Network pharmacology and molecular docking combined with widely targeted metabolomics to elucidate the potential compounds and targets of Euphorbia helioscopia seeds for the treatment of pulmonary fibrosis.

BACKGROUND: The whole herb of Euphorbia helioscopia has been traditionally used for treating pulmonary tuberculosis, malaria, warts, lung cancer and bacillary dysentery for a long time in China. However, E. helioscopia seeds are often discarded and its medicinal value is often ignored, resulting in a waste of resources. METHOD: In this work, widely targeted metabolomics based on UPLC-ESI-QTRAP-MS/MS methods and metware database (MWDB) were firstly used to identify the chemical compositions of EHS. Besides, network pharmacology, molecular docking and molecular dynamics simulation were performed for elucidating the potential compounds and targets of E. helioscopia seeds for the treatment of pulmonary fibrosis via common database (like TCMSP, Genecards, DAVID, STRING) and common software (like Sybyl, Cytoscape, Pymol and Schrödinger). RESULT: The results of widely targeted metabolomics showed 231 compounds including 12 categories were identified. The highest content compositions are lipids (33.89%) followed by amino acids and derivatives (21.78%), nucleotides and derivatives (15.73%), as well as the content of functional ingredients like phenolic acids (7.33%), alkaloids (7.03%) and flavonoids (4.51%) are relatively high. Besides, the results of network pharmacology and molecular docking showed that EHS presented anti-pulmonary fibrosis medicinal value through multi-ingredients, multi-targets and multi-pathways approach. Key ingredients including 9-Hydroxy-12-oxo-15(Z)-octadecenoic acid, Nordihydrocapsiate, 1-O-Salicyl-d-glucose, 9-(Arabinosyl)hypoxanthine, Xanthosine and Galangin-7-O-glucoside. Key targets including SRC, HSP90AA1, AKT1, EGFR, JUN, EP300 and VEGFA, and key signaling pathways mainly related to AGE-RAGE, EGFR tyrosine kinase inhibitor resistance, VEGF and HIF-1 signaling pathway. Molecular dynamics simulation showed that HSP90AA1 and 9-Hydroxy-12-oxo-15(Z)-octadecenoic complex (with the highest docking score) have a stable combination effect. CONCLUSION: In conclusion, this study revealed the chemical compositions of EHS and its anti-pulmonary fibrosis medicinal effect for the first time, it will provide scientific insight for the development of EHS as medicinal resource.

Natural edible pigments: A comprehensive review of resource, chemical classification, biosynthesis pathway, separated methods and application.

Natural edible pigments, high safety and low toxicity, usually possess various nutritional and pharmacological effects, and they have huge practical application value in the market. However, until now, there is no systematic review about the resources, chemical classifications and application about them. Moreover, the extracted methods and biosynthesis pathways which are very important informations for obtaining high-yield and high-purity natural edible pigments from natural resources are still lacking. Therefore, It is necessary to make a comprehensive review of natural edible pigments. In this work, we systematically summarize the resources, chemical classifications, biosynthesis pathways, extraction and separation methods, as well as application of natural edible pigments for the first time. Our work will provide reference data and give the inspiration for further industrial application of natural edible pigments.

Leaves, seeds and exocarp of Ginkgo biloba L. (Ginkgoaceae): A Comprehensive Review of Traditional Uses, phytochemistry, pharmacology, resource utilization and toxicity.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginkgo biloba L. (Ginkgoaceae) is a treasure species with high medicinal value. The Ming Dynasty "Compendium of Materia Medica" and Qing Dynasty "Bencao Fengyuan" in China recorded this herbal medicine can reduce phlegm, clear poison, treat diarrhea and frequent urination, etc. AIM OF THE STUDY: Until now, there is no painstakingly summarized review on leaves, seeds and exocarp of G. biloba simultaneously. This review will systematically summarize and compare current knowledge of G. biloba. MATERIALS AND METHODS: Ample original publications related to traditional uses, phytochemistry, pharmacology, resource utilization and toxicity of G. biloba leaves, seeds and exocarp till the end of 2021 were searched and collected by using various literature databases, including China National Knowledge Infrastructure, PubMed, Elsevier, Springer, Google Scholar and Web of Science database. RESULTS: According to classical Chinese herbal books and Chinese Pharmacopoeia, relieving cough, reducing phlegm, clearing poison and relieving diarrhea are the main pharmacological effects of G. biloba. The common chemical ingredients in different parts of G. biloba are flavonoids, terpenoids, phenolic acids, polysaccharides and endotoxin, etc. Among them, flavonoids and terpenoids are the main bioactive compounds in G. biloba leaves. Phenolic acids are the main bioactive compounds in G. biloba exocarp. G. biloba seeds are rich in nutritional ingredients, such as starch, adipose, protein, etc. Modern pharmacological studies showed that the crude extracts or compounds of G. biloba leaves, seeds and exocarp can be used for treating cardiovascular and cerebrovascular diseases, Alzheimer's disease, atherosclerosis, cancer, asthma, non-alcoholic fatty liver, diabetic complications and other diseases. In daily life, G. biloba seeds were usually used as raw material or additives for commodities, healthy food, drinks, even insecticides and antibacterial agents, etc. G. biloba leaves and seeds have been mainly applied for treating cardiovascular and cerebrovascular diseases, cough and asthma in clinical. However, endotoxins and ginkgolic acids have been identified as the dominating toxic ingredients in different parts of G. biloba. Besides, flavonoids and ginkgolides also have been proved to have toxicity recently. CONCLUSIONS: This review systematically sums up and compares the traditional uses, phytochemistry, pharmacology, resource utilization and toxicity research progress of G. biloba leaves, seeds and exocarp for the first time. It will provide some comprehensive reference data and suggestions for future research on this herbal medicine.

Arenaria kansuensis attenuates pulmonary fibrosis in mice via the activation of Nrf2 pathway and the inhibition of NF-kB/TGF-beta1/Smad2/3 pathway.

Pulmonary fibrosis is a key feature of COVID-19, Chinese herbal medicine Arenaria kansuensis has been used for curing pulmonary disease and antivirus for a long time and it has the potential against COVID-19. In this work, protective effect of A. kansuensis ethanol extract (AE) on pulmonary fibrosis was evaluated through paraquat (PQ)-induced pulmonary fibrosis animal model. Results showed that AE could significantly improve the survival rate, increase the body weight and reduce the lung index of mice at the raw drug doses of 700 and 350 mg/kg. Histopathological observation results showed that the destruction degree of lung tissue structure in mice was significantly improved with the increase of AE dosage. Collagen deposition in lung interstitium was significantly reduced. The marker protein alpha-SMA involved in PF were significantly inhibited through repressing TGF-beta1/Smads pathway. The degree of inflammatory infiltration was significantly reduced and inflammatory cytokines were significantly inhibited in mice through inhibiting the NF-kB-p65. Besides, oxidant stress level including upregulated ROS and down-regulated SOD and GSH was efficiently improved by AE through upregulation of Nrf2 and downregulation of NOX4. In summary, this study firstly showed that the protective effect of AE on pulmonary fibrosis was partly due to activation of Nrf2 pathway and the inhibition of NF-kB/TGF-beta1/Smad2/3 pathway.

ß-carboline alkaloids attenuate bleomycin induced pulmonary fibrosis in mice through inhibiting NF-kb/p65 phosphorylation and epithelial-mesenchymal transition.

ETHNOPHARMACOLOGICAL RELEVANCE: The plant Arenaria kansuensis is used in traditional medicine to treat lung inflammation for a long time. However, the anti-pulmonary fibrosis effect and its corresponding bioactive constituents of this plant have not been studied extensively. AIM OF THE STUDY: The purpose of this study was to investigate the anti-pulmonary fibrosis effect and its corresponding bioactive constituents of A. kansuensis and its possible mechanism. MATERIALS AND METHODS: In vivo experiment, the anti-pulmonary fibrosis effects of the fraction (Part1) enriched from ethyl acetate extracts of the whole plant A. kansuensis were evaluated through bleomycin (BLM)-induced pulmonary fibrosis mice (five groups, n = 10) daily at doses of 50, 100 and 150 mg/kg for 15 days. In vitro experiment, the anti-inflammation and reversed epithelial-mesenchymal transition (EMT) effect of 12 ß-carboline alkaloids isolated from Part1 were evaluated through lipopolysaccharide (LPS)-induced RAW264.7 inflammatory cell model and TGF-ß1 induced A549 cell model. RESULTS: In this study, a fraction named Part1 extracted from Arenaria kansuensis presented strong anti-pulmonary fibrosis effect at the dose of 150 mg/kg. Vivo experiments showed that the survival rate and body weight of mice significantly increased after Part1 treatment. Part1 could significantly inhibit the initial of inflammation, deposition of collagen and expression of TGF-ß1 and α-SMA, moreover, the expression of E-cadherin was significantly elevated after administration of Part1. All the cure effects of Part1 were in dose dependent manner. A total of 12 ß-carboline alkaloids were identified in Part1 and they all showed suppressive effect on inflammatory cytokines including MCP-1, TNF-α, IL-6 and IL-1ß through inhibition of NF-kb/p65 phosphorylation, and that epithelial-mesenchymal transition (EMT) process was reversed by different compounds in different levels. The expression of indicators of EMT including α-SMA, vimentin and E-cadherin was significantly improved after given different ß-carboline alkaloids. CONCLUSIONS: This study showed that antifibrogenic effect of ß-carboline alkaloids was due to inhibiting the initial of inflammation through NF-kb/p65 pathway and reversing the process of EMT.

Purification of Flavonolignan Diastereoisomers from Arenaria kansuensis by Two-Dimensional Liquid Chromatography Combined with Solid-Phase Extraction.

Herbal plants are significant for the reason that they have a great potential in discovering drug precursors. However, how to purify compounds with higher purity from them is a question which needs to be discussed. In present study, an offline 2D reversed-phase (RP) preparative liquid chromatography coupled with solid-phase extraction (SPE) method was successfully developed for the separation of flavonolignan diastereoisomers from Arenaria kansuensis. Based on the analysis of results, the major conclusion that we have drawn from it is a RP-SPE was selected for enriching target flavonolignan sample from A. kansuensis. After that, an ODS preparative column was used for 1D preparation, and the target sample (4.6 g) was divided into five fractions with a recovery of 83.9%. Then, a C18HCE preparative column, a polar-modified RP (polar-copolymerized) type, was used for isolating flavonolignan diastereoisomers in the 2D preparation. By establishing optimal 2D chromatography, hydrophilic interaction chromatography (HILIC) columns and normal-phase (NP) columns were tested simultaneously, and the result showed that diastereoisomers are not suitable for HILIC and NP chromatography mode. Our study resulted in a tricin and five analogous derivative flavonolignans with purity >98% were successfully purified from A. kansuensis. This is the initial report of Salcolin C, Salcolin B, Tricin 4'-O-(C-veratroylglycol) ether and 5'-methoxyhydnocarpin D from A. kansuensis. In addition, it tended to be the first time that Tricin 4'-O-(C-veratroylglycol) ether is isolated from natural resource. This method has great potential for efficiently isolating flavonolignan diastereoisomers from A. kansuensis, and it shows a great prospect for the separation of flavonolignans from complex samples.

Anti-alcohol liver disease effect of Gentianae macrophyllae extract through MAPK/JNK/p38 pathway.

OBJECTIVES: The hepatoprotective effect of Gentianae macrophyllae root extract (GME) on alcoholic liver disease (ALD) was evaluated through ethanol induced ALD animal model. METHODS: Mice were randomly divided into control normal group (10 mice), ethanol-induced ALD model group (10 mice) and GME plus ethanol group (30 mice). Mice in model group were given intragastric administration with 50% (v/v) ethanol aqueous solution (200 µl for each) once daily for 19 days. Mice in control normal group received equal volumes of water. Mice in GME plus ethanol group were given intragastric administration with 50% (v/v) ethanol aqueous solution (200 µl for each) once daily at 10:00 a.m., after 1 h, mice in GME group sequentially were treated with 20, 40 and 100 mg/kg of GME by gastric gavage for 19 days. the average food and water consumed by the mice in every group were recorded every 2 days and body weight of every mouse in every group was measured every 2 days. KEY FINDINGS: Results showed that GME significantly improved alcohol induced liver injury in a dose-dependent manner. The impaired hepatic tissue structure was repaired and the collagen deposition declined after GME administration. Meanwhile, the level of malonaldehyde (MDA), Aspartate transaminase (AST) and alanine transaminase (ALT) (indicators of liver damage) in blood serum were significantly controlled by GME with a dose-dependent manner, moreover, body weight and liver index were also improved after administration of GME. Pro-inflammatory cytokines including MCP-1, TNF-α, IL-1 and IL-6 were detected through RT-PCR and ELISA in experiment and GME can significantly inhibit the expression of TNF-α, IL-1 and IL-6 but have no effect on MCP-1. In order to explore the mechanism of GME on ALD, MAPKs pathway was examined and results indicated that GME attenuated ALD through inhibiting the phosphorylation of JNK and P38 and further suppressing the initiation of inflammation. CONCLUSIONS: GME attenuated ALD through inhibiting the phosphorylation of JNK and P38 and further suppressing the initiation of inflammation.

Efficient Separation of Four Antibacterial Diterpenes from the Roots of Salvia Prattii Using Non-Aqueous Hydrophilic Solid-Phase Extraction Followed by Preparative High-Performance Liquid Chromatography.

An efficient preparative procedure for the separation of four antibacterial diterpenes from a Salvia prattii crude diterpenes-rich sample was developed. Firstly, the XION hydrophilic stationary phase was chosen to separate the antibacterial crude diterpenes-rich sample (18.0 g) into three fractions with a recovery of 46.1%. Then, the antibacterial fractions I (200 mg), II (200 mg), and III (150 g) were separated by the Megress C18 preparative column, and compounds tanshinone IIA (80.0 mg), salvinolone (62.0 mg), cryptotanshinone (70.0 mg), and ferruginol (68.0 mg) were produced with purities greater than 98%. The procedure achieved large-scale preparation of the four diterpenes with high purity, and it could act as a reference for the efficient preparation of active diterpenes from other plant extracts.

Antihypoxic activities of constituents from Arenaria kansuensis.

BACKGROUND: In previous investigation, we have identified antioxidative effects of water-soluble ethanolic extracts (named as AKE) from Arenaria kansuensis and inferred that these extracts or their constituents may also have antihypoxic activity. A. kansuensis has been widely used in traditional Tibetan medicine for altitude sickness (AS) and has been known as the herb of anti-inflammatory and hypoxia resistance for a long time. PURPOSE: The purpose of this study is to evaluate protective effects of AKE and its major constituents against hypoxia-induced lethality in mice and RSC96 cells. STUDY DESIGN AND METHODS: Hypoxia-induced lethality in mice was investigated by 3 experimental animal models of hypoxia. Meanwhile, we established a RSC96 cell model of hypoxia which applied to screen and assess the anti-hypoxic activity of compounds isolated from A. kansuensis. RESULTS: Results indicated that AKE dose-dependently prolonged survival time of hypoxia induced lethality in mice compared to vehicle group and exhibited significantly anti-hypoxic effect. AKE also enhanced the number of red blood cells (RBC) and the concentration of hemoglobin (HB). 8 compounds were bio-guided separated and purified from AKE based on the animal model and cell model of hypoxia. Among which pyrocatechol (C16) and tricin 7-O-ß-d-glucopyranoside (C13) were confirmed to express better protective effects on cell damage induced by hypoxia, suggesting that these two compounds are major active constituents of AKE for anti-hypoxia. CONCLUSION: This study demonstrated that pyrocatechol and tricin 7-O-ß-d-glucopyranoside could be therapeutic candidates for treatment of AS. It is the first time to find the major active constituents of AKE for anti-hypoxia. Meanwhile, a RSC96 cell model of hypoxia was established to screen anti-hypoxic activity of compounds for the first time.

Erythritol Attenuates Postprandial Blood Glucose by Inhibiting α-Glucosidase.

Diabetes mellitus (DM) is a serious metabolic disorder, where impaired postprandial blood glucose regulation often leads to severe health complications. The natural chemical erythritol is a C4 polyol approved by the U.S. Food and Drug Administration for use as a sweetener. Here, we examined a potential role for erythritol in the control of postprandial blood glucose levels in DM. An anti-postprandial hyperglycemia effect upon erythritol administration (500 mg kg-1) was demonstrated in alloxan-induced DM model mice by monitoring changes in blood glucose after intragastric administration of drugs and starch. We also found that erythritol most likely exerts its anti-postprandial hyperglycemic activities by inhibiting α-glucosidase in a competitive manner. This was supported by enzyme activity assays and molecular modeling experiments. In the latter experiments, it was possible to successfully dock erythritol into the catalytic pocket of α-glucosidase, with the resultant interaction likely driven by electrostatic interactions involving Asp215, Asp69, and Arg446 residues. This study suggests that erythritol may not only serve as a glucose substitute but also be a useful agent in the treatment of DM to help manage postprandial blood glucose levels.

Osteon Myospalacem Baileyi attenuates osteoclast differentiation through RANKL induced NFAT pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Osteon Myospalacem Baileyi, known as Sai long gu (Tibetan language, means "blind rat bone"), is the whole skeleton of Tibet plateau rodentia animal Myospalacem Baileyi. Osteon Myospalacem Baileyi had been widely used in the Tibet region as an anti-osteoporosis drug and since 1991 Osteon Myospalacem Baileyi has been listed in the Pharmacopoeia of People's Republic of China as the first-class animal new medical material. However, the mechanism of its anti-osteoporosis activities is still unclear. It is very desirable to solve this problem for further study. MATERIALS AND METHODS: in this study, preparative chromatography was employed to produce the active fraction ET4 from Osteon Myospalacem Baileyi crude. Flow cytometry and MTT assay were used to evaluate the toxicities of ET4. BMM cells were separated from mouse bone marrow to test the inhibition effects of ET4 on osteoclastogenesis. Western blot was used to find out the pathways, through which ET4 could act on osteoclastogenesis. Q-PCR was used to test the osteoclastogenesis marker genes. At last, immunofluorescence confocal microscopy was used to test the osteoclastogenesis master protein NFATc1 nuclei translocation. RESULTS: In this study we report that ET4, at the dose of 60µg/mL, significantly inhibited the formation of osteoclasts. Notably, ET4 did not affect the BMM viability at that dose. In addition, Osteon Myospalacem Baileyi could inhibit the expression of osteoclast marker genes, including cathepsin K (CTSK), nuclear factor of activated T cells cytoplasmic 1 (NFATc1), tartrate resistant acid phosphatase (TRAP, Acp5) dendrite cell-specific transmembrane protein (DC-STAMP), calcitonin receptor (CTR), osteoclast associated and immunoglobulin-like receptor (OSCAR). Mechanistically, ET4 dose- and time-dependently blocked the RANKL-induced activation of ERK and c-Fos as well as the induction of NFATc1 which is essential for OC formation. CONCLUSIONS: These data suggest that ET4 might be a useful alternative therapy in preventing or treating osteolytic diseases.

Trace anti-inflammatory ß-carboline alkaloid identified in Arenaria kansuensis by two-dimensional chromatography coupled with UniElut C18AEX based solid-phase extraction re-enrichment technology.

Traditional Chinese medicine is important for discovery of drug precursors. However, information about trace chemical composition of them is very limited due to the lack of appropriate enrichment and chromatographic purification methods In our work, A. kansuensis was taken as an example, a novel two-dimensional reversed-phase/hydrophilic interaction liquid chromatography coupled with UniElut C18AEX solid-phase extraction re-enrichment method based on anti-inflammatory bioactivity-guided assay was developed for gathering and purifying trace ß-carboline alkaloids with high purity from the ethyl acetate extract of A. kansuensis. Extraction with ethyl acetate as the first enrichment method, then, a UniElut C18AEX column was employed to re-enrich trace fraction which was hardly detected by diode array detector during high performance liquid chromatography analysis, eighteen grams of UniElut C18AEX was used as sorbent material to pack a 60mL pipette tip for the extraction of ß-carboline alkaloids from 100mL of ethyl acetate sample. The whole extraction process was finished in 10min, and the volume of eluent used was only 120mL. The enriching fraction (100mg) was used for the following two-dimensional purification. First-dimensional preparation was carried on a RP-Megress-C18 prep column, and four anti-inflammatory fractions were obtained from the 100mg re-enriching sample with a recovery of 66.9%. A HILIC-XAmide prep column was selected for the second dimensional preparation. Finally, two pair of analogue ß-carboline alkaloids and one other ß-carboline alkaloid were purified from A. kansuensis. The purity of the isolated compounds was ≫>98%, which indicated that the method was efficient to re-enrich and manufacture single trace ß-carboline alkaloids with high purity from A. kansuensis. Additionally, this method showed great potential to serve as a good example for the purification and enrichment of analogue structure anti-inflammation carboline alkaloids from other plant materials.

Anti-inflammatory bioactive equivalence of combinatorial components ß-carboline alkaloids identified in Arenaria kansuensis by two-dimensional chromatography and solid-phase extraction coupled with liquid-liquid extraction enrichment technology.

Bioactive equivalent combinatorial components play a critical role in herbal medicines. However, how to discover and enrich them efficiently is a question for herbal pharmaceuticals researchers. In our work, a novel two-dimensional reversed-phase/hydrophilic interaction high-performance liquid chromatography method was established to perform real-time components trapping and combining for preparation and isolation of coeluting components. Arenaria kansuensis was taken as an example, and solid-phase extraction coupled with liquid-liquid extraction as a simple and efficient method for enriching trace components, reversed phase column coupled with hydrophilic interaction liquid chromatography XAmide column as two-dimensional chromatography technology for isolation and preparation of coeluting constituents, enzyme-linked immune-sorbent assay as bio-guided assay, and anti-inflammatory bioactivity evaluation for bioactive constituents. A combination of 12 ß-carboline alkaloids was identified as anti-inflammatory bioactive equivalent combinatorial components from A. kansuensis, which accounts for 1.9% w/w of original A. kansuensis. This work answers the key question of which are real anti-inflammatory components from A. kansuensis and provides a fast and efficient approach for discovering and enriching trace ß-carboline alkaloids from herbal medicines for the first time. More importantly, the discovery of bioactive equivalent combinatorial components could improve the quality control of herbal products and inspire a herbal medicine based on combinatorial therapeutics.

Two-dimensional chromatography based on on-line HPLC-DPPH bioactivity-guided assay for the preparative isolation of analogue antioxidant compound from Arenaria kansuensis.

Traditional Tibetan medicine is important for discovery of drug precursors. However, information about the chemical composition of traditional Tibetan medicine is very limited due to the lack of appropriate chromatographic purification methods. In the present work, A. kansuensis was taken as an example and a novel two-dimensional reversed-phase/hydrophilic interaction liquid chromatography(HILIC) method based on on-line HPLC-DPPH bioactivity-guided assay was developed for the purification of analogue antioxidant compounds with high purity from the extract of A. kansuensis. Based on the separation results of many different chromatographic stationary phases, the first-dimensional (1D) preparation was carried on a RP-C18HCE prep column, and 2 antioxidant fractions were obtained from the 800mg crude sample with a recovery of 56.7%. A HILIC-XAmide prep column was selected for the second-dimensional (2D) preparation. Finally, a novel antioxidant ß-carboline Alkaloids (Glusodichotomine AK) and 4 known compounds (Tricin, Homoeriodictyol, Luteolin, Glucodichotomine B) were purified from A. kansuensis. The purity of the compounds isolated from the crude extract was >98%, which indicated that the method built in this work was efficient to manufacture single analogue antioxidant compounds of high purity from the extract of A. kansuensis. Additionally, this method showed great potential in the preparation of analogue structure antioxidant compounds and can serve as a good example for the purification of analogue structure antioxidant carboline alkaloids and flavonoids from other plant materials.

Phenolics from Lagotis brevituba Maxim.

A phytochemical investigation on Lagotis brevituba led to the isolation and characterisation of 11 phenolic compounds: p-hydroxy-benzoic acid 1, methyl 3,4-dihydroxybenzoate 2, vanillic acid 3, protocatechuic acid 4, caffeic acid 5, glucose ester of (E)-ferulic acid 6, p-coumaric acid 7, vanillin 8, diosmetin-7-O-ß-d-glucoside 9, chrysoeriol 10 and luteolin 11. Their structures were elucidated using spectroscopic methods and by comparison with data in the literature. Compounds 1-6 were first obtained from the genus Lagotis, and compounds 1-9 were isolated from L. brevituba for the first time. Compound 4 and 11 displayed remarkable antioxidant activities against DPPH radical (IC50 = 5.60 ± 0.09, 27.5 ± 0.06 mg/L, respectively), which were superior to positive control rutin. And compound 11 was also superior to rutin in ABTS assay (IC50 = 2.04 ± 0.13 mg/L).