Obviousis field .intrinsic δ-curvatures of Chen and the extrinsic squared mean curvature of submanifolds in a real space form. Later, these sharp inequalities  have been extended for different types of submanifolds in various ambient spaces, for example, arbitrary Riemannian manifolds, Kähler manifolds, and Kenmotsu space forms .δ-Casorati curvatures, initiated in [extrinsic δ-Casorati curvatures and the intrinsic scalar curvature of submanifolds in real space forms were proved in [Casorati ideal submanifolds. Recently, Lee et al. [Kenmotsu geometry is an area of contact geometry initiated by Kenmotsu in [On the other hand, the study of iated in ,6, offeriated in . Koenderiated in  and Versiated in  studied iated in . A geomeiated in . Very reiated in  gave a groved in ,6. Laterroved in ,17,18,19e et al.  studied motsu in , with mamotsu in . This gemotsu in , where tmotsu in .statistical manifold and conjugate connection in the basic study on information geometry [In 1985, Amari defined the notions of geometry . It is wgeometry . We notigeometry . On the geometry  the conjgeometry  a new fogeometry ,30,31,32geometry , Hessiangeometry , statistgeometry , complexgeometry ,36,37), geometry , and quageometry . For thegeometry  and a gegeometry  for submgeometry . These igeometry  in the sgeometry  proved aKenmotsu statistical manifold, which is locally obtained as the warped product of a holomorphic statistical manifold and a line. They proved that a Kenmotsu statistical manifold of constant Recently, Furuhata et al.  introducstatistical structure [X, Y, ZX, Ytorsion-free.Let tructure  on M¯ ifstatistical manifold [X, Y, Zdual connections.A manifold  is a RieRemark 1.If 1. 2. 3. Any torsion-free affine connection where M be an g the induced metric on M. Then, the Gauss formulas are as follows:h and imbedding curvature tensor of M in , and the imbedding curvature tensor of M in , respectively.Let R and Gauss equation for the submanifold M of Denote by n ∇¯) is (2)g¯g¯Sσ(π)=scalar curvaturep is given bynormalized scalar curvatureM is defined asLet mean curvature vector fields of M, denoted by H and The M, respectively, with respect to the Levi–Civita connection M.We remark that, from Equation , we derisquared mean curvatures of the submanifold M in Then, the Casorati curvatures of the submanifold M in h and The L be an s-dimensional subspace of L. Hence, the Casorati curvatures L are given by:Let normalized δ-Casorati curvaturesThe dual normalized M in Similarly, we can define the generalized normalized δ-Casorati curvaturesM in r, different from The fined in  as:δC generalized normalized totally geodesic with respect to the connection h of M for We recall that a statistical submanifold ntically .almost contact metric manifold with the structure tensors S¯ For any real number r such that where (ii) For any real number r such that where In addition, the equality cases of Equations In additiProof. From Equations –4), we , we 4), Let Equation , we obtaBecause L is a hyperplane of Let L is spanned by the tangent vectors Suppose that the hyperplane quations  and 21)L is spanMoreover, from Equation , we get:Let We study the constrained extremum problemThe first order partial derivatives system is:Q, is the critical point with the expression:The system solution, satisfying the constraint p be an arbitrary point of Q, Q in Let b is a real constant, namelyThe Hessian matrix of Q is totally geodesic in As the hyperplane L of L of However, according to the Remark 2, the critical point quations  and 16)16). FirsWe achieve quations  and 16)hij0α=0, Remark 3.Equation  The normalized δ-Casorati curvatures where (ii) The normalized δ-Casorati curvatures where Moreover, the equality cases of Equations Moreover,Equation , i.e., MProof. The conclusion follows immediately from Theorem 2, taking account of Equations –10).  ☐.  ☐10). Remark 4.Corollary 1 is the statistical counterpart of the Theorem 1 from [m 1 from .Remark 5.Any Kenmotsu manifold can be obtained locally as follows ). Let 1. The triple . Then, it follows that [, Proposition 3.9)).) as in  For any real number r such that where (ii) For any real number r such that where Moreover, the equality cases of Equations Moreover,Equation , i.e., MCorollary 2.Let (i) The normalized δ-Casorati curvatures where (ii) The normalized δ-Casorati curvature where Moreover, the equality cases of Equations Moreover,Equation , i.e., MExample 1.We consider the Kenmotsu manifold  . We recall thatand the structure tensors andξ) from ).=−1Next, letquations  and (26)It is well known that many applications of Amari’s dual geometries involve one or more submanifolds imbedded in a manifold . In partanifolds , Sasakiaanifolds  and cosyanifolds .


Flow cytometry was used to detect the phagocytic function of CD4, CD8 cells, and AM in the bronchopulmonary lavage fluid. The 30 and 100 mg·kg-1 groups had an up-regulation effect on CD8 (p < 0.05), with no significant effect on macrophage phagocytosis. The 300 mg·kg-1 group had an inhibitory effect on CD8 and macrophage phagocytosis (p < 0.05), with no significant difference in CD4 between groups. Further investigation was conducted to evaluate the effect of EO on immune function in rats by detecting blood T, B, and NK cells using flow cytometry, and blood IgA, IgG, IgM, and IFN-γ levels by ELISA. High dosage of eucalyptol significantly reduced the proportion of blood B and NK cells (p < 0.05). IgA was decreased in the 100 and 300 mg·kg-1 groups (p < 0.05). There are no significant differences between the number of T cells and the IgG, IgM, and IFN-γ levels between experimental and control groups. Rational use of EO containing eucalyptol can improve the immune function of the respiratory tract and the body immunity, while high dose could have damaging effects, through modifying the phagocytic function of CD8 cells and reducing the proportion of blood B cells, NK cells, and IgA.Eucalyptol , the major constituent of eucalyptus oil (EO), was used in traditional medicine as a remedy for colds and bronchitis. This study aimed at clarifying the effect of eucalyptol on respiratory immune function of CD8 and CD4 cells, and alveolar macrophages (AM). Thirty male Sprague-Dawley rats were divided into experimental and control groups. The drug was given once a day for 3 weeks and the experimental group was divided according to the eucalyptol dose into: 30, 100, and 300 mg·kg Eucalyptus globulus, commonly known as blue gum tree, is a species of tall, evergreen tree. It has a wide distribution in China, and trees in Yunnan Province in particular has a high content of 1,8-cineole (more than 70%) in leaves, commercially used for the production of essential oils in the pharmaceutical and cosmetic industries -exacerbations , as wellIt thus appears that more details behind these effects need to be clarified. This study aims to explore the effect of EO on the respiratory function of rats to provide a theoretical basis to clarify the effect of the drug on immune function of CD8, CD4 cells, and alveolar macrophages (AM), through analysis of blood T cells, B cells, and NK cells by flow cytometry, and blood IgA, IgG, IgM, and IFN-γ levels by ELISA.-1 of eucalyptol, dose was in eucalyptol, i.e. 30 mg·kg-1 was 36.1 mg·kg-1 EO), and six in the control group . EO or canola oil was administered intra-gastrically once a day for 3 weeks, at the end of which, the left and right main bronchi were separated, and bronchopulmonary lavage was performed separately. Below procedures were carried out to ensure no contamination from circulating blood. Trachea was exposed surgically, and the right primary bronchus was clamped. A tube was inserted to the left primary bronchus, and 2 ml saline (0.025 g·ml-1) was used to flush alveoli and retrieved. The retrieved volume is around 1.5 ml. The flush was repeated three times. Then, same procedure was applied to the right primary bronchus. The retrieved fluids are mixed and combined for the final experiment. Before bronchi collection, 4 ml of heparinized blood was taken via cardiac puncture for testing.Thirty Sprague-Dawley male rats weighing 200–250 g were purchased from Experimental Animal Center of Xi’an Jiaotong University . The experimental design was similar to a previous study, including dosage and period of treatment . InstituE. coli preparation (5 × 108/ml), Percoll separation solution, CD3, CD4, CD8, CD19, and CD56 monoclonal antibodies, allophycocyanin (APC), phycoerythrin (PE), Fluorescein (FITC), goat anti-Mouse IgG (H+L), and sIgA detection kit were purchased from Thermo Fisher Scientific . FACS hemolysin and paraformaldehyde were purchased from Wuhan Booute Biotechnology Co. Ltd. .Extracted EO containing 83% of eucalyptol in canola oil was a gift from Beijing Jiuhe Pharmaceutical Co. Ltd. . It was a light-yellow volatile liquid, and insoluble in water. Red blood cell lysate, 2 incubator from Thermo Fisher Scientific were used in the experiments.A Cytomics FC 500 flow cytometer from Beckman Coulter Inc. , an ELX800TM plate-reader from Biotek Instruments Inc. , and a Forma 381 CO6/ml, and inoculated into a petri dish with a 10 cm diameter. It was incubated at 37°C for 2 h in a CO2 incubator. Non-adherent cells were washed away with D-Hank’s. A bacterial solution of 200 μl was added to each sample (2 ml) and incubated at 37°C for 1 h. Then, 200 μl of the phagocytic bacterial liquid was added, and CD4 and CD8 monoclonal antibodies were added, respectively, and incubated at 4°C for 1 h in the dark. Two ml of FACS hemolysin was added, and the mixture was allowed to stand in the dark for 10 min. It was then centrifuged at 1,500 rpm for 5 min, the supernatant discarded, and 2 ml PBS was added. The mixture was then centrifuged at 1,500 rpm for 5 min, the supernatant discarded, and 2 μl paraformaldehyde solution added for flow cytometry analysis.After isolating the AM in the lavage fluid, its concentration was adjusted to approximately 1 × 106/ml and inoculated onto a Petri dish. It was incubated at 37°C for 2 h, after which non-adherent cells were washed away with D-Hank’s. A bacterial solution of 200 μl was added to each sample and incubated at 37°C for 2 h. Flow cytometry was used to detect phagocytosis.The lavage solution was transferred to a 50 ml centrifuge tube, centrifuged at 1,500 rpm for 5 min, and the pellet was resuspended in RPMI 1640 containing 10% fetal bovine serum with AM at a concentration of 1 × 10CD3, CD3/CD19 goat anti-Mouse IgG, CD3/CD56 goat anti-mouse IgG were added to a 200 μl sample and incubated at 4°C for 1 h in the dark. Two ml of FACS hemolysin was added; the mixture was allowed to stand in the dark for 10 min, and then centrifuged at 3,000 rpm for 5 min. The supernatant was discarded, 2.0 ml of PBS were added, centrifuged at 3,000 rpm for 5 min, with the supernatant discarded, and 100 μl of 2% paraformaldehyde solution were added for flow cytometry analysis. T cells, B cells, NK cells, and other white blood cells were detected and counted by the flow cytometer and percentages T cells, B cells, and NK cells in total white blood cells were calculated by the machine automatically.IgA, IgG, IgM, and IFN-γ in samples were measured according to the ELISA kit manufacturer’s instruction using a microplate reader for final colorimetric reading.post-hoc test to analyze differences between groups using SPSS Statistics software . A P value of <0.05 was considered statistically significant.Data was checked for a normal distribution first and then a one-way analysis of variance (ANOVA) was performed with least significant difference (LSD) The results in The results in The blood IgA levels of the high and medium dose groups were significantly lower than the control group , which may help the body fight inflammation. However, the high dose (300 mg·kg-1) had an inhibitory effect on CD8, which may reduce the body immunity. The high-dose also significantly inhibited the phagocytosis of rat alveolar lavage fluid macrophages (P < 0.05), while the medium (100 mg·kg-1) and low (30 mg·kg-1) doses had no significant effect on macrophage phagocytosis. There is no significant effect on rat alveolar lavage fluid CD4 cells with EO administration.It is suggested that EO, with its dominant ingredient eucalyptol, might be beneficial to cell mediated immunoregulatory agents in infectious diseases, immunosuppressive pathologies, or after cancer therapy . In the -1 groups, and there was no significant change in IgG, IgM, and IFN-γ levels (P > 0.05). This study’s results suggest that the high dose of EO can inhibit the body’s immune function, however, the literature to date showed varied results. For example, Lu and co-workers identified 100 mg·kg-1 of EO as the most effective dose that produced significant decrease in severity of bronchitis symptoms, less inflammatory cells infiltration, and reduced airway mucin secretion in rat chronic bronchitis caused by lipopolysaccharide (LPS) (-1 (The results of this study showed that the high dose of EO had an inhibitory effect on the proportion of B cells and NK cells in the blood (P < 0.05), and no significant effect was detected on the proportion of T cells (P > 0.05). Blood IgA decreased significantly (P < 0.05) in 300 and 100 mg·kgde (LPS) . SimilarLPS) , as well as in vivo in monocytes/granulocytes from peripheral blood of immuno-competent or immuno-suppressed rats (in vivo after EO administration. The combined 5-fluorouracil (5-FU)/EO treatment raised the phagocytic activity of the granulocytic/monocytic system, which was significantly decreased by the chemotherapy and inhibited the 5-FU induced myelotoxicity  in the 300 mg·kgcretions . IgA concretions . Medium in vivo basis in a healthy animal model, which will benefit further clarification of the effect of EO  on the immune function.In conclusion, regulation of immune homeostasis is a delicately and tightly regulated process. The present study reports that high dose of EO can damage the immune function of the respiratory tract and the body immunity, possibly through B cell modulation, which results in an increase of plasma cells, inflammatory intermediates, and immune complexes ; while lThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the ethics committee of 521 Hospital Ethics Committee.Conceptualization, JS, LZ, JL, and YY. Methodology, JS, ZY, YW, QT, and MZ. Validation, JJ, CL, MY, AH, and JL. Formal analysis, JS, ZY, and JL. Investigation, JS, LZ, ZY, and YW. Resources, JS and LZ. Data curation, JS, ZY, YW, and QT. Writing—original draft preparation, JS, AH, and JL; writing—review and editing, JS, WW, and JL.This research was funded by Shaanxi Province Science and Technology Programme project number 2016sf-154.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.


Cryptococcosis is a disease of wide geographic distribution. It is most critical when it affects immunocompromised patients, with AIDS, tuberculosis or other diseases that require prolonged hospitalization.C. neoformans isolated in a patient with non-Hodgkin’s Lymphoma, from Manaus, Brazil.This study described a case report, molecular epidemiology, the phylogenetic relationship, along with antifungal susceptibility test of a new ST 623 of C. neoformans was susceptible to all antifungal drugs tested. Our results showed that ST623 new clone has no evident evolutionary proximity to any other ST of the VNI subtype group identified in Brazil.The new In the context of phylogenetic analysis, this new genotype belongs to VNI subtype, and subsequencing complete genome studies are necessary to better understand the phylogenetic relationships amongst STs in this group. Cryptococcus neoformans and C. gattii [Cryptococcosis is a serious disease possessing a wide geographic distribution, with a global burden of 957,900 cases of cryptococcal meningitis per year, resulting in 624,700 deaths . This di. gattii . They ar. gattii , 5–8.6 inhabitants. In Mexico, the prevalence in patients with immunosuppression caused by different diseases is 21%; while in Venezuela it is 19%; and in Argentina, the prevalence is 20% [6 inhabitants, while in Northeastern Brazil; it is considered endemic [In Latin America, there are records of 2400 deaths per year . In coune is 20% . In the  endemic .C. neoformans (ST 623).As it is considered an opportunistic mycosis, and potentially severe in certain patients, the availability of more sensitive tests for an enhanced routine laboratory diagnosis and treatment efficacy are indispensable for the proper control and monitoring of cryptococcosis. This rapid communications describes the molecular epidemiology, the phylogenetic relationships and the results of antifungal susceptibility test of a new ST (Sequencing Typing) from This study was approved by the Foundation Human Research Ethical Committee (CEP/HEMOAM) under CAAE Nº 73548017.5.0000.0009, and all methods were performed in accordance with the relevant guidelines and regulations. Patients and a parent or guardian of any child participant enrolled in the study provided their written informed consent, before specimens were collected and the results and data were used for the management of each respective patient.A 72 years old, retired farmer, living in the city of Manaus-Amazon-Brazil, sought care at the State Center of Reference in Dermatology—”Alfredo da Matta” Foundation; reported that he observed a tumor approximately 4 cm in diameter in the occipital region, among others distributed diffusely throughout the body. A posterior cervical lymph node biopsy was performed and he was diagnosed with non-Hodgkin lymphoma. During the same period, an abdomen scan was ordered, and he was referred to the Foundation Hospital of Hematology and Hemotherapy Blood Center of Amazonas (HEMOAM), a reference center for Onco-hematologic Diseases.On May 06, 2017, he was treated at HEMOAM with generalized pruritus symptom and palpable liver. He was medicated with antihistamine and antipruritic. On July 06, 2017 after consultations with hematologists, blood tests were performed complementary to the diagnosis to start chemotherapy of LYMPHOMA. On Dezember 06, 2017 the patient presented: abdominal computed tomography (TC Abd): cervical region with expansive/infiltrative lymph node mass, determining almost complete thrombosis of the left internal jugular vein + pulmonary emphysema + multiple mediastinal and axillary lymph node enlargement involving visceral/level and left internal mammary chain, retroperitoneal and mesenteric, inguinal, thoraco-lumbar and internal jugular vein thrombosis (tumor compression); and Hepatitis C (HCV) reagent. The patient was submitted to chemotherapy protocol from August 08, 2017 until October 10, 2017.3; neutrophil: 9.8%; leucocyte: 2400/mm3; positive blood culture for Cryptococcus neoformans, and the diagnosis of Aplasia after chemotherapy. The patient was admitted for parenteral treatment with the antibiotics cefepime + clarithromycin and blood cultures were performed. However, due to his worsening clinical conditions, he was transferred to a University Hospital in Manaus on November 06, 2017.Still under the chemotherapy protocol for lymphoma, the patient presented a fever 37.5 °C (99.5 °F), coryza, sneezing, and elevation of transaminases with rates three times above normal limits. Subsequently, he attended the HEMOAM emergency room reporting weakness, a febrile peak of 38 °C (100.4 °F), vertigo, lack of appetite, nausea and epigastralgia. The laboratory findings were as follows: Haemoglobin (Hb): 12.6 g/L; Hematocrit (Ht) 36.8%; Platelet: 101,000/mmRemaining in the ward of the University Hospital, the patient started treatment with Fluconazol 200 mg for 14 days, after culture result identifying the presence of yeasts. The patient continued in the ward, with worsening clinical evolution of: pulmonary infection + Neurological syndrome  + Hydroelectrolytic disturbance  + Intestinal constipation + Plaquetopenia. Cefepime and Clarithromycin empirically therapy was discontinued, substituted by the Meropenem antibiotic. From November 07, 2017 to November 09, 2017 the clinical condition evolved to cardio-respiratory arrest in Asystole, followed by resuscitation and transferred to the Intensive Care Unit (ICU); still critical under antibiotic therapy, another cardiac arrest on November 09, 2017 and, due to respiratory septic shock, evolved to death.C. neoformans were stored at − 80 degrees Celsisus (°C), in a cryotube with Brain Heat Infusion Broth (BHI)  + 20% Glycerol for further molecular testing.Following the two positives blood cultures , the subculture was carried out in modified Sabouraud dextrose agar medium  and lateSJ01-5´-TTAAGACCTCTGAACACCGCTCC-3´ (10 µM) and 0.5 µl of Taq DNA polymerase (5 U/ml) (Invitrogen), Martins et al. [DNA was extracted using DNeasy Blood & Tissue Kit  according to the manufacturer´s instructions. The molecular identification of the fungus was determined applying the enzymatic restriction protocol (PCR–RFLP). The reaction for amplification of the gene was carried out in a reation volume of 25 µl. Each reaction contained 2 µl of genomic DNA extract (28 ng); 2.5 µl of 10× PCR buffer; 2.5 µl of MgCl2 (50 mM); 5 µl of DNTp mix (2.5 µM) ; 0.5 µl of the primers FW-5´-ATGTCCTCCCAAGC CCTCGACTCCG-3´; s et al. . Reactios et al. .http://mlst.mycologylab.org/). The analysis of Multilocus Sequence Typing (MLST) for C. neoformans was performed according to the site protocol (http://mlst.mycologylab.org/).The PCR product was demonstrated by SYBR™ safe (Invitrogen) DNA gel electrophoresis on 1.5% agar gels. Following this, sequencing was performed using the ABI PRISM 3130 XL Genetic Analyzer , according to manufacturer’s instructions. Sequences generated in the forward and reverse directions were read using Geneious v.11 software. A consensus sequence was extracted after being checked for discrepancies or mutations. The results were compared with the genomic DNA deposited in the site database , totaling 174 samples. The nucleotide sequences of the seven alleles of the 174 samples were edited and aligned by the MEGA X program using the MUSCLE tool [https://itol.embl.de) [Sequences of the MLST genes from CLE tool , 14. Theembl.de) . To recoCryptococcus neoformans. MIC showed susceptibility to all antifungal tested, with values of 1 µg/ml for fluconazole, 2 µg/ml for flucytosine and 0.5 µg/ml for amphotericin B. PCR–RFLP protocol identified the molecular type VNI, and comparative analyzes with the sequences deposited on the MLST website, made it possible to identify a new clone of Cryptococcus neoformansST623. GenBank accession numbers of the C. neoformans allele from our case are MN065812, MN065813, MN065814, MN065815, MN065816, MN065817, and MN065818.The microbiological test realized identified C. neoformans STs results studied by Muñoz et al. [It was also observed that nucleotide diversity Fig.  was highz et al. . Of the A strong positive correlation of r = 0.80 was observed between nucleotide diversity and the number of MLST alleles, as well as the number of haplotypes and the MLST alleles (r = 0.85). The fact that IGS1 corresponds to an intergenic region, contributes to the high nucleotide diversity observed in this region, characterizing it as a hypervariable marker, as described by Muñoz et al. , 17  and the others groups studied. It was observed the proximity of ST623 and STs 341,142, 55, 264, 322, 54 and 84, where these topologies are statistically supported by the high bootstrap values. It was also possible to reconstruct the ancestral relationship of the others monophyletic groups (green) formed by STs 145,153,147,143,145,184,144,149,148,182,171,152,151,183,150 and the others STs of the VNI subtype. Our results showed also that ST623 clone, has no evident evolutionary proximity to any other ST of the VNI subtype group, identified in Brazil by Rocha et al. and Ferreira-Paim et al. , 18, froThe new ST623 genotype was isolated from a patient with non-hodgkin´s lymphoma, which evolved to death. In the evolutionary context of phylogenetic analysis, this genotype belongs to VNI subtype; however, the relationships between the STs in this group only can be elucidated after an approach with complete genome sequencing (WGS) studies.


Corydalis yanhusuo W.T. Wang alkaloids are mainly divided into three categories: protoberberine, prototropine and aporphine alkaloids. Therefore, we have taken into account these three alkaloid contents when extracting and purifying crude drugs, which is essential for the quality control of C. yanhusuo and its derivative products. Herein, we investigated the feasibility of the Q-marker uniform design method in the optimization of the extraction and purification of C. yanhusuo. In this study, Q-marker-based comprehensive scoring (CS) and uniform design methods were used to optimize the extraction and purification of C. yanhusuo. The inspective factors included the solvent concentration, pH, liquid–solid ratio, extraction time and frequency. Then 8 Q-markers, the total alkaloid extraction and the extraction rate were considered as the evaluating indicators during the process. The results indicated that the optimal reflux extraction process of C. yanhusuo was as follows: a total amount of 20 times 70% ethanol (pH = 10 of diluted ammonia), heating and refluxing twice, and extracting each time for 60 min. The results of nine-resin screening exhibited that NKA-9 macroporous adsorption resin had the best separation and purification effect on 8 kinds of C. yanhusuo alkaloids with stronger enrichment. During the optimal enrichment process and elution conditions, the water-soluble impurities were washed off with 5 BV distilled water at a volume flow rate of 2 BV/h, and the elution solvent was 70% ethanol with an elution volume flow rate of 1.5 BV/h and an elution dosage of 12 BV. Additionally, the total alkaloids of the obtained product were over 50%, of which eight quality markers were (+)-corydaline 3.55%, tetrahydropalmatine 3.13%, coptisine 2.79%, palmatine hydrochloride 2.24%, dehydrocorydaline 13.11%, (R)-(+)-corypalmine 2.37%, protopine 2.71% and glaucine 14.03%. Our data demonstrated that the optimal extraction and purification process was stable and feasible, which was expected to provide an experimental basis and reference for the industrial production of C. yanhusuo. Corydalis yanhusuo W.T. Wang is a species in the Corydalis genus, and its tuber has been used as a Traditional Chinese Medicine. C. yanhusuo has a warm nature (Chinese medicine theory) and a pungent and bitter flavor. As an important therapeutic agent, C. yanhusuo can affect blood circulation promotion and air flow in the body  solutions with different concentrations were prepared by accurate drawing and measured according to the above chromatographic conditions. The peak area of the chromatogram was recorded, and the regression equation was obtained by using the peak area value as the ordinate (Y) and the amount of the reference substance as the abscissa (X).2SO4 was added to make 10 mL with the pH 4.0 buffer. Then, 1 mL of the H2SO4-extract solution was placed into a 100 mL separatory funnel and 4.0 mL of pH 4.0 disodium hydrogen phosphate-citrate buffer solution and 7.0 mL of bromocresol green acid dye solution were added. The solution was mixed evenly, 10 activated silica gel and 5 mL of chloroform were added for 1 min; the solution was held for 5 min, and then the lower layer solution was removed to obtain the test solution.The extracts were taken under different processing conditions, filtered and then diluted to 200 mL. Then, 1 mL was placed into a volumetric flask, and 0.5 mL 5% HThe tetrahydropalmatine reference solutions were accurately transferred  to volumetric flasks and then diluted with buffer solution to volume. Each reference solution was taken and processed according to the previously mentioned chromatographic conditions. The absorption value was measured at λ = 417 nm. The linear regression C of the tetrahydropalmatine was considered the X-axis, and the absorption value A was the Y-axis.C. yanhusuo/maximum content × 0.35 + eight Q-markers content M/highest content M × 0.45 + creaming rate/highest creaming rate × 0.25. The total evaluation (M) of the 8 Q-markers was calculated by the mass fraction weight coefficient method [In this study, the comprehensive scoring (CS) method was used to evaluate the assignments, in which total alkaloids, the paste-forming rate and 8 Q-markers were used as indicators. Total evaluation (Y) = total content of t method .M = protopine × 0.05 + coptisine × 0.09 + palmatine hydrochloride × 0.07 + glaucine × 0.12 + dehydrocorydaline × 0.36 + (R)-(+)-corypalmine × 0.06 + tetrahydropalmatine × 0.14 + (+)-corydaline × 0.10.C. yanhusuo powder (50-mesh) was weighed and transferred into an extraction vial. The solvent pH value (X1), liquid–solid ratio (X2), time/min (X3), extraction solvent ethanol concentration V/V (X4), and number of extractions (X5) were used as the main factors. With the U9 (95) uniform design method, total alkaloids (Y1), 8 Q-marker content (Y2) and paste-forming rate (Y3) were used as the evaluation indexes to optimize the alcohol extraction process of C. yanhusuo.Ten grams of C. yanhusuo coarse powder weighing 10 g was extracted according to the optimal process selected by uniform design, concentrated in a water bath, and dried under vacuum at 60 °C under reduced pressure. The final samples were examined by UPLC.Triplicate C. yanhusuo were extracted according to the above optimal extraction method. Briefly, 500 g of C. yanhusuo coarse powder (50 mesh) was weighed, 20 times 70% ethanol (pH 10 of diluted ammonia) was added, refluxed twice, each time for 60 min, and filtered. The filtrate was mixed three times, and ethanol was recovered, diluted with water to 1000 mL  and set aside.The total alkaloids of A suitable amount of macroporous resin was washed with 95% ethanol solution and then the bubbles were removed through soaking and stirring to fully replace them. After resting overnight, the column was packed by the wet packing method. First, the cells were washed with 5 BV ethanol (95%) at 1 BV/h and then washed with water until all ethanol was removed. Second, the column was soaked with 5% hydrochloric acid and 5% sodium hydroxide solution for 6 h and washed with water until the pH reached neutral. Finally, 95% ethanol was used to wash the column until the effluent was turbid with water and set aside for further use.C. yanhusuo Q-markers and total alkaloids, the appropriate types were selected from the following 9 kinds of macroporous resins. One gram of each of the pretreated 9 types of macroporous resin  was placed into a conical flask. A C. yanhusuo sample solution of 25 mL was added and shaken for 24 h in a thermostatic shaker. After achieving adsorption equilibrium, the liquid was loaded to detect the contents. The macroporous resin was dried and transferred to an Erlenmeyer flask and then 50 mL of 70% ethanol was added for desorption. After 24 h, the mass concentration of eight Q-markers in the analytical solution was determined, and the measurement results were evaluated by the CS method. The adsorption rate (AR) and desolation rate (DR) of eight Q-markers were calculated according to the following formula (AR = (C0 − C1)V1/C0V1, DR = C2V2/(C0 − C1)V1)) and CS.Based on the adsorption index and resolution rate of 8 kinds of Each NKA-9, HPD600 and D101 sample was accurately weighed to 1 g and placed in a conical flask. Then, 25 mL of loading buffer was added and transferred to the flasks in a shaking water bath (200 r/min) at 37 °C. The mass concentrations of the 8 Q-markers were measured at 0, 0.5, 1, 3, 5, 7, 9, 12 and 24 h. The adsorption kinetics of each macroporous resin was plotted as the adsorbed time (t) and the comprehensive adsorption amount Q. The above three kinds of statically adsorbed macroporous resins were drained, precisely desorbed by adding 50 mL of 70% ethanol, placed in a constant temperature incubator and shaken. The mass concentrations of the eight components in the analytical process were measured at 0, 0.5, 1, 3, 5, 7, 9, 12 and 24 h. The desorption kinetics of each macroporous resin was plotted according to desorption time (t) and the comprehensive desorption amount Q.Pretreated NKA-9 macroporous adsorption resin (20 mL) was loaded into the column by the wet method while regulating the loading flow rate to 2 BV/h. When the volume reached 10 mL, it was collected as one part. The total amount was 22. The mass concentration of 8 kinds of Q-markers was measured in each leakage liquid. The leakage curve was plotted according to the mass concentration of 8 kinds of index components as the abscissa and the sampling amount as the ordinate.C. yanhusuo was added separately and sampling was carried out according to the volume flow rate of 2 BV/h. After the sample was fully adsorbed, it was washed with 4 BV distilled water. The residual liquid was combined with distilled water. The mass concentration of the eight Q-markers was measured to calculate the adsorption rate.The treated NKA-9 macroporous adsorption resin was packed by the wet packing method according to diameter to height ratios of 1:6, 1:8 and 1:10. Then, 30 mL of total alkaloid extract of Since impurities (such as sugars and pigments) were present in the extraction, the impurity removal treatment was first performed. The column of adsorbed saturated NKA-9 resin was washed with 5 BV distilled water, and then gradient eluted with 5 BV of different ethanol concentrations of 10%, 30%, 50%, 70% and 90%. The eluant was collected in stages and determined to evaluate the elution effect on protopine, coptisine, palmatine hydrochloride, glaucine, dehydrocorydaline, (R)-(+)-corypalmine, tetrahydropalmatine and (+)-corydaline during the elution process. The amount of total alkaloids was calculated by M = CV, where C is the mass concentration of the 8 alkaloids in the eluent (mg/mL) and V is the amount of eluting solvent (mL); the ethanol concentration-elution was plotted.The pretreated NKA-9 macroporous resin was packed in a diameter to height ratio of 1:8 by the wet packing method and then added to the sample solution for adsorption. After 5 BV of distilled water was taken to remove impurities, it was eluted with 30 BV 70% ethanol at 1.5, 2, and 3 BV/h. Each of the resin bed eluents was collected to determine and calculate the total alkaloid content and the cumulative elution amount. The elution amount The pretreated NKA-9 resin was packed at a diameter to height ratio of 1:8, and then the sample solution was added and eluted with 3, 5, 7, 9 and 12 BV distilled water. A mold reaction was used to monitor the washing of impurities such as sugar. A negative Molish’s test indicated that the elution of impurities was complete. To monitor the Molish reaction, the sample was further eluted with 12 BV 70% ethanol at 1.5 BV/h. The paste-forming and comprehensive loss rates of the eight alkaloids were determined and calculated.Through the above investigations, the conditions of the purification process were as follows: NKA-9 macroporous adsorption resin with a column diameter ratio of 1:8; loading volume of 6 BV, water washing of 5 BV; 70% ethanol 12 BV elution; and flow rate of 1.5 BV/h. To further investigate the feasibility and stability of the optimal process for the verification test, a total of 450 mL of the sample solution was added to a column containing 150 mL of NKA-9 resin, and then the operation was carried out according to the process conditions. Finally, the ethanol eluate was collected and recovered by vacuum drying at 60 °C. UPLC and UV were used to measure and determine the content of 8 Q-markers in the product and the amount of total alkaloid, respectively. Furthermore, in consideration of the inherent drawbacks  of bench-scale research, a preliminary scale-up verification test was also carried out.C. yanhusuo. Under the above chromatographic conditions, the samples were further analyzed by UPLC to obtain the chromatogram between the samples and reference substances. In this study, the number of theoretical plates was no less than 5000 per component. As shown in Fig. C. yanhusuo by UPLC.It is important to establish a simultaneous quantitative chromatography analysis method of TCM for optimizing the extraction and purification of To ensure the method accuracy, the linear ranges of eight reference substances were further investigated. The regression equations and linear ranges of the protopine, coptisine, palmatine hydrochloride, glaucine, dehydrocorydaline, (R)-(+)-corypalmine, tetrahydropalmatine and (+)-corydaline reference stock solutions were Y = 4757.5X − 4.304 (r = 0.9999) and 0.0096–0.2100 μg; Y = 4349.7X − 6.606 (r = 0.9998) and 0.0112–0.2610 μg; Y = 7927.2X − 4.130 (r = 0.9998) and 0.0066–0.1441 μg; Y = 8331.4X − 1.378 (r = 0.9998) and 0.0083–0.1834 μg; Y = 7792.6X − 1.872 (r = 0.9999) and 0.0090–0.1983 μg; Y = 4151.7X − 4.248 (r = 0.9998) and 0.0093–1.1871 μg; Y = 4078.7X − 4.883 (r = 0.9998) and 0.0124–0.2508 μg; Y = 3677.3X − 5.653 (r = 0.9998) and 0.0131–0.2586 μg, respectively. The results showed that the linear correlation of each index component fit well in the linear range.C. yanhusuo, extractions through different process conditions were prepared, and the linear range of tetrahydropalmatine was also investigated. Finally, the linear regression equation Y = 8.993X − 0.0539 (R = 0.9996) was obtained by data analysis and linear fit. The results showed that the standard curve had good linear correlation in the linear range of 0.020–0.143 mg/mL.For the content determination of total alkaloids in C. yanhusuo.Total alkaloid (Y1), 8 Q-marker content (Y2) and creaming rate (Y3) were used as the evaluation indexes by the U9 (95) uniform design method to optimize the alcohol extraction process of 1 + 2.164X2 + 0.336X4 + 9.290X5, R = 0.951, F = 9.214 (P = 0.026 < 0.05). The results indicated that the model correlation is good and can be applied to the fitting of the test data. From the equation result it is known that the main factors affecting the absorbance are solvent pH, liquid–solid ratio, solvent concentration and number of extractions. The best conditions obtained by the equation are X1 = 10, X2 = 20, X3 = 60, X4 = 70% and X5 = 2. Therefore, the optimal extraction scheme of total alkaloids of C. yanhusuo was adding 20 times ethanol  as the extraction solvent twice, each time for 60 min. With the extraction scheme, C. yanhusuo could be extracted completely. To validate the feasibility of the U9 (95) uniform design method, the total alkaloid extractions were prepared from C. yanhusuo according to the optimal process selected by uniform design. The results showed that total alkaloids of C. yanhusuo, the amount of 8 Q-markers and the rate of ointment were 18.82 mg/g, 7.59 mg/g, and 22.92%, respectively, which indicated that the extraction process conditions obtained by the uniform test were stable and reliable.As shown, the data in Table C. yanhusuo, 9 types of macroporous resin, AB-8, D101, DM130, HPD600, HPD100, NKA-II, NKA-9, S-8 and X-5, were selected and studied. The results are listed in Table To investigate the purification technology of C. yanhusuo Q-markers  have good effects on the adsorption rate and resolution rate of the 8 kinds of rs Table . TherefoThe adsorption and desorption kinetics of each macroporous resin are shown in Fig. C. yanhusuo Q-markers was better than HPD600 and D101, which was preferable for separating the 8 kinds of C. yanhusuo Q-markers. In addition, NKA-9 reached an adsorption equilibrium at approximately 12 h and complete desorption at approximately 4 h  C. yanhusuo powder was extracted and purified by the optimized conditions. Moreover, the eight Q-markers of the total alkaloids were (+)-corydaline 3.51%, tetrahydropalmatine 2.88%, coptisine 2.44%, palmatine hydrochloride 2.21%, dehydrocorydaline 12.68%, (R)-(+)-corypalmine 2.21%, protopine 2.52%, and glaucine 13.54%. The pilot-scale test results were the same as the bench-scale research results, with some components showing a downward trend, which is consistent with the trend of the leak curve study results.To verify the optimal purification process and investigate its feasibility and stability, we carried out a verification test. The results showed that the total alkaloids of the obtained products reached more than 50%, among which the 8 kinds of quality markers were 3.55% (+)-corydaline, 3.13% tetrahydropalmatine, 2.79% coptisine, 2.24% palmatine hydrochloride, 13.11% dehydrocorydaline, 2.37% (R)-(+)-corypalmine, 2.71% protopine and 14.03% glaucine. All of these results indicated that it is feasible to optimize the extraction and purification process of C. yanhusuo are the total alkaloids and auxiliary medicinal ingredients (polysaccharides and organic acids). Therefore, we selected the total alkaloids, paste-forming rate and 8 Q-markers [Extraction is an indispensable process of producing Chinese medicinal materials and their products, but the final result could be affected by various factors. In addition to the conventional extraction factors, the selection of detective indicators plays an important role in the accuracy of multi-index decision-making, so it is very important to evaluate as many data indicators as possible during data processing. Previous research has shown that the main medicinal substances of -markers , 15 as iC. yanhusuo are mainly 7 alkaloids including (+)-corydaline, tetrahydropalmatine, coptisine, palmatine hydrochloride, dehydrocorydaline, (R)-(+)-corypalmine and protopine. Hence, this study has rationality in the selection of indicators and can fully reflect the quality of C. yanhusuo.Chinese medicine Q-markers are used as an indicative substance to reflect the safety and effectiveness of TCM. Therefore, the quality of a TCM could be better controlled if drug-inherent Q-markers are reasonably selected during the extraction process. Zhang Tiejun et al.  confirmeC. yanhusuo decoction pieces between the intrinsic chemical constituents of total alkaloids. On this basis we established a simultaneous quantitative chromatography analysis method of TCM, which laid the foundation for further optimizing the extraction and purification of C. yanhusuo  method, it is significant to optimize the alcohol extraction process of C. yanhusuo by the U9 (95) uniform design method. In this study, total alkaloid (Y1), 8 Q-marker content (Y2) and paste-forming rate (Y3) were selected as the evaluation indexes. The multiple linear regression equation results indicated that the model correlation is good and can be applied to the fitting of the test data. In addition, further results showed that the main factors affecting the absorbance are solvent pH, liquid–solid ratio, solvent concentration and number of extractions. Using U9 (95) uniform design method, the optimal extraction process of total alkaloids of C. yanhusuo was determined and demonstrated by the verified experiment, which indicated that the extraction process conditions obtained by the uniform test were stable and reliable.Reflux is commonly used for extraction. According to the literature, the pH value of the extraction solvent could affect the dissolution of alkaloids . It was During the purification process, we chose the macroporous resin as the impurity removal material based on previous studies –20, and d-glaucine, etc.). In addition, under the optimized process conditions, the total alkaloid content of C. yanhusuo could reach over 50%. The contents of the 8 quality markers were 3.55%(+)-corydaline, 3.13% tetrahydropalmatine, 2.79% coptisine, 2.24% palmatine hydrochloride, 13.11% dehydrocorydaline, 2.37% (R)-(+)-corypalmine, 2.71% protopine and 14.03% glaucine. These parameters could meet the requirements of the effective content of new Chinese medicines.The adsorption–desorption abilities, static and dynamic adsorptive capacity, water consumption of flushing impurities and diameter to height ratio are the important indicators of the performance and separation characteristics of macroporous resin. To select the optimal purifying process of the macroporous resin more objectively and effectively, we systematically investigated 9 types of macroporous resin, AB-8, D101, DM130, HPD600, HPD100, NKA-II, NKA-9, S-8 and X-5. Since the water content of each resin was different, the final result was converted into the component adsorption or desorption amount per gram of dry resin. Our data confirmed that NKA-9 simultaneously had good adsorption and desorption abilities and static and dynamic adsorptive capacity with high resin utilization rate and low cost compared to scopolamine alkaloids (tertiary amines such as (+)-corydaline, tetrahydropalmatine, (R)-(+)-corypalmine, quaternary ammonium coptisine, dehydrocorydaline and palmatine hydrochloride), protopine alkaloids (protopine) and aporphine alkaloids (Through the above research, we determined the purification techniques of NKA-9 macroporous adsorption resin as follows: column diameter ratio of 1:8; loading volume of 6 BV, water washing of 5 BV; 70% ethanol 12 BV elution; and flow rate of 1.5 BV/h. To further verify the feasibility and stability of the optimal process, a 15X scale-up verification test was carried out. Our initial data showed that the pilot-scale test results were the same as the bench-scale research results, with some components showing a downward trend, which was consistent with the trend of the leak curve study results. Therefore, the purification process has good practical prospects for application.C. yanhusuo. Then, the stepwise statistical method was used to evaluate the existing factors affecting the extraction and purification of C. yanhusuo, including the solvent concentration, pH value, liquid–solid ratio, extraction time and frequency. Then, 8 Q-markers, total alkaloid extraction and extraction rate were considered the evaluating indicators. The results indicated that the optimal extraction and purification process was stable and feasible, which is expected to provide experimental support for the industrial production of C. yanhusuo.To improve treatment efficacy using the least dose and to facilitate processing, the useful components from TCM should be extracted. However, the extraction and purification of TCM has been difficult, and there has been a lack of percise solutions until now. In this investigation, we employed Q-marker-based CS and uniform design methods to optimize the extraction and purification of


Globularia alypum L. colected from the Taza region in northeast Morocco. Additionally, the individual phenolics and volatiles of the extracts were also evaluated. The organic extracts of this plant were obtained by Soxhlet extraction using two different solvents, namely ethyl acetate and chloroform. The antioxidant capacity of leaves extracts was measured using DPPH, ABTS and FRAP assays; the phenolic profile was determined by HPLC-DAD/ESI-MS analysis, whereas the volatile composition was elucidated by GC-MS. The ethyl acetate extract analysis showed a total of 20 phenolic compounds and the determination of phenolic contents showed a significant value of 56.5 ± 0.61 µg GAE/mg of extract in comparison with the chloroform extract (18.9 ± 0.48 µg GAE/mg of extract). Also, the determination of the flavonoid contents revealed that the ethyl acetate extract contained the highest value (30.2 ± 0.55 µg CE/mg of extract) in comparison with the chloroform extract (18.0 ± 0.36 µg CE/mg of extract). Concerning the antioxidant properties, interesting values were attained for the ethyl acetate extract which exhibited higher antioxidant activity, namely IC50 = 12.3 ± 3.83 µg/mL and IC50 = 37.0 ± 2.45 µg/mL for the DPPH and ABTS assays, respectively, whereas a value of 531.1 ± 17.08 (mg AAE/g DW) was obtained for the FRAP assay. Concerning the volatile profile, a total of 73 compounds were positively detected and among them n-hexadecanoic acid (13.5%) was the most abundant one. The results achieved confirm the important role of this plant as a source of natural antioxidants.The Moroccan flora is rich in medicinal plants that are commonly used in folk medicine for the treatment of various diseases. The present study was designed to investigate the total phenolic and flavonoid contents, as well as the antioxidant properties of leaves extracts from  Since ancient times medicinal plants have been used as a treatment to cure several diseases thanks to their curative properties. Nowadays, despite the development of modern medicine and the effectiveness of synthetic drugs in treating different ailments, many people choose to use traditional remedies, especially for their fewer side effects compared to chemical drugs. Medicinal plants are able to produce a large number of diverse bioactive compounds, particularly secondary metabolites. For this reason, extensive studies using different plant extracts have been reported by several scientists, to investigate the antibacterial, anti-inflammatory, analgesic, antioxidant and many other medicinal values of these extracts ,2,3.Reactive oxygen species (ROS) serve as cell signaling molecules for normal biologic processes; in general, the disproportion between production of the ROS and the biological system ability of the normal detoxification leads to oxidative stress. These disturbances in the normal redox state of cells can cause toxic effects through the production of free radicals that react with cellular constituents and caused severe damage to the cells ,5,6. FreVarious aromatic and medicinal plants have been reported as an important source of natural antioxidants due to the activity of their secondary metabolites. To this regard, several studies have revealed that antioxidant compounds play important biological properties that might be used for food conservation, cosmetics and pharmaceutical products, alternative medicine and natural therapies .Globularia alypum L. (G. A.) is a wild perennial shrub, belonging to the Plantaginaceae family which is found throughout the Mediterranean area. The plant, locally named “Ain Larnab” or “Tasselgha”, is known for a variety of purposes in the Moroccan traditional medicine [G. A. are traditionally used as an antidiabetic agent, laxative, stomachic, and purgative [G. A. is one of the most important medicinal plants used in traditional remedies by Algerian people [medicine . Leaves urgative . In Algen people ,14.G. A. and the investigation of the antioxidant activities of these extracts by using three different methods, namely free radical scavenging assays (ABTS and DPPH tests), and reducing power assays (FRAP). In addition, a chemical determination of phenolic compounds and volatile profile of G. A. extract was determinated by the high-performance liquid chromatography coupled to photodiode array and electrospray ionization mass spectrometry (HPLC-DAD/ESI-MS) and gas chromatography coupled to MS detection.The aim of the present study was to determine the total phenolic and flavonoid contents in two different extracts of 2 = 0.999), and the obtained results showed that the highest TPC with (56.5 ± 0.61 µg GAE/mg of extract), was found in the ethyl acetate extract, while the chloroform extract showed the lowest value with (18.9 ± 0.48 µg GAE/mg of extract), (2 = 0.998), and also in this case the ethyl acetate extract yielded the highest flavonoid content (30.2 ± 0.55 µg CE/mg of extract) when compared to the chloroform extract which presents a value of (18.0 ± 0.36 µg CE/mg of extract). By comparison of these results with the available literature on the same species, the obtained values are considerably higher than those reported by Athmouni et al. [G. A. . Also, El khantouch et al.’s study [The total phenolic content (TPC) is reported as gallic acid equivalents (GAE) , . The toti et al.  reportedi et al.  who dete’s study  reported’s study  who dete’s study  in TunisA recent study by Taghzouti et al.  for Fès-On the basis of such considerations, such a species has a significant antioxidant potential, therefore, the analysis of individual phenolic acids and flavonoids, as well as antioxidant assays are important for a better understanding in a view of medicinal and/or pharmacological purposes.G. A. leaves were evaluated by DPPH, ABTS and FRAP assays; the results were expressed as IC50 values  was the most abundant one, followed by oleic acid (12.98%) and linoleic acid (11.58%). The % of similarity ranged from 89 to 98%.The attained results of the GC-MS analysis of the ds, etc. , Table 3G. A. an HPLC-DAD-ESI/MS system was employed. w/w) (DW). The most abundant compound was represented by quercetin-rhamnoside 14.5 ± 1.20 mg/g (w/w) (DW), followed by kaempferol-derivative 10.8 ± 0.50 mg/g (w/w) (DW), which explain the agreement between the obtained results and those on the antioxidant activity and the TPC. The detected compounds have never been reported before as constituents of Moroccan G. A. [In order to provide a phytochemical characterization of an G. A. ,27,28,29Globularia alypum L. was collected from Taza region in Morocco, in the spring season between March and April 2018; plant identification was carried by Professor Mohamed El Kadiri, a botanist of the Abd Al-Malek Essaadi University  and deposited in the herbarium of the Faculty with a voucher specimen code GA-LABP01. The fresh leaves of G. A. were dried on air at room temperature in shade approximately for 2 weeks. Once the leaves were well dried, they were powdered and stocked in glass bottle in the dark for further analyses.2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic) acid (ABTS), L-ascorbic acid, and butylated hydroxytoluene (BHT), were purchased from Sigma . Folin-Ciocalteu phenol reagent and standards  were obtained from Merck Life Science . LC-MS grade methanol, acetonitrile, acetic acid, acetone and water were also purchased from Merck Life Science. All other chemicals were of analytical grade and obtained from Sigma.G. A. were rigorously extracted with 250 mL of each solvent, then the obtained extracts were concentrated and free of solvent under reduce pressure, using rotary evaporator. At the end of the extraction operation two crude extracts were obtained and were subsequently weighed to calculate the yield of the extraction for each solvent and stored in a refrigerator (4 °C) in airtight bottles until used for further analysis.The extraction of samples was conducted by Soxhlet apparatus with chloroform, and ethyl acetate, in order to get two extracts with different polarities. 50 g of dried leaves of n-hexane, and the extraction was performed by sonication in an ultrasound bath (130 kHz) for 45 min. After centrifugation for 5 min, the supernatant was filtered through a paper filter, dried with rotary evaporator, and reconstituted with n-hexane, prior to GC-MS analysis.For GC-MS analysis, five grams of plant powder was defatted three times in 50 mL of G. A. leaves extracts was determined by Folin-Ciocalteu colorimetric method as described by Singleton et al. 1965 [2CO3), were mixed; the mixture was then thoroughly homogenized and incubated for 30 min at 40 °C. After incubation, the absorbance of each sample was measured at 765 nm against methanol as blank and the TPC was calculated by using the gallic acid calibration curve. All assays were performed in triplicate. The TPC was expressed as µg GAE/mg of extract.TPC of al. 1965 ,31 with G. A. leaves extracts was determined using the aluminum chloride colorimetric method, according to the protocol described by Dewanto et al. [2); the mixture was incubated for 6 min at room temperature. Thereafter, 150 µL of aluminum trichloride solution (10% AlCl3) were added and then allowed to stand for 6 min. Afterwards, 500 µL of sodium hydroxide solution (4%NaOH) were dissolved in 275 µL of distilled water. Then, the mixture was thoroughly mixed and allowed to stand for 30 min at room temperature. The absorbance was measured at 510 nm versus reagent blank containing methanol instead of the sample. Catechin was used as a standard compound for the quantification of total flavonoids. All trials were performed in triplicate. Results were expressed as µg of catechin equivalents per mg DW extracts (µg CE/mg).TFC of o et al. . BrieflyG. A. leaves were subjected to screening for their possible antioxidant activity by three test systems namely DPPH, ABTS and FRAP methods.The organic extracts of G. A. were measured using the stable free radical DPPH according to a slightly modified method by Hatano et al. [G. A. . The mixture was vortexed thoroughly and after 30 min incubation time in darkness and at room temperature, the absorbance was measured at 517 nm with a blank containing DPPH and methanol. The butylated hydroxytoluene (BHT) was used as a positive control and the DPPH radical scavenging activity was calculated according to the equation:The radical scavenging activities of the extracts of o et al. . DPPH me50 of DPPH radical was calculated from the line regression of the percentage of remaining DPPH radical against the sample concentration.The IC+ stock solution was diluted with methanol to an absorbance of 0.7 ± 0.02, at 734 nm. 15 µL of the test sample was mixed with 185 µL of ABTS+ diluted solution, and the mixture was incubated for 10 min. The results were expressed as IC50 (µg/mL) and the percentage of inhibition was calculated according to the following equation:0 is the absorbance of the control, and A1 is the absorbance of the sample.ABTS radical scavenging activity was determined according the method of Re et al. . The ABTw/v), and 2.5 mL of phosphate buffer . Then the mixture was incubated for 20 min at 50 °C, and 2.5 mL of trichloroacetic acid (10%) were added. After centrifugation of the mixture for 10 min at 3000 rpm, 2.5 mL were collected from the upper layer, mixed with 2.5 mL of distilled water, and 0.5 mL of FeCl3 (0.1%). Ascorbic acid (10 µg/mL) was used as a standard for the preparation of the calibration curve, and the absorbance was measured at 700 nm by a spectrophotometer. Results were expressed as mg of ascorbic acid equivalents per mg AAE/g DW. All tests were performed in triplicate.The reducing power of extracts was determined according to the method reported by Oyaizu  with minm/z, with an event time of 0.2 s. Relative quantity of the chemical compounds present in each sample was expressed as percentage based on peak area produced in the GC chromatogram. The analysis of the volatile fraction was performed on a GC-MS-QP2020 system  with an “AOC-20i” system auto-injector. The analyses were carried out on an SLB-5ms column . The initial temperature was set at 50 °C, afterwards increased up to 350 °C . GC-MS parameters were as follows: injection temperature: 280 °C; injection volume: 1.0 μL (split ratio: 10:1); pure helium gas (99.9%); linear velocity: 30.0 cm/s; Inlet pressure: 26.7 KPa. EI source temperature: 220 °C; Interface temperature: 250 °C. The acquisition of MS spectra was carried out in full scan mode, in the mass range of 40–660 Compounds were identified by using the “FFNSC 4.0” , and “W11N17” . Each compound was identified applying a MS similarity match and an LRI filter. Linear retention indices (LRI) were calculated by using a C7-C40 saturated alkanes reference mixture . Data files were collected and processed by using “GCMS Solution” software, ver. 4.50 (Shimadzu).LC analyses were carried out on a Shimadzu liquid chromatography system  consisting of a CBM-20A controller, two LC-20AD dual-plunger parallel-flow pumps, a DGU-20A5R degasser, a SIL-20AC autosampler, an SPD-M30A photo diode array detector and an LCMS-2020 mass spectrometer, through an ESI source .v/v, solvent A) and acetonitrile/acetic acid , The flow rate was fixed at 1 mL/min under gradient elution: 0–5 min, 5% B, 5–15 min, 10% B, 15–30 min, 20% B, 30–60 min, 50% B, 60 min, 100% B. DAD detection was applied in the range of λ = 200–400 nm and a wavelength of 280 nm was monitored . MS conditions were as follows: scan range and scan speed were set at m/z 100–800 and 2500 u s−1, respectively, event time: 0.3 s, nebulizing gas (N2) flow rate: 1.5 L min−1, drying gas (N2) flow rate: 15 L min−1, interface temperature: 350 °C, heat block temperature: 300 °C, DL (desolvation line) temperature: 300 °C, DL voltage: 1 V, interface voltage: −4.5 kV. Calibration curves (R2 ≥ 0.997) of three phenolic standards were used for the quantification of the ethyl acetate extract.Chromatographic separations were performed on 150 × 4.6 mm; 2.7 µm Ascentis Express RP C18 column  ,37,38. Tp < 0.05.All data were analysed using IBM SPSS Statistics for Windows, version 21 . The experiments were carried out in triplicates and the results are expressed as the average of the three measurements ± SD. The comparison of means between groups was performed with one-way analysis of variance (ANOVA) followed by Tukey test. Differences were considered significant when Globularia alypum, L. grown in Morocco, were investigated. The phenolic profile revealed a total of 20 compounds including phenolic acids, flavonoids and iridoids in the ethyl acetate extract, whereas the volatile profile showed a total of up to 73 compounds belonging to different chemical classes. The results achieved are very promising and if supported by “in vivo” studies might propose such a species to be used for therapeutic purposes.In this contribution, total phenolic and total flavonoid contents as well as the antioxidant capacity evaluated by ABTS, FRAP and DPPH assays of ethyl acetate and chloroform leaves extracts of


All these results suggested that some metals released from containers had binding affinities with acids, thereby reducing SFB organoleptic stimulation by forming metal‐aroma compound complexes during storage.Storage is essential in improving the quality of strong‐flavor baijiu (SFB). Here, we investigated the release behaviors of metals from containers into SFB and their effects on the evolution of aroma compounds during storage. Twenty‐six metals were identified in SFB samples. The concentrations of Na, K, Ca, Mg, Al, and Fe obviously increased after storing in pottery jar, whereas those of Fe and Cu greatly increased after storing in stainless‐steel vessel. The volatility of most esters, alcohols, ketone, furan, and aldehyde decreased, whereas that of most acids increased after adding the metal ions into fresh SFB. The fluorescence intensity of SFB decreased with increased aging time in pottery jar, whereas the fluorescence intensity of acids was quenched with adding Fe Some metals were dissolved out from containers such as pottery jar and stainless‐steel vessel into strong‐flavor baijiu (SFB) during storage. These metals had binding affinities with aroma compounds, thereby reducing SFB organoleptic stimulation by forming metal‐aroma compound complexes during storage. Baijiu has a distinctive flavor different from other major distilled spirits, such as brandy, whiskey, rum, gin, and vodka , and trace metals Ag, As, Ba, Be, Cd, Cr, Cu, Li, Mn, Ni, Pb, Sr, Ti, Zn, La, Pr, Nd, Sm, Eu, and Gd were determined via inductively coupled plasma‐mass spectrometry (ICP‐MS). Then, changes of aroma compounds in SFB after adding various metal ions were analyzed via headspace solid‐phase microextraction combined with gas chromatography–mass spectrometry (HS‐SPME‐GC‐MS), and the metal‐binding abilities of aroma compounds were evaluated via fluorescence spectroscopy to further understand the effects of metals on aroma compounds of SFB.22.13, and its surface area is approximately 5.8 m2 in comparison to 1 m3 of SFB. The volume of the stainless‐steel vessel is 10 m3, and its surface area is approximately 2.1 m2 in comparison to 1 m3 of SFB. During storage, the pottery jar was sealed and put in a cave at 20°C. The stainless‐steel vessel was sealed and put in the shade at a room temperature which varied with the seasons. Standard solutions of Na, K, Ca, Mg, Al, Fe, Ag, As, Ba, Be, Cd, Cr, Cu, Li, Mn, Ni, Pb, Sr, Ti, Zn, La, Pr, Nd, Sm, Eu, and Gd were purchased from Guobiao Testing & Certification Co., Ltd. Acetic acid, butanoic acid, and hexanoic acid  were purchased from Dr. Ehrenstorfer. Internal standard, methyl hexanoate, was purchased from ANPLE Scientific Instrument Co., Ltd. A C7–C40 n‐alkane mixture (O2SI Smart Solutions) was employed to determine retention indices (RIs). Ethanol , nitric acid, hydrogen peroxide, NaCl, KCl, CaCl2, MgCl2•6H2O, AlCl3•6H2O, FeCl3•6H2O, and CuCl2•2H2O were of analytical grade and purchased from Kelong Chemical Co., Ltd. Milli‐Q water (18.2 MΩ/cm resistivity) was prepared through a Milli‐Q system (Millipore).SFB samples were the same level base baijiu and provided by Luzhoulaojiao Co., Ltd. . Samples were stored in pottery jar or type 304 stainless‐steel vessel for 0, 1, 2, 3, 5, and 10 years. The volume of the pottery jar is 1 m2.2SFB samples (25 ml) were heated at 80°C until their volume was reduced to 2–3 ml. Then, the concentrated samples were digested at 120°C with 5 ml nitric acid and 1 ml hydrogen peroxide. After cooling to room temperature, these digested solutions were diluted to 25 ml with Milli‐Q water. The concentrations of major metals, such as Na, K, Ca, Mg, Al, and Fe, were determined by ICP‐OES , and the concentrations of trace metals, such as Ag, As, Ba, Be, Cd, Cr, Cu, Li, Mn, Ni, Pb, Sr, Ti, Zn, La, Pr, Nd, Sm, Eu, and Gd, were determined by ICP‐MS .2.32, MgCl2, AlCl3, FeCl3, and CuCl2, in which the metal ion has a concentration of 1,000 mg/L, were prepared individually. Different volumes of the above‐mentioned solutions were added into fresh SFB samples to obtain 2 and 10 mg/L of Na+, K+, and Ca2+, 0.5 and 2 mg/L Mg2+, Al3+, Fe3+, and Cu2+. SFB samples with addition of metal were regarded as experimental groups, and SFB sample without addition of metal was the control group. All samples were stored at 25°C for a week.Aqueous solutions of NaCl, KCl, CaClChanges in aroma compounds of fresh SFB after adding various metal ions were analyzed through HS‐SPME‐GC‐MS using the reported method with some modifications . HP‐Innowax column  was used as capillary column. Helium (purity 99.999%) was used as carrier at a constant flow rate of 1 ml/min. Injector was maintained at 250°C. Oven temperature was started at 40°C for 5 min, raised to 100°C at rate of 4°C/min, then ramped to 230°C at a rate of 6°C/min and held for 15 min. For MS analysis, electron ionization was used at 70 eV. Temperatures of the interface and the ion source were set at 250 and 230°C, respectively. The mass range scanned was from 30 to 450 amu at a scan rate of 0.3 s.Volatile aroma compounds were identified by comparing their mass spectra (MS) with the NIST14 mass spectra database and comparing their retention indices (RIs) with those of reference compounds. Net changes were used to evaluate aroma volatility and were calculated according to the equation: Net change (%) = / mean peak area of control × 100%  spectra of the SFB samples stored in pottery jar for 0, 1, 2, 3, 5, and 10 years were measured using a fluorescence spectrometer . Excitation and emission wavelengths were recorded from 200 nm to 350 nm and 200 nm to 450 nm, respectively, with 2.5 nm slit widths.2, MgCl2, AlCl3, FeCl3, and CuCl2 aqueous solutions, in which metal ion concentration was 2000 mg/L, were added into fresh SFB samples to obtain different concentrations of Na+ , K+ , Ca2+ , Mg2+ , Al3+ , Fe3+ , and Cu2+ . Fresh SFB samples with metal ions were stilled for a week. Then, fluorescence intensities at the maximum emission wavelength of the samples were recorded with the maximum excitation wavelength of 280 nm. F0 and F were the fluorescence intensities of the SFB with and without metal ions, respectively.Different volumes of NaCl, KCl, CaCl+, K+, Ca2+, Mg2+, Al3+, Fe3+, or Cu2+) were recorded at maximum excitation wavelength of 220 nm. Acid solutions include 0.5 g/L of acetic acid, butanoic acid, and hexanoic acid in 70% (v/v) ethanol/water. Metal ion concentrations were 0, 0.1, 0.5, 2, 5, 10, 20, and 50 mg/L.Fluorescence emission spectra of acid solutions with different concentrations of a metal ion (Na2.5p < .05).Each sample was analyzed in triplicate for each determination. The results in tables are presented as mean ± standard deviation. One‐way analysis of variance (ANOVA) and Duncan's multiple range tests were carried out with SPSS 25.0 software to evaluate significant differences of assay . When SFB was stored in pottery jar, the concentrations of Na, K, Ca, Mg, Al, and Fe increased considerably, and concentrations of Ba, Be, Cu, Li, Mn, Ni, Sr, Ti, and Zn increased slightly. Pottery jar is made from fired clay and covered with glaze over the surface. Its main components are SiOMetals in alcoholic beverages have a two‐faced role. Metals of proper amount and type are beneficial to modulating the flavor of alcoholic beverages and human health. For example, Fe and Cu can remove bad odors, and K, Ca, Cu, Zn, Fe, and Mg are essential metals in the human body . Esters and acids were the main aroma compounds in baijiu. Total acid content increased and total ester content decreased during the aging period  and SFB. Fluorescence EEM landscapes of fresh and stored SFB samples are shown in Figure +, K+, Ca2+, Mg2+, Al3+, Fe3+, and Cu2+ were separately added into fresh SFB and acid solutions. Changes in SFB fluorescence intensity in the presence of Na+, K+, Ca2+, Mg2+, Al3+, Fe3+, and Cu2+ are shown in Figure +, K+, Ca2+, Mg2+, and Al3+ at a low concentration . These fluorescence quenching data showed that Fe3+ and Cu2+ had higher binding affinity with carboxyl of acetic acid, butanoic acid, and hexanoic acid in 70% (v/v) ethanol/water solution than Na+, K+, Ca2+, Mg2+, and Al3+.Fluorescence quenching is a useful technique to determine binding affinities of metals to target components (De Costa & Jayasinghe, 3+, Al3+, Cu2+, and Ca2+ could bind with a compound derived from nitrilotriacetic acid. Hussain, Rahim, and Farooqui (3+ and Cu2+ had higher stability than those with Cd2+, Zn2+, Ni2+, and Co2+. Their results indicated that these metals, especially Fe3+ and Cu2+, could chelate or bind with different organic compounds, which is consistent with our fluorescence analyses. Besides, Fe3+ and Cu2+ even have oxidative potential and may oxidize aroma compounds in SFB (Benítez, Castro, Antonio Sanchez, Pazo, & Barroso, Irto et al.  reportedFarooqui  showed t4+, K+, Ca2+, Mg2+, Al3+, Fe3+, and Cu2+. These metal cations promoted the decrease in total ester content and the increase in total acid content, indicating that these metal cations had an aging‐advancing function. Furthermore, maximum fluorescence intensity of SFB decreased with increasing aging time. This result might be ascribed to the binding between metals released from the container and aroma components, especially acids in SFB during storage. This binding can promote the formation of metal‐aroma compound complexes or colloidal particles. This finding is helpful to explain the release of metal cations and the formation of aroma compounds of baijiu during storage, which is significant for modulating the flavor and sensory properties of baijiu.Some metals were dissolved out from pottery jar and stainless‐steel vessel into the SFB during storage. Their concentrations were lower than the recommended allowable daily intake. When SFB was stored in pottery jar, the concentrations of Na, K, Ca, Mg, Al, and Fe remarkably increased, and concentrations of Ba, Be, Cu, Li, Mn, Ni, Sr, Ti, and Zn slightly increased. When SFB was stored in stainless‐steel vessel, the concentrations of Fe and Cu greatly increased, and concentrations of Cr, Mn, Ni, Sr, Ti, and Zn slightly increased. Metals released from pottery jar and stainless‐steel vessel into SFB were consistent with the main components of the two containers. It is worth noting that the Cu released in SFB should be the impurity, which was introduced from welding process in production of the stainless‐steel vessel. Headspace concentrations of most esters, alcohols, ketone, furan, and aldehyde in fresh SFB decreased, whereas headspace concentrations of most acids increased after adding NaThe authors declare that they do not have any conflict of interest.This study does not involve any human or animal testing. Click here for additional data file.


Scientific Reportshttps://doi.org/10.1038/s41598-020-66484-x, published online 11 June 2020Correction to: The original version of this Article contained an error in the number of Cetacean Stranding events recorded.As a result of this, in the Abstract,“We identified a total of 389 CS events affecting eight cetacean families, 21 genera, and 35 species, which represent more than 85% of the reported species richness for the country.”now reads:“We identified a total of 436 CS events affecting eight cetacean families, 21 genera, and 35 species, which represent more than 85% of the reported species richness for the country.”Additionally, in the Results section under the subheading “Cetacean Stranding Events”,24.”“Between January 1968 and January 2020, a total of 441 CS events, affecting 1,607 stranded cetaceans, were recorded along the Chilean coast . Most CS events (94.1%) were single . There were also 18 mass strandings  and nine unusually large mass stranding event . Cetacean strandings were reported every month; with 20.2% (n = 89) of total events occurring in January, 13.2% (n = 58) in February, and 8.4% (n = 37) in July. . Spatially, at least one CS event was reported in 15 out of the 16 administrative regions of Chile. When the locations were aggregated by month, it became apparent that most events occurred in the regions of Valparaíso with 17% (n = 75), Magallanes with 14.5% (n = 64), and Coquimbo 11.3% (n = 50). On the opposite end, the region of Araucanía had the least number of reported events with only two strandings . The greatest number of CS events was observed in 2019 with 11.3% (n = 50), followed by the year 2018 with 10.7% (n = 47) and 2015 with 9.3% (n = 41). Regarding the numbers of stranded individuals, the greatest numbers of total stranded cetaceans were reported in March with 37.9% (n = 610), followed by July with 16.5% (n = 266) and April with 10.8% (n = 174) . The Aysén and Magallanes regions accounted for most of the geographical distribution, with a 33.8% (n = 543) and a 31.5% (n = 506) of the total number of stranded individuals, followed by the Coquimbo region with 9.6% (n = 154). The year 2015 also accounted for the highest number of stranding individuals with 25.3% (n = 407) of the total, followed by the years 1989 and 2016 with 11.1% (n = 185) and 9.4% (n = 151) specimens. Overall, the median of stranded cetaceans in an stranding event was 2 with an interquartile range of 3; and the largest number of stranded cetaceans for a given event was 367 Sei whales that were reported at the Golfo de Penas area in the Magallanes region in March 2015now reads:24.”“Between January 1968 and January 2020, a total of 436 CS events, affecting 1,596 stranded cetaceans, were recorded along the Chilean coast . Most CS events (94.1%) were single . There were also 17 MSE  and nine UME . Cetacean strandings were reported every month; with 20.4% (n = 89) of total events occurring in January, 13.3% (n = 58) in February, and 8.5% (n = 37) in July. . Spatially, at least one CS event was reported in 15 out of the 16 administrative regions of Chile. When the locations were aggregated by month, it became apparent that most events occurred in the regions of Valparaíso with 17% (n = 74), Magallanes with 14.4% (n = 63), and Coquimbo 11.5% (n = 50). On the opposite end, the region of Araucanía had the least number of reported events with only two strandings . The greatest number of CS events was observed in 2019 with 11.2% (n = 49), followed by the year 2018 with 10.3% (n = 45) and 2015 with 9.4% (n = 41). Regarding the numbers of stranded individuals, the greatest numbers of total stranded cetaceans were reported in March with 38.2% (n = 610), followed by July with 16.6% (n = 266) and April with 10.9% (n = 174) . The Aysén and Magallanes regions accounted for most of the geographical distribution, with a 34% (n = 543) and a 31.6% (n = 505) of the total number of stranded individuals, followed by the Coquimbo region with 9.6% (n = 154). The year 2015 also accounted for the highest number of stranding individuals with 25.5% (n = 407) of the total, followed by the years 1989 and 2016 with 11.5% (n = 185) and 9.4% (n = 151) specimens. Overall, the median of stranded cetaceans in an stranding event was 1 with an interquartile range of 1; and the largest number of stranded cetaceans for a given event was 367 Sei whales that were reported at the Golfo de Penas area in the Magallanes region in March 2015Also in the Results section, under the subheading “Cetacean species”,Odontoceti and Mysticeti species accounted for 74.8% (n = 330) and 25.2% (n = 111) of CS events, respectively; while odontocetes accounted for 66.8%  and mysticetes for 33.2% (n = 534) of stranded cetaceans. Within stranded odontocetes, most events belonged to the Delphinidae 36.3% (n = 160), followed by the Phocoenidae 15.4% (n = 68) and Physiteridae 10.7% (n = 47) families. Delphinidae had the highest number of stranded individuals (n = 865), 80.6% of the Odontoceti order and 53.8% of all cetaceans. In the Mysticeti suborder, individuals of the Balaenopteridae family were the most frequently stranded with 96.3% (n = 514) of cases, followed by members of the Balaenidae family at 3.4% (n = 18). In term of stranding events, 87.4% (n = 97) were composed of Balaenopterids. If all events are considered together, the Delphinidae and Balaenopteridae families account for 58.3% (n = 257) of all stranding events, and for up to 85.8%  of all cetaceans stranded through the years.”“Cetacean stranding events were reported in eight cetacean families including 21 genera and 35 species. now reads:Odontoceti and Mysticeti species accounted for 74.3% (n = 324) and 24.3% (n = 106) of CS events, respectively; while odontocetes accounted for 67.2%  and mysticetes for 32.7% (n = 523) of stranded cetaceans. Within stranded odontocetes, most events belonged to the Delphinidae 36.3% (n = 160), followed by the Phocoenidae 20.9% (n = 68) and Physiteridae 14.5% (n = 47) families. Delphinidae had the highest number of stranded individuals (n = 865), 80.6% of the Odontoceti order and 53.8% of all cetaceans. In the Mysticeti suborder, individuals of the Balaenopteridae family were the most frequently stranded with 98.2% (n = 514) of cases, followed by members of the Balaenidae family at 1.3% (n = 7). In term of stranding events, 91.5% (n = 97) were composed of Balaenopterids. If all events are considered together, the Delphinidae and Balaenopteridae families account for 58.9% (n = 257) of all stranding events, and for up to 86.4%  of all cetaceans stranded through the years.”“Cetacean stranding events were reported in eight cetacean families including 21 genera and 35 species. and,“Only six odontocetes were classified as undetermined due to their advanced state of decomposition.”now reads:“Only six cetaceans were classified as undetermined due to their advanced state of decomposition.”o Individuals” and “No Events”,In the first row of Table 1 under the headings “N“18” and “12”,now read:“7” and “7”and under the heading “Common name”,“Short-finned Whale” and “Long-finned Whale”now read:“Short-finned Pilot Whale” and “Long-Finned Pilot Whale”.Figure 1 was incorrect in the original version of the Article  occurring between December 2019 and January 2020 (last summer season). All these CS events reported a total of 92 stranded individuals that were extensively distributed in the Chilean coast.”now reads:“We also discuss the within-year variation of reporting, the identification of hot spots in different areas of Chile and the species composition of the dataset. In the last year, there have been 60 CS events, 15 of them (25%) occurring between December 2019 and January 2020 (last summer season). All these CS events reported a total of 90 stranded individuals that were extensively distributed in the Chilean coast.”,26, CS events reported here identified 88.9% of cetacean families, 87.5% and 85.4% of genera and species, respectively.”“In fact, from nine existent families, 24 genera and 41 species that have been reported in Chilean watersnow reads:26, CS events reported here identified 88.9% of cetacean families, 87.5% and 85.3% of genera and species, respectively.”,“In fact, from nine existent families, 24 genera and 41 species that have been reported in Chilean watersP. spinipinnis, reported in 43 events. Phocoena spinipinnis are a group of porpoises that regularly strand in coastal areas worldwide3. It has been described that the leading causes of death are from bycatch and naval presence28. In our study, the strandings of P. spinipinnis took place for the most part in major ports and fisheries located in central Chile. A different situation occurred with B. borealis (n = 387), with 13 reported CS throughout the study period. However, a single event reported about 95% of total reported stranded individuals.“This was observable for most species, but it was unusually high for now reads:P. spinipinnis, reported in 66 events. Phocoena spinipinnis are a group of porpoises that regularly strand in coastal areas worldwide3. It has been described that the leading causes of death are from bycatch and naval presence28. In our study, the strandings of P. spinipinnis took place for the most part in major ports and fisheries located in central Chile. A different situation occurred with B. borealis (n = 414), with 17 reported CS throughout the study period. However, a single event reported about 88.6% of total reported stranded individuals.“This was observable for most species, but it was unusually high for and,“The analysis of the within-year variation indicates that although CS events were reported at all times of the year, the months from February to April (summer and early autumn) and July (winter) account for 41% of all CS events.”now reads:“The analysis of the within-year variation indicates that although CS events were reported at all times of the year, the months from February to April (summer and early autumn) and July (winter) account for 41.9% of all CS events.”Additionally, the original Article contained an error in the legend of Figure 3. As a result of this,a) Stranding events (black dots) along Chile since 1968 to 2020. (b) Heatmap that illustrates density of stranding cases along Chile.”“(now reads:a) Cetacean stranding events (black dots) along Chile since 1968 to 2020. (b) Heatmap that illustrates density of stranding cases along Chile.”“(Additionally, the original Article contained an error in the Author Contributions section, where,“M.A.R. and F.T. led the project, designed the work, collected data and performed the systematic review. M.A.R., F.T., and F.O.M. wrote the paper, and J.E.D., M.S., B.T., C.S.A., J.C., G.C.H., N.Z.V. and A.C.K. helped to improve the manuscript. Time series, spatiotemporal analysis, visualization and plotting results were performed and described by M.A.R., J.E.D. and F.O.M.”now reads:“M.A.R. and F.T. led the project, designed the work, collected data and performed the systematic review. M.A.R., F.T., and F.O.M. wrote the paper, and J.E.D., M.S., C.A., J.C., G.C.H., N.Z.V. and A.C.K. helped to improve the manuscript. Time series, spatiotemporal analysis, visualization and plotting results were performed and described by M.A.R., J.E.D. and F.O.M.”These errors have now been corrected in the HTML and PDF versions of this Article.