Potential Biological Activity Research Articles

Electrochemically Induced, Metal Free Synthesis of 2-substituted chroman-4-ones.

Electrochemically induced, decarboxylative functionalization of chromone-3-carboxylic acids by N-hydroxyphthalimide esters as alkyl radical precursors was studied. Electrochemical protocol offers a sustainable and green approach, obviating the need for catalysts, relying on the direct reduction of NHPI esters using electric current. Developed protocol provides a straightforward route to the synthesis of diverse molecules with potential biological activity.

Marine Cyanobacteria: A Rich Source of Structurally Unique Anti-Infectives for Drug Development

Marine cyanobacteria represent a promising yet underexplored source of novel natural products with potent biological activities. Historically, the focus has been on isolating cytotoxic compounds from marine cyanobacteria, but a substantial number of these photosynthetic microorganisms also produce diverse specialized molecules with significant anti-infective properties. Given the global pressing need for new anti-infective lead compounds, this review provides a concise yet comprehensive overview of the current knowledge on anti-infective secondary metabolites derived from marine cyanobacteria. A majority of these molecules were isolated from free-living filamentous cyanobacteria, while several examples were derived from marine cyanobacterial symbionts. In addition, SAR studies and potent synthetic analogs based on selected molecules will be featured. With more than 200 molecules, this review presents their antibacterial, antifungal, antiviral, antiprotozoal, and molluscicidal activities, with the chemical and biological information covered in the literature up to September 2024.

Peptidomic Analysis of Potential Bioactive Peptides in Mare Milk Under Different Heat Treatment Conditions

Active peptides in mare milk have unique biological activities, but how the bioactive protein in mare’s milk changes under the influence of temperature has not been fully studied. Therefore, in this study, the differential expression of bioactive peptides potentially present in horse milk under different heat treatment conditions was investigated for the first time using peptidomic and bioinformatic techniques. We collected a total of 15 samples. In this study, we divided the samples into five groups, specifically, 65 °C, 30 min; 72 °C, 15 min; 83 °C; 10 min; 95 °C, 5 min; and an untreated group as a control, which involved four different temperature treatments, in order to understand changes in bioactive peptides and to identify the sequence of bioactive peptides. In the experiment, a total of 2341 active peptides were identified. The amino acid composition of the potential active peptides remained stable across different temperatures, but their abundance varied with temperature. In all, 23 peptides from 20 different proteins were identified, with the highest number of active peptides identified at 72 °C. Through database searches, we found that a majority of these peptides were within β-lactoglobulin and immunoglobulin domain proteins, which are known for their potential biological activities. These findings provide a theoretical foundation for the development of peptides with different bioactivities as potential functional foods.

Modification of B-Nor Steroids Mediated by Filamentous Fungus Fusarium culmorum: Focus on 15α-Hydroxylase Activity

The metabolic activities of microorganisms to modify the chemical structures of organic compounds are an effective tool for the production of high-value steroidal drugs or active pharmaceutical ingredients (APIs). The integration of biotransformation into the synthesis of APIs can greatly reduce the number of reaction steps and achieve higher process efficiency, thus enabling their greener production. The current research efforts are focused on either the optimization of existing processes or identification of new potentially useful bioconversions. This study aimed to assess the catalytic abilities of the filamentous fungus Fusarium culmorum AM 282 to transform B-nor analogues (5(6→7)abeo compounds) of steroid hormones: androstenedione (AD), dehydroepiandrosterone (DHEA) and its acetate. Our previous studies have demonstrated that this strain is an active hydroxylating catalyst for many steroidal compounds with diverse structures. The results presented in this work showed that the hydroxylation of B-nor steroids occurred with the regio- and stereoselectivity typical of this strain in relation to the corresponding natural hormones of the standard 6:6 A/B series. After the transformations of B-nor-DHEA and its acetate, 15α-hydroxy-B-nor-DHEA was obtained as the sole product of the reaction, while the transformation of the AD analogue resulted in a mixture of its 15α- and 6α-hydroxy derivatives. A detailed analysis of the transformation course indicated that all the obtained hydroxy derivatives could be the result of the activity of the same enzyme. The presented results may provide a basis for research aimed at understanding the molecular nature of cytochrome P-450 monooxygenase from F. culmorum AM 282 with its ability for 15α-hydroxylation of steroidal compounds. An analysis of the pharmacokinetic and pharmacodynamic properties of the obtained metabolites with cheminformatics tools suggests their potential biological activity.

Cascade [3 + 2]/[4 + 2] Cycloaddition of Enaminones with Vinylene Carbonate for the Synthesis of Functionalized Pyrrolo[2,1-a]isoquinoline Derivatives.

We developed a protocol for the synthesis of functionalized pyrrolo[2,1-a]isoquinoline derivatives (PIQDs) 3 from enaminones 1 using vinylene carbonate 2. This strategy involved [3 + 2] and [4 + 2] cycloadditions via heating a mixture of substrates 1 with vinylene carbonate 2 and DCE at 60 °C, catalyzed by [Cp*RhCl2]2 and oxidized with Cu(OAc)2 and AgSbF6 promoted by NaOAc. As we increased the reaction temperature to 110 °C under the same conditions, we synthesized PIQDs 4 through sequential C-H activation, alkene insertion, migratory insertion, C-N reductive elimination, β-O elimination, and finally dehydration. As a result, a series of PIQDs 3-4 were generated by forming four bonds (2 C-C and 2 C-N bonds) in a single step. This strategy realizes the synthesis of linear molecules with potential biological activity, specifically natural-like heterocycles (3-4). It expands the application of vinylene carbonate as a C2 synthon in the construction of pyrrole and isoquinoline skeletons for the synthesis of functionalized PIQDs in combinatorial and parallel syntheses via one-pot reactions.

Structure analysis and immunomodulatory activity of novel oligosaccharide from Nicotiana tabacum roots

A novel oligosaccharide (NTRP60-W-2) with an average molecular weight of 1377 Da was isolated and purified from Nicotiana tabacum roots. Its structural characteristics and immunomodulatory properties were investigated. Structural analysis revealed that NTRP60-W-2 was composed exclusively of glucose, featuring →1)-α-D-Glcp-(6→ backbone. Immunological assays demonstrated that NTRP60-W-2 significantly enhanced cell viability, nitric oxide production and cytokine secretion (IL-6 and TNF-α) in RAW264.7 cells. These findings provide a foundation for further exploration of Nicotiana tabacum carbohydrates and their potential biological activities.

Resolution and Absolute Configuration of Fargesin Enantiomers.

Fargesin is an important bioactive furofuran lignan isolated from different plant species. Despite presenting potent biological activities, its stereochemical characterization has relied mostly on empirical correlations of optical rotation, an approach considered risky that commonly leads to misassignments and error propagation. Additionally, the enantiomeric purity of fargesin isolates used for biological assays has not been previously investigated. Herein, we report the enantioresolution of a scalemic mixture of fargesin isolated from Aristolochia warmingii along with the first unambiguous determination of the absolute configuration of each enantiomer by means of optical rotatory dispersion, as well as electronic and vibrational circular dichroism aided by quantum-chemical calculations.

Synthesis, Characterization, and Biological Investigation of Antimony(III) Halide Complexes With Different Coordination Architectures Constructed From Thiophene Thiosemicarbazones

ABSTRACTIn this study, we report the synthesis, characterization, and biological investigation of antimony(III) halide complexes with various coordination architectures constructed from thiophene thiosemicarbazones. Antimony(III) thiophene‐2‐carbaldehyde thiosemicarbazone complexes (1, 2, 4, and 5) exhibit a square pyramidal geometry, with ligands coordinated to the central antimony atom in two distinct binding modes. In contrast, antimony(III) 2‐acetylthiophene thiosemicarbazone complexes (3, 6, and 7) adopt a seesaw geometry. These complexes (1–7) represent the first reported examples of antimony(III) halide thiosemicarbazone compounds. The unique coordination environments observed in these complexes are of significant importance within the realm of antimony chemistry. These synthesized complexes exhibit different coordination geometries as well as potential biological activities. The antiproliferative activity against the human breast adenocarcinoma (MCF‐7) cell line and antimicrobial activity against Gram‐positive and Gram‐negative bacteria were assessed. Antimony(III) thiophene‐2‐carbaldehyde thiosemicarbazone complexes (1, 2, 4, and 5) showed significant antiproliferative activity with IC50 values ranging from 8.5 to 19.1 μM, while antimony(III) 2‐acetylthiophene thiosemicarbazone complexes (3, 6, and 7) had higher IC50 values. Additionally, the antimony complexes demonstrated selective antimicrobial activity against Gram‐negative bacteria.

Dimorphandra gardneriana Tul. (Fabaceae): Medicinal and Industrial Potential of a Brazilian Plant with Pharmacological and Bioactive Properties

The traditional use of medicinal plants, particularly in the Middle East and Asia, highlights the historical and contemporary importance of these herbs in the treatment of various diseases and infections. Among these plants, the Fabaceae family stands out for its incredible diversity and significant therapeutic potential, including the genus Dimorphandra, which has demonstrated remarkable efficacy in several studies. Known as fava d'anta, Dimorphandra gardneriana is a plant native to Brazil with significant pharmacological properties, due to the presence of rutin, a flavonoid with anti-inflammatory and antioxidant effects. This study aims to consolidate data on Dimorphandra gardneriana, reviewing its chemical constituents, biological activities, and pharmacological properties to provide a comprehensive overview of its medicinal potential. The review was conducted based on published material and new data collected through databases such as MEDLINE, PUBMED, and SCOPUS. The process included defining the topic, inclusion/exclusion criteria, extraction and classification of studies, evaluation, and interpretation of the results. The analysis revealed that Dimorphandra gardneriana contains several bioactive compounds, such as rutin, galactomannans, and quercetin. These compounds have demonstrated antioxidant, anti-inflammatory, and antimicrobial properties. Combined with their use in advanced controlled release technologies and the application of silver nanoparticles, these properties highlight their value not only in natural medicine but also in industry. These promising characteristics indicate potential future biological activities in various fields of scientific and technological research, with the potential to significantly contribute to the development of new treatments and innovative products in diverse areas of well-being and other sectors, in addition to presenting significant financial value.

Synthesis and Structure of Novel Hybrid Compounds Containing Phthalazin-1(2H)-imine and 4,5-Dihydro-1H-imidazole Cores and Their Sulfonyl Derivatives with Potential Biological Activities.

A novel hybrid compound-2-(4,5-dihydro-1H-imidazol-2-yl)phthalazin-1(2H)-imine (5) was synthesized and converted into di-substituted sulfonamide derivatives 6a-o and phthalazine ring opening products-hydrazonomethylbenzonitriles 7a-m. The newly prepared compounds were characterized using elemental analyses, IR and NMR spectroscopy, as well as mass spectrometry. Single crystal X-ray diffraction data were collected for the representative compounds 5, 6c, 6e, 7g, and 7k. The antiproliferative activity of compound 5, sulfonyl derivatives 6a-o and benzonitriles 7a-m was evaluated on approximately sixty cell lines within nine tumor-type subpanels, including leukemia, lung, colon, CNS, melanoma, ovarian, renal, prostate, and breast. None of the tested compounds showed any activity against the cancer cell lines used. The antioxidant properties of all compounds were assessed using the DPPH, ABTS, and FRAP radical scavenging methods, as well as the β-carotene bleaching test. Antiradical tests revealed that among the investigated compounds, a moderate ABTS antiradical effect was observed for sulfonamide 6j (IC50 = 52.77 µg/mL). Benzonitrile 7i bearing two chlorine atoms on a phenyl ring system showed activity in a β-carotene bleaching test (IC50 = 86.21 µg/mL). Finally, the interaction AGE/RAGE in the presence of the selected phthalazinimines 6a, 6b, 6g, 6m, and hydrazonomethylbenzonitriles 7a, 7c-g, and 7i-k was determined by ELISA assay. A moderate inhibitory potency toward RAGE was found for hydrazonomethylbenzonitriles-7d with an electron-donating methoxy group (R = 3-CH3O-C6H4) and 7f, 7k with an electron-withdrawing substituent (7f, R = 2-Cl-C6H4; 7k, R = 4-NO2-C6H4).

Xanthine Oxidase Inhibition by Monoterpene-Chalcone Conjugates from Kaempferia subglobosa and Molecular Docking Insights.

Kaempferia subglobosa is a perennial medicinal plant in the Zingiberaceae family, identified as a new species in January 2024. To uncover the biological benefits of K. subglobosa and its compounds, investigation of the metabolites of the roots and rhizomes, yielded three new monoterpene-chalcone conjugates, the globosones A-C, representing a rare metabolite group within the Zingiberaceae, along with six known compounds. The biogenetic pathway for the globosones involves an oxidative [3+2] cycloaddition between α-phellandrene and 4'-methoxy-4,2',6'-trihydroxychalcone. Biological testing revealed potent xanthine oxidase (XO) inhibition by globosones A and B, with IC50 values of 7.0±1.0 and 3.0±0.2 μM, respectively, surpassing the positive control drug allopurinol (IC50 7.2±0.1 μM). Globosones A-C also showed good aromatase inhibition (IC50 3.0-3.5 μM). Molecular docking studies indicated that globosones A and B may inhibit xanthine oxidase through binding at the FAD domain site. The physicochemical properties of these isolates suggest that they possess characteristics suitable for additional biological assessment in more advanced test systems. This study enhances an understanding of monoterpene-chalcone conjugate inhibitors of XO, and offers preliminary insights into the metabolites and bioactivities of K. subglobosa, uncovering potent biological activities associated with this newly discovered plant species.

Visible-light-promoted [3 + 2] cycloaddition for the synthesis of spirooxindoles under external photocatalyst-free conditions

A visible-light-promoted [3 + 2] cycloaddition of 3-alkenylindole-2-ones with 2-mercaptoimidazoles for the construction of diverse imidazo[2,1-b]thiazole fused 3,3′-spirooxindoles has been developed. Mechanism studies have shown that this transformation is initiated through visible-light excitation of 3-alkenylindole-2-ones, rather than an electron donor-acceptor (EDA) complexes-promoted process. In summary, we have successfully developed a method for simultaneously constructing C−N and C−S bonds under visible-light conditions, and synthesized over 40 examples of new nitrogen- and sulfur-containing fused heterocyclic compounds with potential biological activity with a yield of up to 99 %. This method exhibits simple operation, mild reaction conditions, high atom economy, good functional group tolerance and does not require the use of any transition metals and external photocatalysts. Gram-scale reaction further demonstrates the practicality of this protocol.

Discovering bioactive phytoconstituents from Citrullus lanatus for antimicrobial and antioxidants therapeutic applications

In recent decades, significant interest in natural bioactive compounds has led many researchers to study plants as primary natural sources of bioactive compounds, mainly when considering a few biological properties, including antioxidant, antimicrobial, and anti-inflammatory activities, and as nutritional supplements with health benefits. We designed this study to explore the phytochemicals profiling of Citrallus lanatus and evaluate their in vitro bioactive properties. The methanol extraction of the watermelon's stem, rind, and leaf parts through Soxhlet extraction was analyzed using Gas Chromatography-Mass Spectrometry. The GC-MS profiling of Citrullus lanatus examines the presence of numerous bioactive phytoconstituents with potential biological activities such as antioxidant, anti-inflammatory, antimicrobial, anticancer, and other properties. We have evidenced that the stem, rind, and leaf extracts with significant antioxidant and antimicrobial activities against clinically relevant pathogens like Salmonella typhi, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus. Overall, our study sheds light on exploring novel bioactive phytoconstituents from Citrullus lanatus and explore, harness these compounds' therapeutic potential for human health.

Study of the Phenolic Compounds and Biological Activities of the Wild Fruits of Vaccinium leucanthum Schltdl.

Around 450 species of blueberries of the genus Vaccinium are known, of which some have gained preferential breeding, such as the ‘Biloxi’ variety. Some little studied species, such as Vaccinium leucanthum Schltdl. located in Mexico, could be a potential source of bioactive compounds. In this study, the phenolic compounds (chlorogenic acid content, hyperoside, phenols, flavonoids, tannins and total anthocyanins content) as well as the potential biological activity (antioxidant, antimicrobial, xanthine oxidase converting enzyme inhibition and angiotensin I inhibition) of Vaccinium leucanthum Schltdl. were studied, making a comparison with the Biloxi variety, which is the most widely cultivated one. The extract of V. leucanthum showed the highest content of flavonoids (4.853 ± 0.341 mg QE/g DW), total anthocyanins (0.303 ± 0.008 mg CGE/g DW), petunidin-3-glucoside (6.92 ± 0.12 mg PGE/g DW), malvidin-3-glucoside (11.80 ± 0.10 mg MGE/g DW) and hyperoside (5.137 ± 0.100 mg HE/g DW). It should be noted that V. leucanthum showed the same total tannin content and the same efficacy in the inhibition of Angiotensin I-converting enzyme as ‘Biloxi’, as well as the same antibacterial effect against the enterobacteria Salmonella choleraesuis ATCC 12022, Escherichia coli ATCC 12792and Shigella flexneri ATCC 10708. These findings demonstrate that V. leucanthum extracts could be an important source of preservatives as well as nutraceutical compounds for use in foods and medicines.

Revealing the Diversity of Sequences, Structures, and Targets of Peptides from South China Sea Macrodactyla doreensis Based on Transcriptomics.

The South China Sea is rich in sea anemone resources, and the protein and peptide components from sea anemone toxins comprise an important treasure trove for researchers to search for leading compounds. This study conducted a comprehensive transcriptomic analysis of the tentacles and column of Macrodactyla doreensis and explored the distribution and diversity of proteins and peptides in depth using bioinformatics, initially constructing a putative protein and peptide database. In this database, typical peptide families are identified through amino acid sequence analysis, and their 3D structures and potential biological activities are revealed through AlphaFold2 modeling and molecular docking. A total of 4239 transcripts were identified, of which the putative protein accounted for 81.53%. The highest content comprised immunoglobulin and a variety of proteases, mainly distributed in the column and related to biological functions. Importantly, the putative peptide accounted for 18.47%, containing ShK domain and Kunitz-type peptides, mainly distributed in the tentacles and related to offensive predatory behavior. Interestingly, 40 putative peptides belonging to eight typical peptide families were identified, and their structures and targets were predicted. This study reveals the diversity and complexity of Macrodactyla doreensis toxins and predicts their structure and targets based on amino acid sequences, providing a feasible approach for research regarding the discovery of peptides with potentially high activity.

Diversity and Bioactivity of Endophytic Actinobacteria Associated with the Roots of Artemisia herba-alba Asso from Algeria.

The isolation of endophytic actinobacteria from the roots of wild populations of Artemisia herba-alba Asso, a medicinal plant collected from the arid lands of Algeria, is reported for the first time. Forty-five actinobacterial isolates were identified by molecular analysis and in vitro evaluated for antimicrobial activity and plant growth-promoting (PGP) abilities (1-Aminocyclopropane-1-carboxylic acid (ACC) deaminase activity, nitrogen fixation, phosphate and potassium solubilization, ammonia, and siderophores production). The phylogenetic relationships based on 16S rRNA gene sequences show that the genus Nocardioides (n = 23) was dominant in the sampled localities. The remaining actinobacterial isolates were identified as Promicromonospora (n = 11), Streptomyces (n = 6), Micromonopora (n = 3), and Saccharothrix (n = 2). Only six (13.33%) strains (five Streptomyces and one Saccharothrix species) were antagonistic in vitro against at least one or more indicator microorganisms. The antimicrobial activity of actinobacterial strains targeted mainly Gram-positive bacteria. The results demonstrate that more than 73% of the isolated strains had ACC deaminase activity, could fix atmospheric nitrogen and were producers of ammonia and siderophores. However, only one (2.22%) strain named Saccharothrix sp. BT79 could solubilize phosphorus and potassium. Overall, many strains exhibited a broad spectrum of PGP abilities. Thus, A. herba-alba provides a source of endophytic actinobacteria that should be explored for their potential biological activities.

Phytochemical Profile and Antioxidant, Enzyme-Inhibitory, and Cytotoxic Potential of Endemic Cynoglottis chetikiana subsp. chetikiana

Background Cynoglottis chetikiana subsp. chetikiana, an endemic Boraginaceae plant growing in Turkey, has not been previously studied in terms of pharmacognosy. However, related species, such as Anchusa, Symphytum, and Rindera, have been known for their pharmacological effects attributed to compounds such as flavonoids, terpenoids, and other phenol derivatives. Purpose This study aimed to reveal the phytochemical composition and potential biological activity of C. chetikiana. Methods Phytochemical profiling analysis was performed by using the liquid chromatography–high-resolution mass spectrometry (LC–HRMS) system. The cytotoxic effects of the extracts were evaluated on the MCF-7 mammalian breast cancer cell line by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. The in vitro α-glucosidase and tyrosinase enzyme inhibitory activities were assessed using literature methods. Additionally, antioxidant properties were investigated, leading to the bioactivity-guided isolation of the most active substance. Results LC–HRMS analysis revealed 27 major phenolic compounds and 2 pyrrolizidine alkaloids in C. chetikiana. Bioactivity-guided studies led to the isolation of a flavonoid, rutin. The n-butanol extract of the plant exhibited high antioxidant capacity and was rich in phenolic compounds. The methanol extract of C. chetikiana demonstrated low-moderate α-glucosidase inhibitory activity (IC50 = 608.62 ± 7.01 µg/mL). Moreover, the methanol extract showed significant anti-tyrosinase enzyme activity (195.84 ± 5.81 mg Kojic acid equivalent/g extract), and the petroleum ether extract displayed the highest cytotoxicity on MCF-7 cells (IC50 = 76.91 ± 3.12 µg/mL). Conclusion This is the first phytomedicinal study report for C. chetikiana, which gives promising insight for further pharmaceutical research.

ESTUDO IN SILICO DOS CONSTITUINTES DO MEL DA AROEIRA (Astronium urundeuva) E A CAPACIDADE DE INTERAÇÃO COM A PROTEINASE GP63 DA LEISHMANIOSE

Neglected tropical diseases represent a significant source of morbidity and mortality in developing countries, representing a global health challenge. Honey produced by bees is an organic food, composed of a mixture rich in nutrients, such as carbohydrates, amino acids, fatty acids, enzymes, minerals and vitamins. Depending on flowering, honey may contain phenolic compounds, flavorings and flavonoids, which may exhibit biological activities. In this in silico study on the constituents of mastic honey (Astronium urundeuva), we sought to predict the physicochemical, pharmacokinetic and toxicological properties, considering the interaction capacity of the constituents with the GP63 receptor, from Leishmaniasis. To this end, a systematic review of the literature was carried out and the molecular structures of the constituents were obtained, predicting ADMET properties and evaluating the binding affinity with molecular docking. Thus, information was obtained on the in silico physicochemical and pharmacological properties of the identified compounds, highlighting the potential biological activity for the treatment of Leishmaniasis, through the ability to interact with the GP63 protease. This focus will enable new investigations and therapeutic advances in the area.

Discovery of sp3-rich diazatricycloundecanes as lysosomotropic autophagy inhibitors

We have discovered lysosomotropic autophagy inhibitors from our compound library of sp3-rich diazatricycloundecane skeletons. Compound 1u was identified as the most potent biological activity for LC3-II protein accumulation through the structure-activity relationships (SARs) for LC3-II protein accumulation and anti-proliferative activity at the three freely available substituents (R1-R3) in the diazatricycloundecane skeleton. Compound 1u inhibited lysosome-dependent degradation without affecting autophagosome formation. Furthermore, compound 1u enlarged lysosomes and raised lysosomal pH similar to lysosomotropic agents such as chloroquine, resulting in inhibiting late-stage autophagy by inducing lysosomal dysfunction. Moreover, compound 1u exhibits excellent drug-like chemical properties, not previously reported for lysosomotropic agents.

The beneficial health effects of puerarin in the treatment of cardiovascular diseases: from mechanisms to therapeutics.

Cardiovascular diseases (CVDs) are the leading causes of death globally that seriously threaten human health. Although novel western medicines have continued to be discovered over the past few decades to inhibit the progression of CVDs, new drug research and development for treating CVDs with less side effects and adverse reactions are continuously being desired. Puerarin is a natural product found in a variety of medicinal plants belonging to the flavonoid family with potent biological and pharmacological activities. Abundant research findings in the literature have suggested that puerarin possesses a promising prospect in treating CVDs. In recent years, numerous new molecular mechanisms of puerarin have been explored in experimental and clinical studies, providing new evidence for this plant metabolite to protect against CVDs. This article systematically introduces the history of use, bioavailability, and various dosage forms of puerarin and further summarizes recently published data on the major research advances and their underlying therapeutic mechanisms in treating CVDs. It may provide references for researchers in the fields of pharmacology, natural products, and internal medicine.