover, minor change was also observed in the amide bond intensity, located at ∼1624 in case of GGSE, indicating its role in CuNPs. No prominent change in the rest of the peaks indicates that the corresponding functional groups help in stabilizing the as synthesized CuNPs.The role of the extracts and the corresponding functional groups towards synthesis and stabilization of CuNPs was studied by recording the FTIR spectra of GGFE, GGLE, GGSE, and PZLE before and after synthesis of CuNPs . The plaα-amylase (α-amylase up to 50.99 ± 4.27% followed by GGFE-CuNPs that showed 50.01 ± 4.19%. CuNPs synthesized using GGSE (32.36 ± 2.71%) and PZLE (33.34 ± 2.79%) showed an inhibition equivalent to the standard drug acarbose (35.30 ± 2.95%).Bioreduced CuNPs showed promising inhibition against porcine pancreatic -amylase . Among tα-glucosidase inhibition up to 88.60 ± 0.78% followed by GGLE-synthesized CuNPs (86.58 ± 3.26%). Standard drug acarbose also showed similar inhibitory potential (81.09 ± 2.82%). CuNPs synthsized by GGFE and PZLE showed comparatively lower inhibitory potential up to 76.20 ± 1.14% and 73.29 ± 0.96%, respectively  to 70°C. Moreover, increase in the reaction temperature evidently led to the synthesis of larger quantities of nanoparticles and, simultaneously, reduction in the size of the nanoparticles [Metal nanoparticles have got wide applications in optoelectronics, semiconductors, sensors, and biomedical applications as well. Medicinal plants are widely explored to synthesize metal nanoparticles. In this study, we found that nd AgNPs –26. Howe extract . In our  extract , 17, 19. extract . Intensi extract . It can  extract . Likewiscapacity –33. High15–30 nm . It is iectively , 35. Thearticles .G. glauca is reported to have potent antioxidant activity owing to its high phenolic and flavonoid content [P. zeylanica has high concentration of phenolics, flavonoids, reducing sugar, citric acid, and plumbagin which might play a significant role in the process of bioreduction and capping [Synthesis and stabilization of the CuNPs by the both the plants may be attributed due to their rich phytochemistry.  content . Althoug content . In our  capping . FTIR anα-amylase and α-glucosidase, which are key enzymes of carbohydrate metabolism. The variation in the extent of inhibition may be attributed due to the variation in size and shape. This fact may be evident from the observation where GGLE-synthesized CuNPs showed the highest inhibition of porcine pancreatic α-amylase while GGSE-synthesized CuNPs exhibited highest α-glucosidase inhibition. Type II diabetes mellitus (T2DM) is associated with postprandial hyperglycemia which can be effectively controlled using α-amylase and α-glucosidase inhibitors. However, the adverse effects associated with the available drugs like biguanides, thiazolidinediones, sulphonylureas, meglitinides, and α-glucosidase inhibitors in addition to insulin includes hepatotoxicity, abdominal pain, flatulence, diarrhoea, and hypoglycaemia [α-amylase and α-glucosidase may be the most predominant mechanism of inhibition. Hereby, these phytogenic CuNPs may serve as novel complementary and alternative antidiabetic nanomedicine for the effective treatment and management of T2DM. Our findings are in close agreement with the previous reports where CuNPs and associated copper complexes were proved to be antidiabetic in nature. Recently, our findings about CuNPs, synthesized by D. bulbifera tuber extract, B. prionitis leaf extract, L. chinensis peel extract, and P. orientalis leaf extract, have shown spectacular success towards considering phytogenic CuNPs as potential candidate for designing antidiabetic nanomedicine [Calotropis procera L. latex is reported to produce highly biocompatible CuNPs which did not show any toxicity even at a concentration as high as 120 µM which substantiates the biocompatibility of phytogenic CuNPs [The phytogenic CuNPs could efficiently inhibit both lycaemia , 39–42. medicine . Similaric CuNPs . The yieic CuNPs .G. glauca- and P. zeylanica-mediated synthesis of CuNPs may prove to be a novel, rapid, and efficient route to fabricate spherical nanoparicles of smaller dimensions. The extracts rich in diverse phytochemicals not only bioreduce but also stabilize the nanoparticles as well. Elevated temperature was found to be suitable for synthesis. Phenolics and flavonoids might play a key role in the synthesis process. CuNPs could effectively inhibit α-amylase and α-glucosidase. The mechanism of enzyme inhibition was established to be alteration of the secondary structures in the enzyme leading to conformational change. In view of the background, it can be concluded that phytogenic CuNPs reported herein may lead to development of environmentally benign route for rational designing of safe and effective antidiabetic nanomedicine.


Scientists search for potential antimicrobial sources that can cope with antibiotic resistance. Plants used in traditional medicine can be identified as potential candidates for the synthesis of novel drug compounds to act against antibiotic-resistant bacteria.  Antibiotic resistance is one of the greatest threats of the 21 To determine the potential antimicrobial effects of ethanol, aqueous, and hexane extracts of five Sri Lankan medicinal plants against four human pathogens. Asparagus falcatus (tubers), Asteracantha longifolia (whole plant), Vetiveria zizanioides (roots), Epaltes divaricata (whole plant), and Coriandrum sativum (seeds) were used in the study. Plant extracts were screened against four clinically important Gram-positive and Gram-negative bacterial strains, Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Klebsiella pneumoniae (ATCC 700603). Antibacterial activity of plant extracts were monitored using the agar disc diffusion method. Eight concentrations of each positive plant extract were used to determine the minimum inhibitory concentration (MIC) by 5-fold dilution of plant extracts yielding a serial dilution of the original extract. E. divaricata gave the maximum zones of inhibition of 16.3 mm, 7.4 mm, and 13.7 mm and MIC values of 0.48 mg/ml, 1.2 mg/ml, and 1.6 mg/ml, respectively, against S. aureus. Ethanol and hexane extracts of V. zizanioides gave the maximum zones of inhibition of 12.1 mm and 11.4 mm and MIC values 2.4 mg/ml and 0.003 mg/ml, respectively, against S. aureus. None of the other plants were effective against any microorganism used for the study.  Ethanol, aqueous, and hexane extracts of E. divaricata and V. zizanioides crude ethanol, aqueous, and hexane extracts exhibited significant in vitro antibacterial activity against S. aureus, and the active compounds isolated from them can be potential sources for the synthesis of antibacterial drugs. It can be concluded that  Infectious diseases have increased to a great extent during the recent years , and theIn the recent past, a lot of evidence emerged that human pathogenic microorganisms have developed antibiotic resistance . The exiMicroorganisms, fungi, algae, symbiotic lichens and mosses, and higher plants are used to develop antimicrobial agents with novel mechanism of action against microorganisms . Throughin vitro antimicrobial activity against Gram-positive and Gram-negative pathogenic bacteria  were determined by the simple and standard qualitative methods described by Trease and Evans  and Sofo(1) Solvent ExtractionEthanol Extract. Ethanol extracts of all the five medicinal plants were prepared by dissolving 20 g of coarse powder of each plant material separately in 200 ml of ethanol. The contents were kept in a mechanical shaker for 72 hr at 25°C. Then, the extracts were filtered, and the solvent was evaporated using a rotary evaporator. The extracts were resolubilized with 10% DMSO and stored at 4°C in air tight bottles for further studies.Hexane Extract. 10 g portions of dried plant material were extracted with 100 ml of n-hexane using a mechanical shaker for 72 hr at 25°C. Then, the extracts were filtered, and the solvent was evaporated using a rotary evaporator. The extracts were resolubilized with 10% DMSO and stored at 4°C in air tight bottles for further studies.(2) Aqueous Extraction. 2.62 g of dried plant material was refluxed in 60 ml of distilled water for 3 hr. Extract was filtered, evaporated, resolubilized with 10% DMSO, and stored at 4°C in air tight bottles for further studies.Escherichila coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853), and Klebsiella pneumoniae (ATCC 700603) were used to check the effectiveness of medicinal plants. Standard bacterial cultures were obtained from Microbiology Department, Faculty of Medicine, University of Ruhuna.CLSI control strains of 2SO4 in distilled water and 0.05 ml 1% BaCl2 in distilled water in order to estimate bacterial density [McFarland number 0.5 standard was prepared by mixing 9.95 ml 1% H density . The pre(1) Disk Diffusion Assay. The antimicrobial susceptibility was initially assayed by the agar disk diffusion method [8 bacterial/ml inoculum. Each bacterial suspension was inoculated on Mueller-Hinton agar plates, and the plates were then allowed to dry for 5 minutes. The sterile filter paper disks  were soaked in 10 μl of each plant extract. The extract-soaked filter paper disks were then placed on the inoculated Mueller-Hinton agar plates. Cefotaxime (30 μg) disk was used as the positive control, and 10% DMSO-soaked filter paper disk was used as the negative control. Plates were incubated for 18 hr at 35 ± 2°C. After incubation, the zones of inhibition were recorded as the diameter of the growth-free zones measured in mm using a Vernier caliper.n method . Three c(2) Minimum Inhibitory Concentration (MIC). Plant extracts that gave a positive result for the disk diffusion assay were used to determine MIC using the microplate dilution method [8 bacterial/ml. 150 μl of plant extract was added to each well of the 96-well microplate. 50 μl of bacterial suspension was added to each well except the negative controls. Cefotaxime IV drug was used as the positive control. 10% DMSO and plant extracts without bacterial suspension were used as the negative controls. Microtiter plates were incubated at 35 ± 2°C for 24 hr. Antimicrobial activity was assessed by measuring absorbance at 630 nm of wave length.n method . Serial Studies were performed in triplicate. Data were expressed as mean ± SEM.Asparagus falcatus. Saponins were present in all other plant extracts except Coriandrum sativum. Cyanogenic glycosides and reducing sugars were absent in all five plant extracts , while the minimum yield was given by the hexane extract of Asparagus falcatus (0.5%).The yield of extraction given by different plant extracts studied is shown in Epaltes divaricata showed a significant inhibition zone in all three extracts  and Vetiveria zizanioides showed a significant inhibition zone in two extracts (ethanol and hexane) against S. aureus , and minimum zone of inhibition was given by the aqueous extract of Epaltes divaricata (7.4 mm). Aqueous extract of Epaltes divaricata gave 7.4 ± 0.2 mm zone of inhibition for crude extract against S. aureus. Ethanol extract of Epaltes divaricata gave corresponding zones of inhibition 16.3 ± 0.2, 13.4 ± 0.2, and 7.6 ± 0.1 mm, respectively, for crude extract, 10-fold dilution of crude extract, and 100-fold dilution of crude extract while Vetiveria zizanioides gave 12.1 ± 0.2 and 7.3 ± 0.3 mm, respectively, for crude extract and 10-fold dilution of crude extract against S. aureus. Hexane extract of Epaltes divaricata gave 13.7 ± 0.1 mm zone of inhibition for crude extract while Vetiveria zizanioides gave 11.4 ± 0.2 mm for crude extract against S. aureus. Positive control cefotaxime (30 μg) gave corresponding zones of inhibition 31.2 ± 0.3, 32.9 ± 0.3, 33.7 ± 0.2, and 18.6 ± 0.3 mm, respectively, against S. aureus, E. coli, K. pneumoniae, and P. aeruginosa. However, due to the differences in concentrations of extracts, effectiveness of the plant extracts cannot be accurately compared by comparing the respective diameters obtained in the disc diffusion assay. Hence, minimum inhibitory concentration was used to determine the effectiveness of the plant extracts accurately.Among the five plants studied, . aureus . The con. aureus . The antE. divaricata gave MIC values of 0.48 mg/ml, 1.2 mg/ml, and 1.6 mg/ml, respectively, against S. aureus. Ethanol and hexane extracts of V. zizanioides gave MIC values of 2.4 mg/ml and 0.003 mg/ml, respectively, against S. aureus. Therefore, the highest antimicrobial activity was observed for the hexane extract of Vetiveria zizanioides.The MIC values obtained from plants exhibited antibacterial activity ranged between 0.003 and 2.4 mg/ml . EthanolSearch for potential antimicrobial drugs increased during the past few decades due to concomitant rise of antibiotic resistant bacterial strains . StudiesAsparagus falcatus, Asteracantha longifolia, Vetiveria zizanioides, Epaltes divaricata, and Coriandrum sativum extracts. But, those reports are also from other countries where the soil composition of the respective region may alter the composition of active compounds and hence the antimicrobial activity present in each plant extract. In the current study, extracts from two plant species gave positive results during the initial antibacterial screening against S. aureus. None of the plant extracts were effective against Escherichila coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Ethanol, aqueous, and hexane extracts of Epaltes divaricata gave maximum zones of inhibition 16.3 mm, 7.4 mm, and 13.7 mm, respectively, against S. aureus. Ethanol and hexane extracts of Vetiveria zizanioides gave maximum zones of inhibition 12.1 mm and 11.4 mm, respectively, against S. aureus. Zones of inhibition observed with the positive control, cefotaxime, for the organisms used in the study were 31.5 ± 0.2 for Staphylococcus aureus, 33.1 ± 0.2 for Escherichia coli, and 18.8 ± 0.2 for Pseudomonas aeruginosa and were compatible with the published zones of inhibitions such as 25–31 mm, 29–35 mm, and 18–22 mm, respectively.There are limited data published on the antimicrobial activity of Epaltes divaricata gave MIC values of 0.48 mg/ml, 1.2 mg/ml, and 1.6 mg/ml, respectively, for ethanol, aqueous, and hexane extracts against S. aureus. Ethanol and hexane extracts of V. zizanioides gave MIC values of 2.4 mg/ml and 0.003 mg/ml, respectively, against S. aureus. According to literature, cefotaxime, the positive control used for this study was given a MIC value between 1 and 4 μg/ml.The MIC was determined against selected bacteria to quantitate the activity of these extracts. E. divaricata and V. zizanioides have previously been documented as effective against the growth of S. aureus, E. coli, and P. aeruginosa [E. divaricata and V. zizanioides) exhibited a considerable activity against Gram-positive bacteria. None of the plant species that have been tested in the present study were effective against Gram-negative bacteria.ruginosa . These dIn a study by Wigmore et al. , they haAccording to the WHO basic criteria, plants should be nontoxic for the use as therapeutic agents. Five plants used in the study were previously evaluated for subchronic toxicity in mice, and it has already been documented that they are nontoxic in ICR mice .E. divaricata and V. zizanioides have exhibited the antibacterial activity on the Gram-positive bacteria than Gram-negative bacteria. The difference in morphological constituents between Gram-positive and Gram-negative bacteria may be the reason for the differences in antibacterial sensitivity. The structural lipopolysaccharide components in outer phospholipid membrane of Gram-negative bacteria cause the impermeability of cell wall to antimicrobial chemical substances. The Gram-positive bacteria have an outer peptidoglycan layer, which makes the cell wall more permeable to antimicrobial substances than lipopolysaccharide layer. Therefore, the complexity of the cell walls of Gram-negative bacteria is higher than that of Gram-positive bacteria. Therefore, Gram-negative bacteria are less susceptible to antimicrobial chemical substances than Gram-positive bacteria [In the present study, bacteria .V. zizanioides hexane extract. This MIC value is almost similar to the MIC value shown by cefotaxime, the positive control used for the study. When consider E. divaricata, the ethanol extract exhibited the lowest MIC value. According to the results of the present study, less polar compounds exhibited more antibacterial activity compared to more polar compounds.Compounds extracted during the extraction procedure are mainly dependent on the type of the solvent. Water is the primary solvent used in the traditional medicine, but in the present study, compounds extracted in organic solvents (ethanol and hexane) have exhibited more significant antibacterial activity compared to those extracted in water. Among the extracts investigated, hexane extract exhibited the highest antibacterial effect followed by the ethanol extract. These observations can be due to the polarity of the compounds which were extracted by each solvent and the ability of extracts to diffuse and dissolve in different culture media used in the study. Among the three solvents used in the study, water is the most polar solvent and hexane is the least polar solvent. The lowest MIC value was given by Absence of antimicrobial activity does not mean that the bioactive compounds are not present in the plant or the plant has no activity against microorganisms. Presence of inadequate quantities of active constituent or constituents in the extract to exhibit the antimicrobial activity can be the reason for the negative results.In vitro studies show that flavonoids also have antiallergic, anti-inflammatory, antimicrobial, anticancer, and antidiarrheal activities [Phytochemical studies have shown that plants with antimicrobial activity contain bioactive constituents such as tannins, flavonoids, alkaloids, and saponins . Polyphetivities . Saponintivities . TherefoE. divaricata and V. zizanioides and aqueous extract of E. divaricata exhibited beneficial antibacterial activity against S. aureus (ATCC 25923). None of the other plant extracts were active against any of the microorganisms tested. The highest antimicrobial activity was observed for hexane extract of Vetiveria zizanioides which showed a MIC value in the same range as the positive control used. It can be concluded that a low polar active compound present in the Vetiveria extract may be responsible for the significant antimicrobial activity. Many polyphenolic constituents and alkaloids derived from plants are known to have antimicrobial activity. Therefore, presence of these compounds in the above plant extracts may be responsible for their antimicrobial action. Further scientific evaluation of these plants should be done including fractionation and further characterization of phytochemicals to identify the active components responsible for the antimicrobial activity, as well as to adjudge the in vivo activities of these constituents.The results obtained from this study provided evidence that the ethanol and hexane extracts of the Sri Lankan traditional medicinal plants


The data presented are related to the article entitled “Six Sigma quality approach for HPLC-UV method optimization” Ibrahim et al., 2019. The raw data of HPLC analysis of ascorbic acid (AS), paracetamol (PA) and guaifenesin (GU) are presented. Calibration standards were prepared at six concentrations levels  each day and measured in triplicate. Validation standards were prepared at four concentration levels  each day and measured in quintet. Three different series were used for method validation and prepared at the rate of one series per day. V2 was the type of the modified protocol.Calibration standards were prepared at six concentrations levels  each day and measured in triplicate.Validation standards were prepared at four concentration levels  each day and measured in quintet.Three different series were used for method validation and prepared at the rate of one series per day.−1 of AS, 0.325 mg ml−1 of PA, and 0.10 mg ml−1 of GU at optimal condition.2−1, respectively.AS, PA and GU were supplied by NODCAR. Methanol (HPLC grade), potassium dihydrogen phosphate, and ortho-phosphoric acid 85% were purchased from Merck. A validation matrix solution was prepared by dissolving sodium bicarbonate, citric acid anhydrous, tartaric acid anhydrous, povidone K25, aspartam, and disodium edetate in methanol in order to obtain 24.2, 9.6, 12.3, 1.2, 0.7 and 03 mg ml® CN column, , using an isocratic mode with a mixture of 50 mM potassium dihydrogen phosphate buffer adjusted with ortho-phosphoric acid to pH 2.2 as the aqueous component of the mobile phase and methanol in the ratio 85.9:14.1 (v/v) at 1 ml/min flow rate, with UV detector at 275 nm and a column temperature of 45 °C. The injected volume was 20 μL.The separation was performed on A Phenomenex Luna


Total temporomandibular joint (TMJ) prosthesis is an effective and reliable method of joint reconstruction. However, there is still an urgent need to design a new TMJ prosthesis because of no commercially available TMJ prosthesis appropriate for the clinical application on the Chinese population. This study was introduced to prospectively confirm the safety and effectiveness of a new TMJ prosthesis with customized design and 3D printing additive fabrication in clinical application.Patients with unilateral end-stage TMJ osteoarthrosis were recruited in this study from Nov 2016 to Mar 2017. Computed tomography scans for all patients were obtained and transformed into three-dimensional (3D) reconstruction models. The customized TMJ prosthesis consisted of three components including the fossa, condylar head, and mandibular handle units, which were designed based on the anatomy of the TMJ and were fabricated using the 3D printing technology. The prominent characters of the prosthesis were the customized design of the fossa component with a single ultra-high-molecular-weight polyethylene and the connection mechanism between the condylar head  and mandibular handle components . The clinical follow-up, radiographic evaluation and laboratory indices were all done to analyze the prosthesis’ outcomes in the clinical application.12 consecutive patients were included in the study. There were no complications  found after surgery. Pain, diet, mandibular function, and maximal interincisal opening showed significant improvements after surgery. But the lateral movement was limited to the non-operated side and the mandible deviated towards the operated side on opening mouth following surgery.The presented TMJ prosthesis is considered an innovative product in TMJ Yang’s system, which is unique compared to other prostheses for the special design and 3D printing additive manufacture. Moreover, the prosthesis is very safe and efficient for clinical use.Trial registration Prospective reports on Chinese customized total temporomandibular joint prosthesis reconstruction cases, ChiCTR-ONC-16009712. Registered 22 Nov 2016, http://www.chictr.org.cn/showproj.aspx?proj=16091 Temporomandibular joint (TMJ) is often affected by a wide spectrum of disorders, including extra-articular and intra-articular pathologies which usually present with various clinical symptoms including pain in the preauricular region, limitation of mouth opening, malocclusion, or jaw deformity , 2. The Total alloplastic TMJ prosthesis is one of the effective and efficient methods of joint reconstruction , 5. WithUndoubtedly, there is a great need for the research and development of Chinese TMJ prosthesis in domestic TMJ surgery. Based on previous experiences, the authors have designed a newly customized TMJ prosthesis Fig. c, which This was a prospective clinical study conducted at the Department of Oral Surgery, Shanghai Ninth People’s Hospital between Nov 2016 and Mar 2017. In this study, patients were recruited for TMJ reconstruction with a new prosthesis based on the indications and contraindications as follows.Unilateral end-stage TMJ osteoarthrosis (stage V of Wilkes-Bronstein Classification) with a stable occlusion relationship .Allergy to prosthetic components materials.Uncontrollable masticatory muscle hyperfunction or parafunctional habits (clenching or grinding).Active or even suspected infections in or around the implantation site of the prosthesis.History of previous TMJ surgeries.Systemic diseases contraindicating the use of the artificial prosthesis –24.This study was approved by Shanghai Ninth People’s Hospital Human Research Ethics Committee. Moreover, the principles outlined in the Declaration of Helsinki were followed in the study as well. All patients were informed about the surgical purpose, management protocol, recovery period, and possible complications. An informed consent was obtained from all participants.CT scan  of the entire mandible, maxilla, and TMJ for all patients (0.625 mm slice thickness).Processing CT data with DICOM format to create the 3D craniomaxillofacial model in Mimics software 18.0 .Cutting the lower part of the eminence and entire condyle with the aid of Mimics software.Designing the prosthesis  by using 3-Matic research software 9.0 . The main principles of the TMJ prosthesis design are focusing on the following points: (1) the dimension and slope of the articular surface of the fossa component are ascertained based on the Chinese TMJ anatomy database in our previous study ; (2) theManufacturing the three components of the prosthesis: The fossa component is fabricated from ultra-high-molecular-weight polyethylene  by 5-axis milling device . The condylar head component is fabricated from the cobalt-chromium-molybdenum alloy  by 5-axis milling device. The mandibular component is fabricated from titanium alloy  by a 3D-printing machine . Then, all of the components are polished and the medial surface of the mandibular handle is treated with the sandblasting technique.Fitting the prosthesis in the 3D skull model before sterilizing and packaging: The three components of the prosthesis are fitted in the 3D model to check whether the stability and accuracy of each individual component are the same with the models in Mimics software.Sterilization and packaging of the prosthesis: All TMJ prosthesis components are provided clean and non-sterile and therefore, no additional cleaning prior to sterilization is needed. The glenoid fossa component is sterilized utilizing ethylene oxide gas sterilization, and the condylar head and mandibular components are sterilized using steaming sterilization. Afterward, The TMJ prosthesis components are repackaged again. The simple processing procedure is showed in Fig. All patients received general anesthesia through nasal intubation.A modified preauricular approach was performed in all patients to expose the zygomatic arch, eminence, condyle, and lateral mandibular ramus.The entire condyle Fig. a and theThe fossa components were fixed with titanium screws firstly  based on the orientation holes of the templates Fig. c.The mandibular handle with condylar head components facilitated the insertion of the titanium screws with the previous holes in the template. Then, the lower resultant holes of the mandibular component were implanted with the screws with the help of the endoscope (accessed through the preauricular incision) and transbuccal retractor (inserted through a 3 mm incision in parotideo-masseteric region) after the occlusion was guaranteed as stable as preoperatively  infection, (b) dental malocclusion, and (c) incision healing in 1 week, 1, 3, 6, and 12 months postoperatively.The displacement, breakage, or loosening of the prosthesis components were checked in CT scans at 1 week and 12 months postoperatively , 14.The laboratory indices included (a) routine blood tests; (b) kidney function tests; (c) liver function tests; (d) routine urine tests; and (e) routine stool tests. These tests were performed and recorded 1 week preoperatively, 1, and 12 months postoperatively.The subjective and objective indices were used to assess the clinical efficacy. These data were collected using a standardized data collection format 1 week preoperatively, 1, 3, 6, and 12 months postoperatively. Quantitative measurements were performed by two oral and maxillofacial surgeons together. When there was a disagreement, the consensus was reached by a discussion.Subjective data including (a) pain, (b) functions of the mandible, and (c) diet, were obtained using a 10-length visual analog scale (VAS). The pain scale ranged from no pain at 0 to worst pain at 10. The mandibular functions scale ranged from no loss at 0 to complete loss of functions at 10. The diet scale ranged from no restriction at 0 to only liquids at 10 –24.Objective measurements of the mandibular range of motion, including (a) maximal interincisal opening, (b) lateral movements (left and right), (c) forward movement, and d) mandibular deviation when opening the mouth, were recorded in millimeters.  side; MNS means movement to normal (non-operated) side, MFM means mandible forward movement, MOD means mouth opening deviation to the diseased side) , 23.Data were analyzed using the Statistical Package for Social Sciences software package, version 17.0 . The subjective and objective assessment indices before and after surgery were compared using the paired t-test of one-way analysis of variance. A P value of less than 0.05 was considered statistically significant .12 consecutive patients were included in the study based on the inclusion and exclusion criteria. There were 7 females and 5 males. Their mean age was 47.8 years , and the mean duration of the disease was 4.9 years . The left side was affected in 5 patients and the right side in 7  Fig. .Fig. 4SuThe mean preoperative MIO was 26.42 ± 9.30 mm and the postoperative values were 31.42 ± 7.62, 36.92 ± 6.16, 38.67 ± 6.08, and 39.25 ± 5.17 mm at 1, 3, 6 and 12 months after surgery. There were statistically significant improvements for MIO at 3, 6, and 12 months follow-up points (P < 0.01).The mean preoperative MOD was 1.67 ± 1.37 mm with postoperative means of 4.71 ± 2.16, 3.83 ± 0.56, 4.13 ± 1.11, and 3.83 ± 0.98 mm. There was a statistically significant deviation to the normal side after surgery (P < 0.01).The mean preoperative MDS was 4.79 ± 2.17 mm with postoperative means of 6.50 ± 1.88, 7.08 ± 1.82, 7.42 ± 1.69, and 7.50 ± 1.54 mm demonstrating statistically significant increases after surgery (P < 0.05). Regarding MNS, The mean preoperative value was 7.25 ± 2.21 mm, while the postoperative means were 3.13 ± 1.48, 2.96 ± 1.25, 2.92 ± 1.47, and 3.54 ± 1.10 mm revealing statistically significant decreases after surgery (P < 0.0001).The mean preoperative MFM was 6.33 ± 2.14 mm with postoperative means of 3.96 ± 1.86, 4.00 ± 6.16, 4.42 ± 1.72, and 4.63 ± 1.75 mm at the corresponding follow-up points. There were statistically significant decreases for MFM after surgery (P < 0.05) , we realized that the safety of the prosthesis has been verified. However, Mercuri et al. reported the serum metal levels in patients who underwent different maxillofacial implanted metallic objects. The results showed the possibility of the increases of the metal levels including the cobalt, titanium, or chromium in the bloodstream after dental implant placement, orthognathic surgery using rigid metal fixation plates and screws, and total TMJ prosthesis. But they did not elucidate the clinical symptoms resulting from the metal level increases . NeverthMoreover, completely postoperatively subjective and objective indices have been measured to confirm the efficacy of the prosthesis in 12 patients without missing case or in compliance for more than one year following surgery. The methods of follow-up, which have been widely used in previous studies for the clinical applications of Zimmer Biomet or TMJ Concepts products, were referred to the common criteria confirmed by Kent et al. in 1993 , 23, 33.The presented TMJ prosthesis is an innovative product in TMJ Yang’s system due to its special design, 3D printing additive fabrication, and corresponding surgical procedure with the help of surgical templates and endoscopy. Moreover, this prospective self-control study proved the safety and efficacy of the prosthesis by clinical, radiological and laboratory examinations and comparisons in detail. This study will support the evidence for an extensive clinical application of the innovative prosthesis in the future.


Drynariae rhizoma (Kunze ex Mett.) J. Sm. has been used to treat osteoporosis and bone resorption for 2500 years. Based on the previous study and literature references, flavonoids were proved to be the most abundant and main active compounds of Drynariae rhizoma for osteoporosis treatment. In order to make good and rational use of Drynariae rhizoma in future, a rapid, sensitive, and selective ultraperformance liquid chromatography-mass spectrometry (UPLC-MS/MS) method was developed to investigate the pharmacokinetics of eight main flavonoids in rat plasma after oral administration of the Drynariae rhizoma extract, including neoeriocitrin, luteolin-7-O-β-D-glucoside, astragalin, naringin, eriodictyol, luteolin, naringenin, and kaempferol. Plasma samples' pretreatment involved a solid-phase extraction column. The separation was performed on an ACQUITY UPLCTM BEH C18 column with a gradient mobile-phase system of acetonitrile and 1% acetic acid in water. The detection was performed using a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization interface (ESI) by multiple reaction monitoring (MRM) in the positive ion mode. All calibration curves exhibited good linearity (r2 > 0.9990) over the measured ranges. The intraday and interday precisions (RSD) were within 13.87%, and the accuracy (RE) ranged from −14.57% to −0.25% at three quality control levels. Extraction recovery, matrix effect, and stability were satisfactory. The pharmacokinetic characteristics of the eight flavonoids of interest were clearly elucidated.As a traditional Chinese medicine,  Drynariae rhizoma (Kunze ex Mett.) J. Sm., commonly known as Gu-Sui-Bu, is a fern plant widely distributed in southern China. Drynariae rhizoma is effective for the treatment of osteoporosis and bone resorption [Drynariae rhizoma contains various types of chemical constituents, including flavonoids, triterpenes, phenolic acids, and their glycosides. But flavonoids and their glycosides are the most abundant constituents of Drynariae rhizoma [Drynariae rhizoma could activate the estrogen receptors and have the trend of replacing estrogen for clinical use [Drynariae rhizoma.The traditional Chinese medicine sorption . Drynari rhizoma , 3. Furt rhizoma –10. Totaical use , 12. FlaDrynariae rhizoma, the key issue is how to study the effective substance of Drynariae rhizoma to play a key role in osteoporosis. Wang suggested a conceptual framework for illuminating the absorbed bioactive compounds in herb medicines [in vivo after oral administration. Thus, it is necessary to measure the absorbed bioactive compounds in plasma to understand the effective substances in the herb. In our pilot study, eight flavonoids were detected in plasma after oral administration of Drynariae rhizoma extract (DRE), including neoeriocitrin, luteolin-7-O-β-D-glucoside, astragalin, naringin, eriodictyol, luteolin, naringenin, and kaempferol  by multiple reaction monitoring (MRM) in the positive ion mode. The newly described UPLC-MS/MS method was validated and successfully applied to pharmacokinetic study of eight flavonoids in rats after oral administration of DRE.In the present study, a rapid, sensitive, and selective UPLC-MS/MS method was developed and validated for the simultaneous determination of eight flavonoids from the DRE in rat plasma. Plasma samples were pretreated with the CDrynariae rhizoma naturally grown in Jiangxi Province, China, was purchased from Anguo Herb Market . Neoeriocitrin, luteolin-7-O-β-D-glucoside, astragalin, naringin, eriodictyol, luteolin, naringenin, and kaempferol and the internal standard  were purchased from Bailingwei Technology Co., Ltd.  with purities >98%. Methanol and acetonitrile of the HPLC grade were obtained from Dikma . Acetic acid (HPLC grade) was purchased from Scharlau Chemie S. A. . Water was purified by redistillation and a Milli-Q® ultrapure water system . Other chemicals and solvents used were of analytical grade.TM system , which included a cooling autosampler, column oven, and two pumps. The chromatographic separation was performed using an ACQUITY UPLCTM BEH C18 column  at 35°C. The mobile phase consisted of 1% acetic acid in water as the aqueous phase (A) and 100% acetonitrile as the organic phase (B). The gradient elution program was 0–3 min, 10–30% B; 3–4.5 min, 30–40% B; 4.5–6 min, 40–70% B; and 6-7 min, 70–90% B. The flow rate was 0.3 ml/min, the temperature of the autosampler was 4°C, and the sample injection volume was 5 μl.Liquid chromatography analysis was performed on an ACQUITY UPLC−1, respectively. Argon was used as the collision gas at a pressure of approximately 2.61 × 10−3 mbar. Quantitative analysis was performed in multiple reaction monitoring (MRM) mode, and the parent ion, daughter ion, cone voltage, and collision energies of eight analytes and the IS were optimized. + MS spectra of the eight flavonoids of interest and the IS and their fragmentation pathways. All data collected in the centroid mode were processed using MassLynx™ NT 4.1 software with the QuanLynx™ program (Waters Corp.). The structure of the eight analytes are displayed in Mass spectrometric detection was performed using a Waters® Micromass® Quattro Premier™ XE triple quadrupole tandem mass spectrometer equipped with an electrospray ionization interface in the positive ion mode. The ESI + source operation optimal parameters were capillary voltage 3.5 kV, source temperature 120°C, and desolvation temperature 300°C. Nitrogen was used as the desolvation and cone gas with flow rates of 600 and 30 L·hμg/ml was prepared in methanol. The internal standard (quercetin) stock solution of 200 ng/ml was also dissolved with methanol. The plasma calibration standard solutions were prepared at concentrations in the range of 3.749–3749 ng/ml for neoeriocitrin, 1.856–4230 ng/ml for luteolin-7-O-β-D-glucoside, 1.317–1400 ng/ml for astragalin, 1.237–6370 ng/ml for naringin, 0.135–1040 ng/ml for eriodictyol, 2.742–3780 ng/ml for luteolin, 0.121–1210 ng/ml for naringenin, and 5.328–1209 ng/ml for kaempferol.Stock solutions of the standards for eight flavonoids and quercetin (IS) solution were prepared in a 10 ml volumetric flask. Each standard stock solution of 100 O-β-D-glucoside, 5.1, 102, 1020 ng/ml for astragalin, 10.2, 408, 6120 ng/ml for naringin, 1.02, 40.8, 816 ng/ml for eriodictyol, 10.1, 40.4, 1010 ng/ml for luteolin, 5.2, 41.6, 208 ng/ml for naringenin, and 1.06, 42.4, 424 ng/ml for kaempferol) were used for quality control (QC) evaluation in UPLC-MS/MS analysis. All the solutions were stored at 4°C before use.Three concentrations  of each analyte solution in drug-free plasma  were powdered and extracted three times under reflux in 15 times volume of methanol for every 2 hours. The solution was filtered and evaporated under reduced pressure in a Rotavapor R-3 rotary evaporator . Subsequently, the concentrated extract was dried in an oven, and the final weight of the DRE was 198.9 g with a yield of 19.9%. The contents of the main flavonoid constituents in DRE were quantitatively determined with the method described above, with the results of neoeriocitrin (5.1 mg/g), luteolin-7-O-β-D-glucoside (1.2 mg/g), astragalin (0.96 mg/g), naringin (8.5 mg/g), eriodictyol (0.66 mg/g), luteolin (0.11 mg/g), naringenin (2.71 mg/g), and kaempferol (0.14 mg/g), respectively.The dried roots of μl) were placed in a 10 ml centrifuge tube; 50 μl of internal standard solution (200 ng/ml) and 50 μl acetic acid were added. The mixture was vortexed for 1.0 min and then loaded onto an activated SPE C18 column . After rinsing with 2 ml purified water, the SPE column was eluted with 4.5 ml of methanol and the eluate was evaporated to dryness under a gentle stream of nitrogen. The residue was reconstituted in 140 μl methanol and centrifuged at 16000 × g for 15 min; then, a 5 μl aliquot was injected into the UPLC-MS/MS system for analysis.Aliquots of plasma samples , lower limit of quantification (LLOQ), accuracy and precision, extraction recovery, stability, and matrix effects based on the method validation procedure in the previous work .We compared the chromatograms of blank plasma from six individual rats with those of corresponding plasma samples spiked with eight mixed standard samples and IS and plasma samples after oral administration of DRE.Calibration curves for the eight standards were constructed by plotting the peak area ratios of each analyte to that of the IS versus the corresponding concentration. The lower limit of detection (LLOD) was defined as the lowest concentration with a signal-to-noise ratio of 3 : 1. The lower limit of quantification (LLOQ) was determined as the lowest concentration of the calibration curve with a signal-to-noise ratio of 10 : 1.Intraday precision and interday precision and accuracy were assessed for low-, middle-, and high-concentration QC samples in six replicates on the same day and once a day for three consecutive days, respectively. Each tested sample was related to the calibration curve. The precision was calculated as the relative standard deviation (RSD%) and the accuracy as relative error (RE%). These results indicated that the precision and accuracy of the method were within acceptable limits. The intraday precision and interday precision and accuracy values for lowest acceptable reproducibility concentrations were denied as being within ±15%, and the precision and accuracy were within 80%–120%.The recovery and matrix effects of analytes were determined for low-, middle-, and high-concentration QC samples with six replicates. Extraction recovery was determined at the three QC levels by comparing the peak areas of analytes between plasma samples spiked with analytes before and after extraction. Matrix effects were evaluated at the three QC levels by comparing the peak areas of analytes obtained from plasma samples spiked with analytes after extraction to those of pure standard solutions at the same concentrations; the acceptable range was 80–120%.The stability of determination was assessed by analysis of low-, middle-, and high-concentration QC samples with three replicates. The short-term stability was assessed by analyzing the QC samples kept in the autosampler (4°C) for 36 h. To assess long-term stability, the QC samples were stored at −20°C for 30 days. Freeze-thaw stability was evaluated by subjecting the QC plasma samples to three complete freeze/thaw cycles from −20°C to room temperature.Female Sprague Dawley rats (280–350 g) were supplied by the Animal Safety Evaluation Center of Heilongjiang University of Chinese Medicine . All protocols for animal experiments were approved in accordance with the Regulations of Experimental Animal Administration issued by the State Commission of Science and Technology of the People's Republic of China. The rats were housed at 24 ± 2°C, and relative humidity was 60 ± 5%, with a 12 h-12 h light-dark cycle. Water and food were supplied freely.The UPLC-MS/MS method was successfully applied in a pharmacokinetic study of eight flavonoids in rats after oral administration of the DRE. The dosing solutions were freshly prepared DRE administrated via an oral gavage to the rats at a single dose of 4 g/kg. The animals had free access to water during the experiment. A series of blood samples were collected in 1.5 ml heparinized polythene tubes from the suborbital venous lexus of each rat at 0.08, 0.33, 0.5, 0.67, 1, 2, 4, 6, 8, 12, and 24 h after administration. The blood samples were centrifuged at 4500 × g for 10 min, and the supernatants were collected immediately and stored at −20°C until analysis.Pharmacokinetic parameters were determined using a noncompartmental model and analyzed using pharmacokinetic software WinNonlin Standard Edition, version 1.1. Data are shown as the mean ± standard deviation (SD) for each parameter.O-β-D-glucoside, astragalin, naringin, eriodictyol, luteolin, naringenin, kaempferol, and quercetin were monitored across a full scan in both positive and negative modes. The signal intensity and fragment stability in the positive mode were better than those in the negative mode for all the analytes. Therefore, the positive mode was used for analysis. The parent ion and daughter ion for each compound were obtained  and 100% acetonitrile as the organic phase (B). In the optimized UPLC-MS/MS conditions for simultaneous determination of the eight compounds, all analytes were eluted rapidly with 5.0 min.O-β-D-glucoside, astragalin, naringin, eriodictyol, luteolin, naringenin, kaempferol, and IS were approximately 2.20 min, 2.36 min, 2.60 min, 2.69 min, 3.31 min, 3.63 min, 4.34 min, 4.43 min, and 3.42 min, respectively. The representative chromatograms of blank plasma, blank plasma spiked with reference standards and IS, and plasma obtained after the oral administration of DRE are shown in The retention times of neoeriocitrin, luteolin-7-r2 ≥ 0.9990) over the measured ranges. The calibration curves were linear over the concentrations in the range of 3.75–3749 ng/ml for neoeriocitrin, 1.86–4230 ng/ml for luteolin-7-O-β-D-glucoside, 1.32–1400 ng/ml for astragalin, 1.24–6370 ng/ml for naringin, 0.14–1040 ng/ml for eriodictyol, 2.74–3780 ng/ml for luteolin, 0.12–1210 ng/ml for naringenin, and 5.33–1209 ng/ml for kaempferol, respectively. The linearity regression equation, correlation coefficients, and linear ranges of the eight analytes are shown in All calibration curves exhibited good linearity  (4414.18 ± 360.38 ng/ml and 1490.98 ± 124.54 ng/ml) and high area under the plasma concentration-time curve from 0 h to infinity AUC (0–∞) (8760.77 ± 347.83 and 3340.34 ± 237.36). Neoeriocitrin still exhibited a shortest Tmax to reach the maximum drug concentration (Tmax = 0.33 h). In the plasma concentration time profile of naringin, another small peak could be seen at 8 h . The slow elimination may be due to the fact that orally administrated naringin can be metabolized into naringenin and naringenin glucuronide [O-β-D-glucoside, eriodictyol, and kaempferol were lower, with small area under curve (AUC) and Cmax. The Tmax values for luteolin-7-O-β-D-glucoside and luteolin were 1 h and 0.5 h, respectively, which were in line with a previous report [O-β-D-glucoside, the plasma concentration of the former is greater than that of the latter. This may due to the conversion from glycoside to aglucone after oral DRE.Naringenin, the aglycone of naringin, was absorbed into blood with curonide . Pharmaccuronide . The plas report , 19. LutCompared with synthetic drugs, ingredients of plant medicines are complex and have synergistic activity. In this work, we found that most of the analytes reached their maximum concentrations around 2 h after oral administration of DRE. This phenomenon may allow the DRE to exert its greatest pharmacological activity in clinical application.In summary, a rapid, sensitive, and selective UPLC-MS/MS method was successfully applied in a pharmacokinetic study for analyzing the active compounds in Chinese herbal medicines. As the main active constituents of DRE, the major pharmacokinetic parameters of eight flavonoids were described for first time. The high efficiency, sensitivity, and selectivity of this multiple components quantitation were guaranteed by UPLC coupled ESI-MS. MRM quantitative analysis mode and two best MRM transitions enhanced the accuracy for each analyte. Results showed that this method could be applied for pharmacokinetic study of the DRE. The present results may provide useful information for better understanding of the absorption of the major constituents of DRE, as well as their potential clinical value.


I) and the mean expected heterozygosity were 2.528 and 0.879, respectively. At the population level, the regions of 42°N and 124°E had the highest genetic diversity among all latitudes and longitudes. The greater the difference in latitude was, the greater the genetic distance was, whereas a similar trend was not found in longitude populations. Three main clusters  were assigned to populations. AMOVA analysis showed that the genetic differentiation among latitude and longitude populations was 0.088 and 0.058, respectively, and the majority of genetic variation occurred within populations. The Mantel test revealed that genetic distance was significantly correlated with geographical distance . Furthermore, spatial autocorrelation analysis showed that there was a spatial structure  and the correlation coefficient (r) decreased as distance increased within a radius of 250 km.In this study, the genetic diversity and population structure of 205 wild soybean core collections in Northeast China from nine latitude populations and nine longitude populations were evaluated using SSR markers. A total of 973 alleles were detected by 43 SSR loci, and the average number of alleles per locus was 22.628. The mean Shannon information index ( Glycine soja Sieb. and Zucc.), the direct progenitor of the cultivated soybean (Glycine max (Linn.) Merr.), is a predominantly self-pollinated annual plant species [The annual wild soybean , Tris-HCl (pH 8.3), 500 mmol/L KCl, 200 mmol/L MgCl2 , 0.4 μL dNTP (2.5 mmol/L), 2.0 μL each of forward and reverse primers (10 pmol/L), 2.0 μL total DNA (20 ng/μL), 1.0 μL Taq DNA polymerase (2 U/μL), and 15.1 μL ddH2O. PCR was performed using the T100™ thermal cycler  with the following cycle conditions: an initial denaturing at 95°C for 5 min, followed by 35 cycles of 95°C denaturing for 45 s, 52–57°C annealing for 45 s, 68°C extension for 45 s, and a final extension at 72°C for 10 min.The 25 Amplified products were fractionated by electrophoresis through 6% denaturing polyacrylamide gels and stained with silver staining, which could detect 60 bp to 500 bp fragments and had a high resolution of 2 bp. The size of the stained band was analyzed based on its migration distance relative to the 100 bp DNA ladder (MBI Fermentas) using AlphaView software (version 1.3.0.7).Na), Shannon-Weaver index (I), expected heterozygosity (He), observed heterozygosity (H0), fixation index (Fis), genetic differentiation coefficient (Fst), genetic identity and genetic distance, molecular variation analysis of variance (AMOVA), Mantel tests, and spatial autocorrelation coefficients were computed by GenAIEx v6.5 [I) and expected heterozygosity (He) for evaluating the diversity were measured according to the formulas I = −1 × sum (pi × Ln (pi)) and He = 1–sum pi2, where pi is the frequency of the ith allele. H0 is generally lower than He due to inbreeding, and Fis = (1 − H0)/He. Outcrossing rate (t) was calculated using the equation t = (1 − Fis)/(1 + Fis) [The amplification fragments of genomic DNA by each SSR marker were scored based on the migration difference. The data format was converted accordingly in Microsoft Excel. The number of alleles (IEx v6.5 . The Sha + Fis) . Based o + Fis) , the den + Fis) . Spatialk value was set to 1 to 12, and ten runs were performed for each value of k to test stability of the results. The MCMC (Markov chain Monte Carlo) value was set as 100,000 burn-in with 200,000 iterations. The correct number of genetic clusters was inferred according to a value of Δk (Δk = mean|lnP(k + 1) − 2lnP(k) + lnP(k − 1)|/Sd|lnP(k)|) [The population genetic structure was predicted by STRUCTURE 2.3.4 . The deflnP(k)|) .I) and expected heterozygosity (He) were 2.528 and 0.879, respectively  to 36 (Satt286), with an average of 22.628. The mean Shannon information index (vely see .I) of nine latitude populations ranged from 1.300 to 2.419, with an average of 1.716; the expected heterozygosity (He) ranged from 0.661 to 0.884, with an average of 0.756. The region of 42°N had the highest genetic diversity among all latitudes. The Shannon information index (I) of nine longitudes varied from 1.659 to 2.368, averaging 1.919; the expected heterozygosity (He) varied from 0.751 to 0.880, averaging 0.805. The region of 124°E had the highest genetic diversity among all longitudes. As shown in Tables At the population level see , the Shak was 3, Δk was the highest, which indicated that 3 main clusters had been identified  [The  < 0.15)  was obser = 0.207, p < 0.05), which suggests that geographic distance limits gene flow among populations and influences the genetic structure. For further analyses, spatial autocorrelation analysis was performed by distance classes of 100 km, and a general decline was found in the correlation coefficient (r) with distance. The correlation values were negative and significant up to 250 km. This revealed that there is a clinal spatial structure in wild soybeans in Northeast China  (see The Mantel test indicated that there was a positive correlation between geographic and genetic distance (.01) see .I), and expected heterozygosity (He) were 22.628, 2.528, and 0.879, respectively, which were significantly higher than previously reported results [The wild soybean in Northeast China is an important ecotype, and its genetic diversity has been widely studied by using phenotypic traits , 30, 31  results –34. The  results , 36. On  results . These a results .Fst value was 0.088) and longitude populations  occurred   see , which cr = 0.207, p < 0.05), which indicates that geographical isolation has also been an important factor in forming the current genetic structure of wild soybeans in Northeast China. Spatial autocorrelation analysis revealed that the correlation between geographical distance and genetic distance is limited.The Mantel test revealed that there was a positive relationship between geographic and genetic distance (The wild soybean in Northeast China has become one of the most severely endangered wild plant species due to the interference of human activities . The genIn summary, the genetic diversity and geographic population structure of the wild soybean in Northeast China were fully investigated as a single population, or at different latitude or longitude populations for the first time. The distribution pattern of genetic variation is related to latitude, and the highest level of genetic diversity was found at 42°N, and protection in areas with higher genetic diversity should be prioritized. This study disclosed that natural selection to adapt temperature and photoperiod, selfing mating reproductive system, and distance isolation resulted in the current population structure of wild soybean in Northeast China.


A total of 90 LAB were isolated, of which 4 (4.44%) isolates showed 45.35–97.11% and 38.40–90.49% survival rates at pH values  for 3 and 6 h, in that order. The four acid-tolerant isolates were found tolerant to 0.3% bile salt for 24 h with 91.37 to 97.22% rate of survival. The acid-and-bile salt-tolerant LAB isolates were found inhibiting some food-borne test pathogenic bacteria to varying degrees. All acid-and-bile-tolerant isolates displayed varying sensitivity to different antibiotics. The in vitro adherence to stainless steel plates of the 4 screened probiotic LAB isolates were ranged from 32.75 to 36.30% adhesion rate. The four efficient probiotic LAB isolates that belonged to Lactobacillus species were identified to the strain level using 16S rDNA gene sequence comparisons and, namely, were Lactobacillus plantarum strain CIP 103151, Lactobacillus paracasei subsp. tolerans strain NBRC 15906, Lactobacillus paracasei strain NBRC 15889, and Lactobacillus plantarum strain JCM 1149. The four Lactobacillus strains were found to be potentially useful to produce probiotic products.Probiotics are live microorganisms which when consumed in large number together with a food promote the health of the consumer. The aim of this study was to evaluate  Worldwide, a variety of fermented food products are produced, which contribute significantly to the diets of many people . Fermentin vitro studies indicate that the growth of food-borne pathogenic microbes is inhibited by probiotic lactic acid bacteria ) in 2.5 mol/L potassium hydroxide, were used in order to test ammonia production from arginine. MRS containing 0.3% L-arginine hydrochloride was transferred into duplicate tubes with 5 ml amount and inoculated with each of 1% overnight cultures of LAB. Thereafter, tubes were incubated at +37°C for 24 h. A 100 µl of cultures were transferred onto a white background. Thereafter, the same amount of Nessler's reagent was pipetted to the cultures. The change in color to bright orange indicates a positive reaction, while the yellow color indicates the negative reaction. A negative control, which does not contain arginine, was used as a negative control.Arginine containing MRS medium and Nessler's reagent (HgI2 production from glucose was determined in modified MRS broth containing inverted Durham tubes with 1% glucose. MRS broth (8 ml) in separate tubes containing 1% glucose with inverted Durham tubes was prepared and inoculated separately with 50 µl of 1% overnight fresh LAB culture. Then the test tubes were incubated at +37°C for 5 days. The presence of gas in Durham tubes during 5 days of observation indicates CO2 production from glucose.In order to determine the homofermentative and heterofermentative characteristics of LAB isolates, COn = 4) of potential probiotic LAB. Separately, 1 ml of each pure liquid culture was centrifuged for 3 min at 10000 rpm. The supernatant was removed, and the cells were resuspended in 300 µl buffer . To the suspension, 3 µl lysozyme (10 mg/ml) was added, and cells were lysed at 37°C for 60 min under occasional stirring of the tube content by overturning it. Lysing buffer  of 300 µl and 3 µl RNAse (10 mg/ml) was added to the mixture. The mixture was incubated at 37°C for 30 min and then cooled on ice for 1 min. Then, 100 µl solution of ammonium acetate (7.5 M) was added to the mixture, mixed on a vortex for 20 seconds and was centrifuged at 13000 rpm for 5 min. The supernatant was transferred into clean 1.5 ml tubes, and 300 μl isopropanol was added. Thereafter, the mixture was mixed by overturning for 1 min and stored at −20°C for 30 min. The mixture was centrifuged at 13000 rpm for 5 min. The supernatant was accurately decanted, and the tubes were placed overturned on a clean filter. Then, 400 µl of 70% ethanol was added and mixed several times by overturning to wash the DNA sediment. Finally, the sediment was dried at 37°C for 15 min till ethanol drops disappeared completely. The dried sediment was dissolved in 30 µl TE buffer.Genomic DNA was extracted from pure cultures , forward primer 1 µl, reverse primer 1 µl, and UPH2O 22 µl. Then, 49 µl of the mixtures was added to a sterile PCR tube, and 1 µl of the gDNA was used as a template; the amplification reactions were carried out in a thermal cycler (Bio-Rad Mycycler).For the amplification of the 16S rDNA gene, the specific primers AMP_F 5′- GAG AGT TTG ATY CTG GCT CAG -3′ and AMP_R 5′- AAG GAG GTG ATC CAR CCG CA -3′ were used. PCR reaction mixture was prepared by mixing 25 PCR products were separated in a 1% agarose gel and stained with ethidium bromide followed by examination on a UV illuminator, and images were captured by a digital camera.A 16S rRNA PCR amplification and sequencing was performed by Eurofins, Novogene (Hong Kong). The V4 hypervariable region of the 16S rRNA was amplified using specific primers 515F and 806R. All the PCR reactions were carried out with Phusion® High-Fidelity PCR Master Mix (New England Biolabs). The libraries generated with TruSeq DNA PCR-Free Sample Preparation Kit were sequenced using paired-end Illumina sequencing (2 × 250 bp) on the HiSeq2500 platform .Forward and reverse sequences were assembled and edited using BioEdit Sequence Alignment Editor Version 5.0.9. Sequence similarity was estimated by searching the homology in the Genbank DNA database using BLAST. Finally, the isolate was then identified based upon the sequence.The evolutionary history was inferred using the neighbor-joining method . The optp < 0.05.All the measurements were performed in triplicate, and the results were expressed as mean standard deviation (SD). Data were analyzed by the one-way ANOVA plus post hoc Duncan's test by SAS software . The phylogenetic tree was prepared using MEGA7 (version 7.0). Statistical significance was determined at Teff dough, Ergo, and Kocho). Among them, 56 (62.22%) isolates were found Gram-positive, endospore-negative, and catalase-negative.A total of 90 (30 from each sample) lactic acid bacteria were isolated from three different traditionally fermented Ethiopian food products  produced gas from glucose . Thus, tOut of 56 isolates, 4 isolates (7.14%), 4 isolates (7.14%), and 9 isolates (16.07%) tolerated pH values of 2, 2.5, and 3 for 3 h, respectively . Upon fup < 0.05  LAB cultures were isolated from Ergo samples. In general, the survival rate of the isolates was ranged from 38.40 to 97.11% at different pH values for 3 and 6 h incubation periods  isolates survived pH 2.0, 2.5, and 3.0 upon exposure for 3 and 6 hours, and the mean value of the treatments was significantly different at p < 0.05 . Among t periods . Isolate periods . Though All of the 4 LAB isolates  were able to survive above 90% in the presence of 0.3% of bile salt . IsolateStaphylococcus aureus ATCC 25923, Listeria monocytogenes , E. coli ATCC 25922, and Salmonella enterica Typhimurium with the inhibition zone ranged from 19.33 to 21 mm in diameters. However, E052 showed a minimum inhibition zone of diameter ranging from 17 to 19 mm against the indicator microorganisms  isolates showed tolerance to pH 2 for 6 h . Thus, tr-Eddine  have demu et al.  and Rajou et al.  who have for 3 h . Akalu e for 3 h  verifiedo et al. . The autLactobacillus strains at pH 2.5 and 3 for 3 and 6 h was found to be tolerant with a different survival rate (77.98–97.11%) and (65.58–90.49%), respectively (Shamita and Kocho), 17 lactobacilli showed 82 to 97% and 81 to 91% survival rate at pH 2.5 and 3 for 3 and 6 h, respectively. This is also similar to the report of Oh and Jung [Lactobacillus species in pH 2.5 and 3 for 3 h that were isolated from Omegisool, a traditionally fermented millet alcoholic beverage of North Korea. As reported by Haghshenas et al. [Lactobacillus strains isolated from Iranian fermented dairy products survived (71% to 76%) at pH 2.5 for 3 h. From the previous investigations, it has generally been accepted that an isolate with full tolerance to pH 3.0 for 3 h can be considered as high-acid-resistant strain with promising probiotic properties [Lactobacillus plantarum strains recovered from the faeces of breast-feeding piglets were found to be highly tolerant to pH 3 for 3 h.In this study, out of the 56 probiotic LAB isolates, only 4 (7.14%) of the isolates were tolerant to pH 2.5 for 3 and 6 h . Similarectively . Similarand Jung  who haves et al. , Lactobaoperties , 38. Veroperties  have repLactobacillus species at pH 2.5 and 3.0 for 3 h. The same authors indicated that the maximum survival rate of the six strains were 22.5% at pH 3 for 6 h. Nevertheless, complete loss of viability of lactobacilli isolated from traditional Ethiopian ergo was recorded at pH 2.5 for 6 h [Lactobacillus plantarum at pH 3 for 2 and 6 h exposure [Lactobacillus species isolated from Omegisool, a traditionally fermented millet alcoholic beverage in Korea showed considerable tolerance to bile salt [Lactobacillus strains ranged from 88% to 92% [Lactobacillus isolates obtained from Ethiopian traditionally fermented Shamita and Kocho showed remarkably high tolerance to an environment containing 0.3% bile salt. On the contrary, Boke et al. [Lactobacillus strains B3, G12, A13, and 22 exhibited low level of tolerance to 0.3% bile salts with survival rates of 36%, 33%, 3%, and 3%, respectively. In line with this, Handa [The present results are in contrast with those of Mamo et al.  who have for 6 h . In addiexposure . The 4 aile salt . Similar% to 92% . In a re% to 92%  have alse et al.  have inds, Handa  has repoLactobacillus plantarum strain JCM 1149, Lactobacillus plantarum strain CIP 103151, Lactobacillus paracasei subsp. tolerans strain NBRC 15906, and Lactobacillus paracasei strain NBRC 15889) exhibited varying degree of antagonism against Staphylococcus aureus, Listeria monocytogenes, Salmonella Typimurium, and Escherichia coli (n = 4) exhibited strong antimicrobial activity against the food-borne pathogens, where T035 (Lactobacillus plantarum strain CIP 103151) displayed the highest antagonistic activity against Staphylococcus aureus, Listeria monocytogenes, E. coli, and S. typhimurium with the inhibition zone ranged from 19.33 to 21 mm in diameters.The selected four potential probiotic lactic acid bacterial strains  originated from Borde and Shamita belonging to the genera Lactobacillus, Lactococcus, Leuconostoc, and Streptococcus were found to inhibit the growth of the test strains such as S. aureus, Salmonella spp., and E. coli O157 : H7 with inhibition zones that ranged from 15 to 17 mm in diameters. In line with this, Choi et al. [Lactobacillus strain has completely inhibited the growth of food-borne pathogens, E. coli O157 : H7 ATCC 35150, Salmonella enteritidis KCCM 12021, Salmonella typhimurium KCTC 1925, and S. aureus. Tigu et al. [Datta and Awaze, 2 Lactobacillus isolates inhibited the growth of Salmonella typimurium and Escherichia coli with inhibition zones ranging from 10.3 to 14.3 mm in diameters. In line with this, Haghshenas et al. [Lactobacillus species, particularly Lb. plantarum 15HN, showed the most efficient antagonistic activity against Staphylococcus aureus, Listeria monocytogenes, Salmonella typimurium, and Escherichia coli with inhibition zones of 11.7, 13.7, 12.3, and 12.3 mm diameters, respectively. Likewise, Rajoka et al. [Lactobacillus rhamnosus isolated from human milk inhibited the growth of Staphylococcus aureus, Salmonella typimurium, and Escherichia coli using the agar-well diffusion method with variable diameters (6 mm to 14 mm).In accordance to the current study, Bassyouni et al.  have deme et al.  have veri et al.  have repu et al.  have alss et al.  have repa et al.  have verDisparity in the antagonism activity against different pathogens indicates that probiotic strains are highly pathogen-specific and a prerequisite for probiotic potential. In general, the antimicrobial activity of probiotics might be caused by the production of antimicrobial compounds such as organic acids, ethanol, carbon dioxide, hydrogen peroxide, short-chain fatty acids, and bacteriocins. Therefore, by producing these antimicrobial compounds, probiotic microorganisms gain an advantage over other microorganisms to survive in the adverse conditions of the gastrointestinal tract .Lactobacillus strains were found to be resistant to streptomycin and kanamycin but sensitive towards tetracycline, ampicillin, and erythromycin  tract mucosa.Among the main important characteristics of probiotic bacteria, adhesion to the intestinal mucosa is required. The current results showed that the screened probiotic LAB isolates possessed o 36.30% . In agreLactobacillus, and the identified species were Lactobacillus plantarum strain JCM 1149, Lactobacillus paracasei strain NBRC 15889, Lactobacillus plantarum strain CIP 103151, and Lactobacillus paracasei subsp. tolerans strain NBRC 15906 . According to Shokryazdan et al. [L. acidophilus, L. fermentum, L. buchneri, and L. casei. In addition, Cho, Lee, and Hahm [Lactobacillus strains with potential probiotic properties from the faeces of breast-feeding piglets using 16S rRNA genes analysis. Similarly, other recent studies (Dowarah et al. [The phylogenetic analysis and the 16S rDNA sequencing assigned all the LAB isolates with probiotic properties to genus n et al. , the resLactobacillus isolates from Ergo, Kocho, and Teff dough were found to have potentially probiotic characteristics. It is suggested that these strains can be good candidates for food industries as prospective probiotic cultures with other human health benefits. However, further research work is needed to evaluate the in vivo probiotic characteristics of these potential lactic acid bacteria.In the present study, four


To improve the quality and safety of brown fermented milk (BFM), the formation and alterations of potentially harmful Maillard reaction products (MRPs), including 3-deoxyglucosone (3-DG), methylglyoxal (MGO), 5-(hydroxymethyl)-2-furfural (HMF), acrylamide and flavour components were investigated during the browning, fermentation and commercial storage. MRPs were shown to be produced mainly during the browning stage. The levels of different substances varied during the fermentation and commercial storage stage. The proportion and type of carboxylic acids in the flavour components significantly increased during the fermentation stage. Browning index of milk during the browning stage was shown to be positively associated with the 3-DG (Pearson’s r = 0.9632), MGO (Pearson’s r = 0.9915), HMF (Pearson’s r = 0.9772), and acrylamide (Pearson’s r = 0.7910) levels and the total percentage of the flavour components from four different categories (Pearson’s r = 0.7407). Changes in physicochemical properties of BFM during production not only contribute to predict the formation of potentially unhealthy MRPs, but also Lactobacillus species used for the fermentation should be carefully selected to improve the quality of this product. Lactobacillus casei fermentation [Lactobacillus casei has been widely used in other dairy products such as cheese and ice cream [Lactobacillus casei have positive effect on consumer’s health, such as modulates immune function and improves constipation [Brown fermented milk (BFM) is a fermented beverage made from the skimmed milk powder and reducing sugar, which is produced using Maillard browning and entation . BFM hasentation . Lactobace cream ,3. Studitipation ,5.Colour and flavour of many food items, such as bread, coffee, chocolate and cakes, are based on the Maillard reaction during food manufacturing ,7. The bα-DCs are highly reactive intermediates in the Maillard reaction, which can convert amino acid and protein modifications to advanced glycation end products (AGEs). AGEs have been shown to reduce the nutritional quality of foods and to be involved in the development of several diseases, such as neurodegenerative diseases, diabetes and others ,15,16. HMicroorganisms may affect the MRPs during fermentation ,21. MethTherefore, the aim of this study was to investigate the formation and alterations of 3-DG, MGO, HMF, acrylamide and flavour components during the manufacturing and commercial storage of BFM, in order to help improve the quality and safety of BFM and to assist in developing a regulatory strategy for this food item.−1 at 180 min after the initiation of the reaction, showing that these compounds are produced during the BFM browning. Additionally, 3-DG content was approximately 20-fold higher compared with that of MGO, indicating that the α-DCs with C6 skeleton are formed at higher levels during browning. Our results are in agreement with the reports examining the browning of dairy products, biscuits, and other foods [−1 with time, which supports previous studies analysing biscuit baking [The formation of 3-DG, MGO, HMF and acrylamide during the browning, fermentation and storage of BFM is presented in er foods ,27,28. Ht baking . Howevert baking ,31,32,33During fermentation, we observed a decrease in 3-DG concentration with time, which was shown to be significantly different. Similar results were reported by , who demDuring the commercial BFM storage , 3-DG levels increased during the first five days and decreased afterwards, which was comparable to its previously reported changes in a probiotic drink, but its levels in other dairy products were shown to be lower ,27. MGO Total levels of flavour components were identified during the browning (180 min) and fermentation (72 h) of BFM. Browning stage flavours components mainly included carboxylic acids, followed by aldehydes, alcohols and ketones . The proThe number of flavour components identified in the fermentation stage was higher than that in the browning stage, of which the carboxylic acid species were particularly numerous, such as acetic and butanoic acids. The total proportion of carboxylic acids, aldehydes, alcohols, and ketones gradually increased during fermentation B. These a*, b*, L*, ΔE* and BI). During browning stage, a* value increased from −2.16 ± 0.15 to 4.19 ± 0.06, while b* increased from 10.32 ± 0.08 to 16.65 ± 0.05 with the browning time, indicating that reconstituted milk lost greenness and blueness and became more red and yellow, respectively, during browning. The L* value, indicating the lightness of the sample, decreased from 88.79 ± 0.2 to 80.41 ± 0.04 with the browning time, indicating that the samples became darker in colour. Due to these results, the overall ΔE* values and BIs increased as well. The browning of the milk is primarily due to the Maillard browning of lysine and reducing sugars in the browned milk [The values of the colour parameters obtained during BFM browning and fermentation are presented in ned milk . No signColour is an important indicator of browning, and it is usually used to determine the finalisation of the browning process. Maillard reactions are directly involved in the browning of BFM. BIs, MRP and flavour component proportions change with the temperature during Maillard reaction, as shown in Positive correlations between the BIs and 3-DG levels (Pearson’s r = 0.9632), MGO (Pearson’s r = 0.9915), HMF (Pearson’s r = 0.9772) and acrylamide levels (Pearson’s r = 0.7910) were observed, together with that between the BIs and the total percentage of flavour components from four described categories (Pearson’s r = 0.7407), indicating a strong correlation (|r| > 0.95) between the BIs and 3-DG, MGO and HMF levels during the browning of BFM. However, the correlations observed between the BIs and acrylamide and flavour components were weaker, indicating that the degree of browning can directly reflect the levels of potentially harmful MRPs during BFM browning. This may help improve the monitoring of MRP formation during the BFM manufacturing. Previous studies support the conclusion that the MRP levels correlate with the degree of browning in foods [The results of these MRPs, flavour and colour changes during BFM manufacturing and commercial storage provide a fundamental basis for improving the quality and safety of BFM, ensuring a stable, sustainable and healthy development of the dairy industry.3-Acrylamide was supplied by Cambridge Isotope Labs , while o-phenylenediamine (OPD) was obtained from J & K Scientific . Potassium hexacyanoferrate and zinc acetate were supplied by Sinopharm . Water (HPLC grade) was prepared using a Milli-Q Integral purification system . Acetonitrile (HPLC grade) was supplied by Merck . Skimmed milk powder  was purchased from the local supermarket. Lactobacillus casei 01 was supplied by Chr. Hansen . Oasis HLB solid phase extraction (SPE) cartridges  were supplied by Waters .MGO, acetic acid (HPLC grade) and HMF were purchased from Sigma-Aldrich . Acrylamide and 3-DG were supplied by TRC . d−1) in water at 40 °C, and then glucose (40 g·kg−1) was added. The mixture was gently stirred for 30 min and heated to 65 °C. The sample was mixed homogenously at 20 MPa and browned at 95 °C for 180 min, which was followed by decreasing the temperature to 37 °C with cold water.Skimmed milk powder was reconstituted to skim milk samples . Afterward, the fermented curd was broken and cooled rapidly to 4 °C. Finally, the fermented milk, syrup and flavouring were mixed and homogenised at 25 MPa and stored in a refrigerator at 4–7 °C.The browned sample was inoculated with the BFM preparation trials were performed in triplicate, and the mean values were reported.−1) were added to a capped brown bottle. The mixture was incubated in water at 60 °C and stirred at 50 rpm. Following a 30-min incubation, the mixture was purified and concentrated using preconditioned Oasis HLB SPE cartridges. The samples were centrifuged at 10,000× g for 5 min and filtered through 0.22 μm nylon for LC/MS/MS analysis.Preparation of 3-DG and MGO was performed as previously described , with mi−1 at 350 °C) and collision gas. The capillary voltage was 4 kV, while the dwell time was 80 ms for each transition. Chromatographic separations were performed on a Polar-RP 80A column  maintained at 25 °C. A flow rate of 0.3 mL min−1 was used. The mobile phase consisted of phase A (0.1% formic acid aqueous solution) and phase B (methanol). The gradient was 20–80% B from 0–15 min and 80–20% B from 15–30 min. The sample injection volume was 5 μL. Transitions: 3-DG: m/z 199.1→m/z 145.1 and MGO: m/z 145.1→m/z 77. LOQs of 3-DG and MGO in samples were 11.5 μg L−1 and 1.5 μg L−1, respectively, while the LODs of 3-DG and MGO were 3.5 μg L−1 and 0.5 μg L−1, respectively. The recovery of these compounds was 86.6–92.7% and 84.8–90.2%, respectively. RSDs were 1.3–2.0% and 2.1–4.7%, respectively.The derivatised and purified samples were analysed using an Agilent 1200 Series HPLC system coupled with Agilent 6410 triple-quadrupole MS  with electrospray ion source in positive ion mode and MRM mode. Nitrogen was used as the nebuliser , drying  and analysed with HPLC.Samples (3 mL) were mixed with water (6 mL) and treated with 0.5 mL Carrez I and II solutions, each. The mixtures were vortexed for 3 min and then centrifuged at 4 °C for 15 min at 6000× −1, and acetonitrile:water mixture  was used as a mobile phase for 20 min. The injection volume was 20 μL, and the UV detection wavelength was 284 nm. LOD and LOQ of the samples were 0.04 mg·L−1 and 0.12 mg·L−1, respectively. Recovery rates and RSDs were 80.2–85.7% and 4.0–5.5%, respectively.HMF was determined as previously described , with sog for 5 min to obtain the clear supernatants. Acrylamide was purified from the supernatants using the preconditioned Oasis HLB SPE cartridges. Supernatants were centrifuged at 10,000× g for 5 min, filtered through a 0.22-μm nylon filter and analysed using LC/MS/MS.Acrylamide was prepared as previously described , with miv/v), used for 15 min, and the flow rate was 0.2 mL min−1. The injection volume was 2 μL. Transitions used were acrylamide: m/z 72→m/z 55 and d3-acrylamide: m/z 75→m/z 58. LOD in samples was 3.0 μg L−1 and LOQ was 9.1 μg L−1. Recovery rates and RSDs were 83.8–89.7% and 1.9–4.5%, respectively.Acrylamide quantitative analysis was conducted according to the method developed previously ,43. SampΔE* . Each sample was analysed six times. The ΔE* and BI (browning index) were calculated using the following equations [Colour measurements were obtained using Konica Minolta Spectrophotometer CM-5 colorimeter . The measurements were performed using a cuvette, colour parameters were CIE-L* (lightness), a* (greenness/redness), b* (blueness/yellowness) and quations .(1)ΔE*=−1, transfer line temperature was 280 °C and the ionisation voltage was 70 eV. A full-scan mode was run from m/z 30 to 500. Oven temperature was 40 °C for 3 min, followed by an increase to 150 °C at the rate of 3 °C·min−1, maintained at 150 °C for 2 min, increased to 250 °C and then maintained at 250 °C for 5 min. Mass spectra of unknown compounds were identified using the NIST MS Search 2.0 database. The percentages of compounds were calculated by the area normalisation method without considering, response factors.Eight millilitres of samples were placed in a 50 mL vial. Volatile compounds were extracted by headspace solid-phase microextraction (HS-SPME) for 30 min at 55 °C using a DVD/Car/PDMS fibre  with a multipurpose sampler. This method has been widely used to extract volatile and semivolatile components from dairy products . Then, sp < 0.05.Data were analysed using the analysis of variance (ANOVA) and Duncan test. Statistical analyses were performed using IBM SPSS 19.0 for Windows . Correlations between the results were performed using OriginPro 2016 . The values were considered statistically significant at Here, for the first time, the changes in the levels of potentially harmful MRPs, flavour components and colour were monitored during the manufacturing and storage of BFM. During BFM browning, MRPs  were shown to be generated through the Maillard reaction and their levels significantly increase with the browning time. The changes in the MRP levels were demonstrated to correlate with the changes in the BIs, showing that these indices reflect the accumulation of potentially harmful MRPs in BFM and can be used for the monitoring of their levels, in order to improve the quality and safety of BFM. During fermentation, 3-DG levels in BFM decreased significantly, while the MGO and HMF levels increased; however, the levels of the MRPs did not significantly change during the commercial storage of BFM. Although alcohols, carboxylic acids, ketones and aldehydes are formed during the browning and fermentation stages, the most abundant components—carboxylic acids—are formed during fermentation. These results indicate that Lactobacillus fermentation plays an important role in the alterations of the MRP levels and flavours during BFM processing and storage, which depends on the metabolism of Lactobacillus species used for the production. The production of α-DCs was shown to be affected by Lactobacillus strains , and the