Oligonucleotide conjugates are versatile scaffolds that can be applied in DNA-based screening platforms and ligand display or as therapeutics. Several different chemical approaches are available for functionalizing oligonucleotides, which are often carried out on the 5′ or 3′ end. Modifying oligonucleotides in the middle of the sequence opens the possibility to ligate the conjugates and create DNA strands bearing multiple different ligands. Our goal was to establish a complete workflow that can be applied for such purposes from monomer synthesis to templated ligation. To achieve this, a monomer is required with an orthogonal functional group that can be incorporated internally into the oligonucleotide sequence. This is followed by conjugation with different molecules and ligation with the help of a complementary template. Here, we show the synthesis and the application of a thiol-modified thymidine nucleoside phosphoramidite to prepare ligatable oligonucleotide conjugates. The conjugations were performed both in solution and on solid phase, resulting in conjugates that can be assembled into multivalent oligonucleotides decorated with tissue-targeting peptides using templated ligation. The ability of specifically recognizing complementary DNA strands makes oligonucleotides attractive tools for diagnostics, therapeutics, or platforms for displaying molecules. Decorating an oligonucleotide strand with molecules while keeping its ability to hybridize specifically to a target sequence has numerous applications. Oligonucleotides conjugated with cell-penetrating peptides, targeting moieties or antibodies, influence their pharmacokinetic properties that allow for their use as therapeutics or detection reagents ,2,3,4. OMany different synthetic approaches are available to functionalize oligonucleotide sequences ,11,12,13tert-butyl disulfide (t-BuSSR) could overcome these issues O-[tert-butyl(dimethyl)silyl]-2’-deoxyuridine (4)5-(Azidomethyl)-3’,5’-bis-2 was dissolved in 100 mL of anhydrous CCl4, then 3.54 g  of NBS and 0.82 g  of AIBN was added. The reaction mixture was stirred at 70 °C under Ar atmosphere for 3 h. The TLC showed incomplete conversion of the starting material, therefore another 0.89 g  of NBS and 0.82 g  of AIBN were added. The mixture was stirred for another 3 h when the TLC showed an almost complete conversion. The excess of NBS and the succinimide were filtered off and 100 mL of anhydrous DMF was added to the solution. After evaporation of the CCl4, 1.63 g  of NaN3 was added and the mixture was stirred at room temperature overnight. The DMF was evaporated and the residue was dissolved in EtOAc (100 mL) and washed twice with 100 mL of aqueous NaHSO4 (1%). The organic phase was dried on Na2SO4 and after the removal of the solvent under reduced pressure, the crude product was chromatographed on silica gel using 10–20% (v/v) of EtOAc in hexanes to obtain an oily residue, yield: 3.07 g (60%).A modified procedure by Gubu et al.  has been1H NMR (Chloroform-d) δ 7.69 , 6.22 , 4.37 , 3.91 , 3.76 , 3.70 , 2.27 , 2.10–2.02 , 0.89 3C), and 0.13–0.02 .13C NMR (Chloroform-d) δ 164.34 (4-C), 150.19 (2-C), 139.30 (6-CH), 110.02 (5-C), 88.09 (4′-C), 85.53 (1′-CH), 72.48 (3′-CH), 63.23 (5′-CH2), 41.06 (2′-CH2), 38.17 (CH2NH2), 26.05 ((CH3)3C), 25.89 ((CH3)3C), 18.50 ((CH3)3C), 18.09 ((CH3)3C), −4.49 (CH3Si), −4.70 (CH3Si), −5.17 (CH3Si), and −5.27 (CH3Si).+), calculated for C22H44N3O5Si2+ 486.2814, measured 486.2811 (ΔM: −0.6 ppm).The HRMS (ESIO-[tert-butyl(dimethyl)silyl]-2′-deoxyuridine (5)5-(Aminomethyl)-3′,5′-bis-4 was dissolved in 60 mL of THF, then 3.14 g  of Ph3P and 2.16 mL  water was added. The reaction mixture was kept at 60 °C for 3 h, then the solvent was evaporated and the residue was column chromatographed on silica using 5–15% (v/v) MeOH in EtOAc. The yield was 2.3 g, white foam (78%).First, 3.07 g (6 mmol) of azide 1H NMR (Chloroform-d) δ 9.56 , 7.68 , 6.24 , 4.38 , 4.08 , 3.93 , 3.77 , 2.93–2.88 , 2.56–2.47 , 2.27 , 2.08–2.04 , 1.30 3CS), 0.89 3CSi), and 0.09 .13C NMR (Chloroform-d) δ 170.96 (CONH), 164.09 (4-C), 150.23 (2-C), 138.90 (6-CH), 111.15 (5-C), 88.16 (4′-CH), 85.61 (1′-CH), 72.52 (3′-CH), 63.26 (5′-CH), 48.02 ((CH3)3CS), 41.16 (2′-CH2), 36.83 (5-CH2), 36.17 (CH2CH2), 35.78 (CH2CH2), 30.05 ((CH3)3CS), 26.08 ((CH3)3CSi), 25.86 ((CH3)3CSi), 18.52, 18.09 (CH3)3CSi), −4.54 (CH3Si), −4.71 (CH3Si), −5.17 (CH3Si), and −5.31 (CH3Si).+), calculated for C29H56N3O6S2Si2+ 662.3144, measured 662.3140 (ΔM: −0.6 ppm).HRMS (ESIO-[tert-butyl(dimethyl)silyl]-5-{[3-(tert-butyldisulfanyl)propanamido]-methyl}-2′-deoxyuridine (8)3′,5′-Bis-tert-butyldisulfanyl)propanoic acid (7), 1.16 g (7.6 mmol) of HOBt, 1.36 mL (8 mmol) of DIPEA, and 2.84 g (7.6 mmol) HBTU were dissolved in 40 mL of anhydrous DMF. In 10 min, 3.04 g  of amine 5 was dissolved in 30 mL of anhydrous DMF and was mixed with the activated carboxylic acid 7 solution; the mixture was stirred for 2 h. The TLC did not show any ninhydrin-active spots; therefore, the reaction mixture was evaporated, dissolved in EtOAc (100 mL), washed twice with 1% NaHSO4 solution (2 × 100 mL), dried over Na2SO4, and evaporated. The crude oily material was column chromatographed using 30–40% (v/v) EtOAc in hexanes eluent. The yield was 3.0 g (73%) pale yellow oil.First, 1.46 g (7.6 mmol) of 3- δ 9.56 , 7.68 , 6.24 , 4.38 , 4.08 , 3.93 , 3.77 , 2.93–2.88 , 2.56 – 2.47 , 2.27 , 2.08–2.04 , 1.30 3CS), 0.89 3CSi), 0.09 .13C NMR  δ 170.96 (CONH), 164.09 (4-C), 150.23 (2-C), 138.90 (6-CH), 111.15 (5-C), 88.16 (4′-CH), 85.61 (1’-CH), 72.52 (3′-CH), 63.26 (5′-CH), 48.02 ((CH3)3CS), 41.16 (2′-CH2), 36.83 (5-CH2), 36.17 (CH2CH2), 35.78 (CH2CH2), 30.05 ((CH3)3CS), 26.08 ((CH3)3CSi), 25.86 ((CH3)3CSi), 18.52, 18.09 (CH3)3CSi), −4.54 (CH3Si), −4.71 (CH3Si), −5.17 (CH3Si), −5.31 (CH3Si).+): Calculated for C29H56N3O6S2Si2+ 662.3144, measured 662.3140 (ΔM: −0.6 ppm).HRMS (ESItert-Butyldisulfanyl)propanamido]methyl}-2’-deoxyuridine (9)5-{-5-{[3-(tert-butyldisulfanyl)-propanamido]methyl}-2′-deoxyuridine (10)5′-9 was dissolved in 15 mL of pyridine and 0.94 g  of 4,4′-dimethoxytrityl chloride was added. The reaction mixture was stirred at room temperature until it became homogeneous then left at room temperature overnight. The TLC showed almost full conversion; therefore, the pyridine was evaporated and the residue was dissolved in EtOAc (80 mL) and washed three times with 80 mL of saturated sodium bicarbonate solution. After drying the organic phase on Na2SO4, the crude product was evaporated and column chromatographed on silica using 50–0% (v/v) hexanes in EtOAc-triethylamine 98:2 (v/v) eluent system. The yield was 0.99 g (73%) of white foam.First, 0.8 g (1.85 mmol) of nucleoside 1H NMR (Methanol-d4) δ 7.74 , 7.52–7.12 , 6.92–6.80 , 6.27 , 4.44 , 4.00 , 3.78 , 3.66 , 3.37 , 2.84 , 2.41 , 2.35–2.28 , and 1.30 3).13C NMR (Methanol-d4) δ 173.44 (CONH), 165.08 (4-C), 160.22 (CH3OC), 152.08 (2-C), 146.13 (DMTr-Cq), 140.32 (6-CH), 137.12 (DMTr-Cq), 136.95 (DMT-CH), 131.39 (DMT-CH), 131.36 (DMT-CH), 129.41 (DMT-CH), 128.93 (DMT-CH), 128.02 (DMT-CH), 114.26 (DMT-CH), 111.91 (5-C), 87.94 (DMTr-Cq), 87.65 (4′-CH), 86.47 (1′-CH), 72.52 (3′-CH), 64.99 (5′-CH2), 55.77 (CH3O), 48.55 ((CH3)3C), 41.19 (2′-CH2), 37.32 (CH2CH2), 37.03 (CH2CH2), 36.56 (CONHCH2), and 30.30 ((CH3)3).+), calculated for C38H45N3O8S2Li+ 742.2803, measured 742.2790 (ΔM: −1.8 ppm).HRMS (ESIO-[Bis(4-methoxyphenyl)(phenyl)methyl]-5-{[3-(tert-butyldisulfanyl)-propanamido]methyl}-3′-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phosphanyl}-2′-deoxyuridine (11)5′-10 was dissolved in dry acetonitrile (30 mL) and under argon atmosphere 308 µL  of N,N-diisopropylethylamine followed by 329 µL  of 2-cyanoethyl N,N-di(propan-2-yl)phosphoramidochloridite were added. The reaction mixture was stirred at room temperature for 30 min when the TLC showed the complete conversion of the starting nucleoside. The reaction mixture was evaporated and the residue was dissolved in a small amount of EtOAc (2–4 mL) and introduced to a short silica column to subject column chromatography. Before the chromatography, the column was washed with 2 column volume of hexanes-EtOAc-triethylamine 50:50:2 (v/v), then 5 column volume of hexanes-EtOAc 50:50 (v/v) to remove the excess of triethylamine. The elution was implemented using 25–0% (v/v) hexanes in EtOAc and the product-containing UV active fractions were evaporated and co-evaporated using acetonitrile (to remove the traces of triethylamine). The yield was 783 mg (71%) of white foam.First, 0.87 g (1.18 mmol) of nucleoside 1H NMR (C6D6) δ 10.34 , 7.88 , 7.73–7.66 , 7.56 , 7.28 , 7.12 , 6.94–6.87 , 6.40 , 4.71 , 4.20 , 3.98–3.79 , 3.66–3.46 , 3.45–3.42 , 3.38–3.19 2), 2.94 , 2.36 , 2.29 , 1.94 , 1.20 3C), 1.11 2), and 1.03 2).13C NMR (C6D6) δ 170.75/170.73 (CONH), 164.34/164.32 (4-C), 159.35/159.33 (CH3OC), 150.75/150.67 (2-C), 145.64/145.60 (DMTr-Cq), 139.28/139.22 (6-CH), 136.29/136.25 (DMTr-Cq), 130.86/130.83, 130.81/130.79, 128.83/128.81, 128.40/128.35 and 127.26/127.24 (DMTr-CHs), 117.81/117.60 (CN), 113.87 (5-C), 111.95/111.93 (DMTr-CH), 87.25 (DMTr-Cq), 85.83/85.80, 85.66/85.64 and 85.60/85.55 (4′-C and 1′-C), 73.59/73.45/73.33/73.20 (3′-CH), 63.58/63.51 (5′-CH2), 60.09 (CH2CN), 58.84/58.69 (POCH2), 55.00/54.97 (CH3O), 47.73 ((CH3)3C), 43.64/43.60 and 43.54/43.50 (CH(CH3)2), 40.23/39.92 (2′-CH), 36.86 (CONHCH2), 36.51 and 36.15 (CH2CH2), 30.02 ((CH3)3C), and 24.75/24.72/24.70/24.67/24.66/24.64/24.61 (CH(CH3)2).31P NMR (C6D6) δ were 148.98 and 148.71.+), calculated for C47H62N5O9PS2Li2+ 942.3881, measured 942.3881 (ΔM: 0 ppm).HRMS  using standard β-cyanoethyl phosphoramidite chemistry at a nominal scale of 0.2 µmol. The phosphorylation was carried out using solid chemical phosphorylating reagent . The 5′ fluorescein modification was carried out using 6-Carboxyfluoresceinamide phosphoramidite Pro .t-Bu-SS-protected thymidine phosphoramidite 11 were prepared using an ETT coupling reagent, using a 10 min coupling time. The cleavage was performed using concentrated NH3 or AMA (ammonium hydroxide/40% aqueous methylamine 1:1 v/v) for 24 h yielding the t-Bu-SS-protected oligonucleotide. The oligonucleotides with S-Bz-Thiol-Modifier C6-dT  were prepared using Ac-C and dmf g using DCI as the coupling reagent. Before cleavage, the synthesis column was treated with 20% diethylamine in acetonitrile to cleave the cyanoethyl protecting groups . Ov. Ovt-Bu-11 in the fifth position (O1-3(SH), t-Bu-SS protection group was removed in solution using tris(2-carboxyethyl)phosphine (TCEP) [Since our goal was to prepare ligatable oligonucleotide conjugates three 5′ phosphorylated 10mer oligonucleotides were synthesized using e (TCEP) . FollowiHAIYPRH (T7) sequence, which is widely utilized to create tumor-targeting delivery systems [GRGDSP containing the RGD recognition motif targeting cellular adhesion receptors [ASSLNIA, which has been shown to target muscle cells specifically [O1-3(SH)  the transferrin receptor binding  systems ,54; (ii)eceptors ; and ASSifically ,57. All O1-3(SH) . We obsenversion b, whereanversion . One of nversion . Therefot-Bu-SS-protected monomer is that the protection group can be selectively removed using reducing reagents while the oligonucleotide is still attached to the solid phase, leaving the other protection groups intact. This opens the possibility to perform conjugation on the solid phase for molecules that can withstand the ammonia cleavage conditions, resulting in the efficient removal of reagents during synthesis and fewer purification steps. To demonstrate this application, the t-Bu-SS protection group was removed using TCEP and conjugated to 3-maleimidopropionic acid on the solid phase (A further advantage of the id phase . We obseid phase  separablO6) containing two 15mer extension sequences on both ends  to ease the detection of the ligated product.With the peptide-functionalized conjugates in hand, our next step was to connect them to a single strand using ligation. T4 ligase can be used to attach short oligonucleotide conjugates to one another, thereby creating DNA strands with multiple functionalities and their arrangement governed by a complementary template sequence . To asseO1 was omitted from the mixture, where a non-specific 30 bp product formation was formed, but this did not interfere with the ligation when all the other components were present. The presence of peptides did not influence the ligation efficiency; we observed similar yields and product distribution, with product sizes slightly higher than the non-functionalized oligonucleotides, which is expected with the covalently attached peptides. No ligation product could be observed when the template or the ligase was excluded from the reaction, indicating that there are no non-specific interactions.The ligation was first performed with three-fold oligonucleotide excess compared to the template at room temperature, without or with the peptides attached, which led to incomplete ligation products b. One exSince the ligation buffer includes reducing agents and heat is applied, we tested whether the thiosuccinimide bond is stable under these conditions, for which the peptide-maleimides were reacted with beta-mercaptoethanol first and then subjected to the same ligation conditions. Apart from a small amount of hydrolyzed product, no change could be observed , indicatt-Bu-SS protection group allowed solid-phase conjugation, resulting in fewer purification steps. The prepared peptide–oligonucleotide conjugates were successfully hybridized and ligated on a template resulting in a DNA strand bearing multiple functionalities. Using the presented monomer, it is possible to directly functionalize nucleotides internally and perform conjugations both on solid and in solution phases using thiol reactive reagents. The conjugated moieties do not influence ligase binding, and with the help of a template, virtually endless modifications can be incorporated into spatially defined arrangements, which can be desirable for oligonucleotides used as therapeutics, detections reagents, or in DNA-based display methods.In summary, we have shown the applicability of a thiol-modified nucleoside phosphoramidite from its synthesis through conjugation to the preparation of ligatable functionalized oligonucleotides. The monomer presented here was synthesized through a greener, metal-free chemistry and successfully incorporated into oligonucleotide sequences using standard synthesis and cleavage protocols, without the risk of degradation. The thiol-containing oligonucleotides were directly functionalized with peptide-maleimides, without the need for any additional crosslinkers. Furthermore, the selective removal of the


All synthesized compounds 7a–o were screened as possible α-glucosidase inhibitors and exhibited good inhibitory activity with IC50 values in the range of 110.6 ± 6.0 to 453.0 ± 4.7 µM in comparison with acarbose as the positive control (750.0 ± 10.5 µM). An exception in this trend came back to a compound 7k with IC50 value > 750 µM. Furthermore, the most potent derivative 7e bearing 3-fluorophenyl moiety was further explored by kinetic studies and showed the competitive type of inhibition. Additionally, the molecular docking of all derivatives was performed to get an insight into the binding mode of these derivatives within the active site of the enzyme. In silico assessments exhibited that 7e was well occupied in the binding pocket of the enzyme through favorable interactions with residues, correlating to the experimental results.A novel series of diphenylquinoxaline-6-carbohydrazide hybrids The online version contains supplementary material available at 10.1186/s13065-022-00848-4. Diabetes mellitus (DM) can be construed as a chronic metabolic disorder identified by insistent hyperglycemia –3. It caIn this regard, α-glucosidase is one of the most important enzyme found in the digestive system , 12. TheQuinoxaline, also called benzo[b]diazine or benzopyrazine, and its derivatives are an integral part of medicinal chemistry owing to their wide range of biological activity, including antithrombotic, anti-tubercular, antitumor, antimalarial, antiplasmodial, antiprotozoal, AMPA receptor antagonist, and antiviral activities –31. BesiAcyl hydrazone moiety has been known as a privileged structure in drug discovery due to its easy synthetic procedure via condensation of hydrazides and aldehydes or ketones under acid, base catalysis, or microwave irradiation . This unIn the current study, novel biphenylquinoxaline derivatives bearing different acyl hydrazone were designed, synthesized, and evaluated against the α-glucosidase. The most potent compound was then subjected to kinetic study and molecular docking assessments.A  as an effective α-glucosidase inhibitor ) and reciprocal of enzyme rate (1/V) over various inhibitor concentrations. The experimental inhibitor constant (Ki) value was constructed by secondary plots of the inhibitor concentration [I] versus Km [The mode of inhibition of the most active compound  [The molecular docking investigation of all derivatives were performed using the maestro molecular modeling platform (version 10.5), Schrödinger suites . X-ray csb.org/) . A protesb.org/) . Acarbossb.org/) , 43.Additional file 1: Fig. S1. (E)-N'-benzylidene-2,3-diphenylquinoxaline-6-carbohydrazide (7a). Fig. S2. (E)-N'-(2-nitrobenzylidene)-2,3-diphenylquinoxaline-6-carbohydrazide (7b). Fig. S3. (E)-N'-(3-nitrobenzylidene)-2,3-diphenylquinoxaline-6-carbohydrazide (7c). Fig. S4. (E)-N'-(4-nitrobenzylidene)-2,3-diphenylquinoxaline-6-carbohydrazide (7d). Fig. S5. (E)-N'-(3-fluorobenzylidene)-2,3-diphenylquinoxaline-6-carbohydrazide (7e). Fig. S6. (E)-N'-(4-chlorobenzylidene)-2,3-diphenylquinoxaline-6-carbohydrazide (7f). Fig. S7. (E)-N'-(4-methoxybenzylidene)-2,3-diphenylquinoxaline-6-carbohydrazide (7g). Fig. S8. (E)-N'-(3-methoxy-2-nitrobenzylidene)-2,3-diphenylquinoxaline-6-carbohydrazide (7h). Fig. S9. (E)-N'-(2-chloro-5-nitrobenzylidene)-2,3-diphenylquinoxaline-6-carbohydrazide (7i). Fig. S10. (E)-N'-(4-hydroxy-3-methoxybenzylidene)-2,3-diphenylquinoxaline-6-carbohydrazide (7j). Fig. S11. (E)-2,3-diphenyl-N'-quinoxaline-6-carbohydrazide (7k). Fig. S12. (E)-N'-(3-phenoxybenzylidene)-2,3-diphenylquinoxaline-6-carbohydrazide (7l). Fig. S13. (E)-N'-methylene)-2,3-diphenylquinoxaline-6-carbohydrazide (7m). Fig. S14. (E)-N'--2,3-diphenylquinoxaline-6-carbohydrazide (7n). Fig. S15. (E)-2,3-diphenyl-N'-(thiophen-2-ylmethylene)quinoxaline-6-carbohydrazide (7o).


Ilex rotunda Thunb., has been used to treat common cold, tonsillitis, and eczema. It is also a source of antioxidants. However, information regarding its antioxidative phytochemical composition is still incomplete and limited. In this present study, we initially determined DPPH radical scavenging activity of the extracts of I. rotunda fruits, twigs, and leaves. Among them, the twig extract exhibited a potential of antioxidant capacity. Based on antioxidant effect guided experiments, extraction condition using 80% EtOH was then optimized. DPPH and ABTS radical scavenging assays were also performed for fractions. The n-butanol fraction showed the highest antioxidant effect. Using chromatographic methods, eight marker compounds (1–8) were further isolated. Their structures were determined by spectroscopic and mass data. Method validation was employed to quantitate contents of these eight marker compounds. Subsequently, the HPLC-DPPH method was used to evaluate the contribution of certain compounds to total antioxidant activity of the extract. Lastly, parallel artificial membrane permeability assay for blood–brain barrier (PAMPA-BBB) was applied to investigate brain-penetrable antioxidants from I. rotunda extract. As a result, compound 7  showed significant antioxidant activity and penetration across the BBB via transcellular passive diffusion. Our findings suggested that compound 7 can be used as a therapeutic potential candidate in natural product-based central nervous system (CNS) drug discovery. Oxidative stress occurs when there is an imbalance between free radicals and antioxidants in the body . The bodOxidative stress can lead to various diseases, including inflammation, aging, cancer, diabetes, cardiovascular, and hypertension ,9,10. ItIlex rotunda belongs to genus Ilex of Aquifoliaceae family. This plant is distributed in the east Asia region including China, Japan, Taiwan, and Korea. Previous studies have reported that this plant contains triterpenes and their saponins, sesquiterpenes, hemiterpene glycosides, flavonoid glycosides, and aromatic compounds + (C17H24O9Na); 1H NMR : δ 6.74 , 6.54 , 6.32 , 4.88 , 4.21 , 3.85 , 3.20–3.79 ; 13C NMR : δ 154.35 , 135.8 (C-7), 135.2 (C-1), 131.3 (C-4), 130.0 (C-8), 105.4 (C-3 and C-5), 105.3 (C-1’), 78.3 (C-5’), 77.8 (C-3’), 75.7 (C-2’), 71.3 (C-4’), 63.5 (C-9), 62.5 (C-6’), 57.0 .Syringin (2): White amorphous powder; ESI-MS: 353.01 [M-H]- (C16H17O9); 1H NMR : δ 7.58 , 7.04 , 6.93 , 6.77 , 6.26 , 5.34 , 4.16 , 3.63 , 2.00–2.00 ; 13C NMR : δ 177.0 (C-7), 168.60 (C-9′), 149.4 (C-4′), 146.80 (C-7′), 146.79 (C-3′), 127.9 (C-1′), 122.9 (C-6′), 116.4 (C-5′), 115.8 (C-8′), 115.1 (C-2′), 76.1 (C-1), 73.4 (C-4), 71.9 (C-3), 71.3 (C-5), 38.7 (C-6), 38.1 (C-2).Chlorogenic acid (3): Yellow amorphous powder; ESI-MS: 609.05 [M-H]- (C27H29O16); 1H NMR : δ 7.66 , 7.63 , 6.87 , 6.39 , 6.20 , 5.11 , 4.51 , 3.80 , 3.63 , 3.54 , 3.26–3.48 , 3.44 , 3.39 , 3.27 , 1.12 ; 13C NMR : δ 179.4 (C-4), 166.1 (C-7), 163.0 (C-5), 159.3 (C-9), 158.5 (C-2), 149.8 (C-4′), 145.8 (C-3′), 135.6 (C-3), 123.5 (C-6′), 123.1 (C-1′), 117.7 (C-2′), 116.1 (C-5′), 105.6 (C-10), 104.7 (C-1′′), 102.4 (C-1′′′), 99.9 (C-6), 94.8 (C-8), 78.2 (C-5′′), 77.2 (C-3′′), 75.7 (C-2′′), 73.9 (C-4′′′), 72.2 (C-3′′′), 72.1 (C-2′′′), 71.4 (C-4′′), 69.7 (C-5′′′), 68.5 (C-6′′), 17.8 (C-6′′′).Rutin (4): Yellow amorphous powder; ESI-MS: 425.06 [M-H]- (C20H25O10); 1H NMR : δ 7.53 , 7.02 , 6.92 , 6.75 , 6.25 , 5.67 , 4.57 , 4.41 , 4.27 , 4.24 , 3.85–3.17 ; 13C NMR : δ 168.8 (C-9), 149.5 (C-3), 147.2 (C-4), 146.7 (C-7), 135.9 (C-3′), 127.6 (C-1), 124.9 (C-6), 123.0 (C-2′), 116.5 (C-5), 115.1 (C-2), 114.8 (C-8), 103.1 (C-1′′), 78.0 (C-3′′), 77.9 (C-5′′), 74.9 (C-2′′), 71.5 (C-4′′), 69.6 (C-4′), 66.0 (C-1′), 62.7 (C-6′′), 14.2 (C-5′).Rotundarpenoside B (5): Yellow amorphous powder; ESI-MS: 515.05 [M-H]- (C25H23O12); 1H NMR : δ 7.57 , 7.52 , 7.02 , 7.01 , 6.90 , 6.88 , 6.74 , 6.72 , 6.26 , 6.25 , 5.62 , 5.07 , 4.26 , 2.34 , 2.13 ; 13C NMR : δ 178.2 (C-7), 168.5 (C-9’), 168.4 (C-9’’), 149.6 , 147.3 (C-4’), 147.2 (C-4’’), 146.8 , 127.7 , 123.2 , 116.5 , 115.3 (C-8’), 115.0 (C-8’’), 114.9 , 78.3 (C-1), 74.0 (C-4), 70.0 (C-3), 67.1 (C-5), 37.9 (C-2), 35.7 (C-6)3,4-Dicaffeoylquinic acid (6): Yellow amorphous powder; ESI-MS: 515.05 [M-H]- (C25H23O12); 1H NMR : δ 7.62 , 7.58 , 7.07 , 6.96 , 6.78 , 6.35 , 6.27 , 5.43 , 5.39 , 3.97 , 2.31–2.15 ; 13 C NMR : δ 177.7 (C-7), 168.9 (C-9’), 168.4 (C-9’’), 149.6 (C-4’), 149.5 (C-4’’), 147.3 (C-7’), 147.1 (C-7’’), 146.8 , 127.9 (C-1’), 127.8 (C-1’’), 123.1 (C-6’), 123.0 (C-6’’), 116.5 , 115.6 (C-2’’), 115.2 (C-2’), 115.1 , 74.8 (C-1), 72.6 (C-5), 72.1 (C-3), 70.7 (C-4), 37.8 (C-2), 36.1 (C-6).3,5-Dicaffeoylquinic acid (7): Yellow amorphous powder; ESI-MS: 515.05 [M-H]- (C25H23O12); 1H NMR : δ 7.48 , 7.42 , 7.02 , 6.96 , 6.74 , 6.23 , 6.15 , 5.38 , 4.94 , 4.17 , 2.31–2.15 ; 13C NMR : δ 174.9 (C-7), 166.1 (C-9’’), 165.7 (C-9’), 148.5 , 145.6 , 145.5 , 125.4 , 121.5 (C-6’), 121.4 (C-6’’), 115.8 (C-2’), 115.7 (C-2’’), 114.8 , 113.8 (C-8’), 113.6 (C-8’’), 73.7 (C-1), 71.5 (C-4), 67.7 (C-5), 66.7 (C-3), 37.5 (C-2), 35.9 (C-6).4,5-Dicaffeoylquinic acid (8): Brown amorphous powder; ESI-MS: 677.16 [M-H]- (C34H29O15); 1H NMR : δ 7.62 , 7.55 , 7.53 , 7.05 , 7.01 , 6.99 , 6.92 , 6.90 , 6.85 , 6.78 , 6.76 , 6.71 , 6.31 , 6.21 , 6.19 , 5.65 , 5.32 , 2.45~2.20 ; 13C NMR : δ 174.2 (C-7), 168.5 (C-9’), 168.1 (C-9’’), 168.1 (C-9’’’), 149.7 (C-3’) 149.6 (C-3’’), 149.3 (C-3’’’), 147.9 (C-4’), 147.7 (C-4’’), 147.6 (C-4’’’), 146.7 , 127.8 (C-1’), 127.6 (C-1’’), 127.5 (C-1’’’), 123.3 , 116.5 (C-5’), 116.4 , 115.2 (C-8’), 115.1 (C-8’’), 115.1 (C-8’’’), 115.0 (C-2’), 114.6 (C-2’’), 114.3 (C-2’’’), 74.7 (C-1), 70.0 (C-4), 69.1 (C-3), 67.9 (C-5), 36.7 (C-2), 35.8 (C-6).3,4,5-Tricaffeoylquinic acid  [2, tR 15.69 min) [3, tR 24.96 min) [4, tR 27.73 min) [5, tR 28.43 min) [6, tR 30.96 min) [7, tR 32.84 min) [8, tR 37.18 min) [Eight marker compounds were identified as syringin (.19 min) , chlorog.69 min) , rutin (.96 min) , rotunda.96 min) , 4,5-dic.18 min)  based onI. rotunda was obtained by optimizing analytical factors including column, mobile phase, gradient elution, flow rate, and wavelength detection. A Triart C18 column  was chosen because it produced more selective and sharper peaks. A mobile phase with pure water containing 0.1% formic acid (A) and acetonitrile (B) was chosen and run according to the programmed gradient elution. Formic acid was the most effective buffer in the aqueous phase. This solvent system produced the high resolution of peak separation in the chromatograms. The column temperature was set at 35 °C to ensure precision. UV detection wavelengths were selected at 254 and 326 nm during experiments because these wavelengths were the most sensitive ones. Finally, the HPLC analytical method was successfully established. As shown in 1–8 exhibited well-separated peaks with a high resolution. Thus, this optimal chromatographic condition was employed to validate marker compounds 1–8 obtained from the extract of I. rotunda twigs.The chromatographic profile of I. rotunda twigs.HPLC experiments for linearity, precision, and repeatability were performed to ensure that the present method was sensitive, selective, precise, and accurate. Subsequently, the established method was used to quantify the eight marker compounds obtained from the extract of 2) using calibration curves of each compound. The linearity of the eight compounds showed the best R2 values (≥ 0.9993) with the following concentration ranges: 6.25–200 μg/mL for 1 and 3; 12.5–400 μg/mL for 2, 4, 5 and 8; and 25–800 μg/mL for 6 and 7. The LOD and LOQ of these eight compounds were 0.13–0.65 and 0.42–1.98 μg/mL, respectively  of standard compounds from the spiked extract solution versus the nonspiked extract sample. As a results, recoveries of these eight compounds were in the range of 96.60–104.7%  were 0.40–1.15 and 2.48–3.65%, respectively  were spiked to the 0–104.7% , demonstectively .I. rotunda twigs. Compound 7  showed the highest content (93.43 mg/g) in the twig extract, followed by compound 6  at 72.77 mg/g, compound 8  at 50.02 mg/g, compound 2 (chlorogenic acid) at 38.54 mg/g, compound 5  at 35.45 mg/g, compound 4 (rotundarpenoside B) at 35.43 mg/g, compound 1 (syringin) at 16.72 mg/g, and compound 3 (rutin) at 8.62 mg/g, respectively  showed the highest antioxidant capacity with a reduction peak area of 83.67%. Compound 8 , compound 2 (chlorogenic acid), and compound 6  showed significant antioxidant activities with reduction peak areas of 67.25%, 60.51%, and 58.88%, respectively (The HPLC-DPPH method can be used to rapidly assess pure antioxidant compounds in complex mixtures . The aboectively .2 and 6–8) was further verified using ELISA. Results revealed that compounds 6–8 exhibited significant antioxidant activities with EC50 values ranging from 10.88 to 13.84 μM, stronger than compound 2 with EC50 value of 35.50 μM.  was detected in the acceptor solution with BBB permeability log Pe value of −5.80, showing PAMPA-BBB potential penetrability based on the study of Könczöl et al. [Pe range for classifying CNS drug candidates with moderate BBB permeation potential  [Permeability assessment of small molecules through the blood–brain barrier (BBB) plays a significant role in the development of effective central nervous system (CNS) drug candidates . For thil et al. . These f > −5.0) .1–8 were further tested using the PAMPA-BBB method at the same concentration of 10 mM because the content of each compound was not consistent in the extract solution. Coumarin and caffeic acid were positive and negative controls, respectively. As can be seen in 7  showed similar log Pe value of −5.85 compared to previous experiment, whereas compounds 1–6 and 8 were not detected in the acceptor solution. Coumarin and caffeic acid showed log Pe values of −4.54 and −9.08, respectively  exhibited stronger radical scavenging activity than the fruit extract (42.4%) and leaf extract (42.2%) at 50 µg/mL   . 1–8). This analytical method was adapted to optimize analytical factors with a high resolution and efficiency (1), chlorogenic acid (2), rutin (3), rotundarpenoside B (4), 3,4-dicaffeoylquinic acid (5), 3,5-dicaffeoylquinic acid (6), 4,5-dicaffeoylquinic acid (7), and 3,4,5-tricaffeoylquinic acid (8) at amounts of 8.62–93.43 mg/g. The established method was validated to have appropriate sensitivity, repeatability, and precision  peak area reduction than those with DPPH-free group  in I. rotunda extract. The HPLC-DPPH method can provide bioactive evaluation and quantitative information [The above analytical method was also employed to screen antioxidant properties of marker compounds derived from twig extract via peak areas reduction using a screening HPLC-DPPH method. Natural antioxidants often decrease during their isolation and purification due to decomposition . An HPLCee group . In conto 67.25% . These a2 and 6–8 can also be found in the following literature. Chlorogenic acid (CGA) (2) is widely recognized to have antioxidant activity. It exists in most abundant quantity in different foods, coffee, and vegetables [6), 4,5-di-CQA (7), and 3,4,5-tri-CQA (8)) also reported as antioxidant due to the presence of high numbers of 3,4-dihydroxyl groups [6–8 showed stronger antioxidant activities than compound 2, suggesting that the presence of more 3,4-dihydroxy moiety contributed to the free radicals scavenging ability.Antioxidant effects of active compounds getables . An intagetables . 3,4-Dihgetables . Three il groups . As showI. rotunda extract. The brain with a high oxygen consumption is highly sensitive to oxidative stress [I. rotunda extract. As shown in 7  was detected in the acceptor solution. Subsequently, the log Pe value for 7 was calculated by the above-described equation. The concentrations of acceptor (Ct)A( = 6.62 μg/mL) and donor (Ct)D( = 287.86 μg/mL) solutions were calculated based on the peak area and regression equation for 7, respectively. Other parameters were used as follows: A = 0.3 cm2, VD = 150 μL, VA = 300 μL, and t =14,400 s. Finally, the log Pe value (−5.80) for 7 was determined. Compounds 1–6 and 8 were not detected in the acceptor area. Thus, the permeability values were not calculated. The detailed calculation procedure can also be found in the 7 also showed similar log Pe value of −5.85, whereas other compounds  were not detected (7 was confirmed to have a moderate BBB permeability. A previous study showed that chlorogenic acid (2) and rutin (4) have poor permeability [1, 3, and 5–8) were firstly tested for BBB permeability in this experiment. Some authors have noted the importance of polar surface area (PSA), lipophilicity, molecular weight, and hydrogen bond donors in natural molecules for BBB permeability [7) with a high molecular weight can also cross the BBB, although less efficiently. Furthermore, parallel artificial membrane permeability assay for the blood–brain barrier (PAMPA-BBB) was applied to investigate brain-penetrable antioxidants from e stress . When ROe stress . Naturale stress . For thidetected . Thus, ceability . Other ceability . Nevertheability . For theI. rotunda twigs by measuring DPPH and ABTS radical scavenging assays. BuOH fraction showed the most potent inhibitory activity. It subsequently afforded eight marker compounds (1–8) via isolation and structure determination. The established method was successfully applied to quantify levels of marker compounds and applied to evaluate their antioxidant activities with a rapid screening HPLC-DPPH method. Significant active marker compounds 2 and 6–8 were further verified using ELISA. Furthermore, the PAMPA-BBB method was applied to investigate brain-penetrable antioxidants from the I. rotunda extract. As a result, 4,5-dicaffeoylquinic acid (7) was able to penetrate across the blood–brain barrier via transcellular passive diffusion. Our findings suggest that compound 7 can be used as a therapeutic potential candidate in natural product-based CNS  drug discovery. Further in silico modeling and in vivo study are needed in the future to better understand the exact mechanisms of action of this compoundIn conclusion, we evaluated antioxidant effects of fractions and compounds from the extract of


Cycas rumphii Miq. leaves for toxoplasmocidal effect, using Viruluent RH Toxoplasma gondii, and cytotoxic activity against HEPG-2, HCT-116 and HELA cancer cell lines using MTT assay. Among the tested extracts, the ethyl acetate fraction was the most effective against T. gondii, with an EC50 of 3.51 ± 0.2 µg/mL compared to cotrimoxazole (4.18 ± 0.01 µg/mL) and was the most potent against the tested cell lines, especially HEPG-2, with an IC50 of 6.98 ± 0.5 µg/mL compared to doxorubicin (4.50 ± 0.2 µg/mL). Seven compounds were isolated from the ethyl acetate fraction by extensive chromatographic techniques and fully elucidated using different spectroscopies. Compound (7) is an undescribed 4′, 4′′′ biapigenin di-C-glucoside, which showed a strong cytotoxic activity. Four known biflavonoids  in addition to a phenolic acid ester (3) and a flavonoid glycoside (6) were also isolated. Compounds  were reported for the first time from C. rumphii.Toxoplasmosis and cancer are serious worldwide diseases, and the available drugs cause serious side effects. Investigation for new alternative therapies from natural sources is now an increasing concern. Herein, we carried out, for the first time, an in vitro screening of  Toxoplasma gondii, which is an obligate parasite  β-D-glucopyranoside-8-C-β-D-glucopyranoside (7) is +36.45 . To our knowledge, this is the first report of 4′-O-4′′′ biapigenin di-C-glucosides from plants.Compound (ound (7)  showed tspectrum  showed telations , four caectively ,26, thusand C-4′ . The 1H-e moiety ,26. The  9.6 Hz) ,29 and bound (7)  indicateranoside . The spe1–7) were evaluated for the cytotoxic activity against the HEPG-2, HELA and HCT-116 cell lines. The results showed that compound (3) was the most potent against HEPG-2, HELA and HCT-116, with IC50 values of 8.67 ± 0.6, 10.08 ± 0.9 and 6.24 ± 0.5 μg/mL, respectively, followed by compound (6), which exhibited a strong cytotoxic activity, with IC50 values of 14.49 ± 1.1, 23.32 ± 2.0 and 11.63 ± 0.9 μg/mL, respectively. Finally, compound (7) showed a strong cytotoxic activity, with IC50 values of 21.47 ± 1.8, 15.66 ± 1.3 and 18.17 ± 1.4 μg/mL, respectively (3) showed higher selectivity for the cancer cells than the normal cells compared to doxorubicin.Compounds , dimethyl sulfoxide (DMSO), doxorubicin HCl, phosphate buffer saline and trypan blue were obtained from Sigma Co. . Fetal bovine serum was purchased from Gibco Co.  and cotrimoxazole  from GlaxoSmithKline.Solvents used were of HPLC analytical grade ≥99.9% and were purchased from Sigma Co. 1H and 100 MHz for 13C and DEPT-Q NMR. ESI-MS spectra were recorded by Advion compact mass spectrometer (CMS) . High-resolution mass spectra were measured using the Q-TOF-LC/HRMS system (6530) from Agilent Technologies Co.  equipped with an autosampler (G7129A) with an ESI ionization source. Melting point determination was carried out using a Gallenkamp melting point apparatus from Hanon Co. . Optical rotation was measured using a Polax-2L Polarimeter . UV spectra were recorded using a UV/Vis spectrophotometer . IR spectra were measured as KBr discs using an FT/IR-6100 spectophotometer from Jasco Co. . An ELISA Processor II Microplate Reader EXL800 from Biotek Co.  was used for the cytotoxic assessment.NMR experiments were performed using a Bruker Avance III spectrometer , with 400 MHz for 254 were purchased from Merck Co. , while Sephadex LH-20 was obtained from Sigma-Aldrich Chemical Co. . Authentic samples of amentoflavone and bilobetin for Co-TLC and IR fingerprint experiments were provided by the Department of Pharmacognosy, Faculty of Pharmacy, Tanta University, Egypt. AlCl3 (5%) and 10% H2SO4 spray reagents were used for detection on TLC. Solvent systems used for TLC were CH2Cl2-MeOH (9:1) “S1”, CH2Cl2-MeOH (8:2) “S1” and CH2Cl2-MeOH-H2O (7:3:0.5) “S3”.Silica gel (70–230 mesh) and precoated TLC sheets of silica gel FC. rumphii Miq. were collected from El-Abd Garden at 68 kilos from the desert Cairo-Alexandria Road in July 2018. It was kindly provided and identified by the researcher Rabea Sharawy . A voucher sample (No. PGG-012) was deposited at the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Tanta University, Egypt.The leaves of n-butanol to yield 38.03 g, 8.10 g, 20.1 g and 59.20 g, respectively.The plant material was dried in shade, reduced to powder and stored in tightly closed containers. The plant powder (5 Kg) was extracted with methanol by cold maceration till exhaustion. The total methanol extract was evaporated under reduced pressure at 40 °C to yield a green residue (294 g). The methanol extract residue (274 g) was suspended in 50% aqueous methanol (1.5 L) and was successively fractionated with petroleum ether (40–60 °C), methylene chloride, ethyl acetate and 2Cl2, and the polarity was increased using MeOH. Fractions (50 mL) were collected, and similar fractions, on TLC, were combined to afford five groups of fractions (F1 to F5). F1, eluted with CH2Cl2:MeOH (95:5), gave a yellow-colored residue (135 mg), which was chromatographed further on a silica gel column  using a gradient elution of CH2Cl2 and MeOH to obtain three subfractions F1-1 to F1-3. F1-1 (78 mg) was re-chromatographed on a Sephadex LH-20 column  using MeOH (HPLC grade) to give compound (1) (8 mg).The ethyl acetate fraction (10 g) was chromatographed on a silica gel column  using a gradient elution, starting with pure CH2Cl2:MeOH (90:10), gave a brown-colored residue (342 mg), which was re-chromatographed on a silica gel column  using CH2Cl2 and MeOH in a gradient elution to give two subfractions F2-1 and F2-2. F2-1 (92 mg) was purified on a Sephadex LH-20  using MeOH (HPLC grade) to give compound (2) (22 mg). F2-2 (125 mg) was also purified further on a Sephadex LH-20  using MeOH (HPLC grade) to afford compounds (3) and (4) .F2, eluted with CH2Cl2:MeOH (85:15), yielded a yellow-colored residue (571 mg), which was chromatographed further on a silica gel column  using a gradient elution of CH2Cl2 and MeOH to afford three subfractions, F3-1 to F3-3. F3-3 (50 mg) was re-chromatographed on a Sephadex LH-20 column  using MeOH (HPLC grade) to afford compound (5) (38 mg).F3, eluted with CH2Cl2:MeOH (80:20), produced a dark brown-colored sticky residue (1.3 g). Further chromatography on a silica gel column  using a gradient elution with CH2Cl2 and MeOH resulted in three subfractions, F4-1 to F4-3. F4-3 (140 mg) was purified on a Sephadex LH-20 column  using MeOH (HPLC grade) to give compound (6) (21 mg).F4, eluted with CH2Cl2:MeOH (75:25), yielded a brown-colored residue (1.7 g). Further chromatography on a silica gel column  using the same gradient elution system afforded three subfractions, F5-1 to F5-3. F5-3 (130 mg) was purified on a Sephadex LH-20 column  using MeOH (HPLC grade) to afford compound (7) (42 mg).F5, eluted with CH7):Acid hydrolysis of compound (7 (1 mg) was dissolved in 2 N HCl/methanol mixture  and heated at 100 °C for 1 h. Then, the solution was evaporated to remove the residual methanol, and the left solution was neutralized with NaHCO3. The sugar in the hydrolysis product was detected by paper chromatography alongside authentic sample of β-D-glucose using the solvent system n-butanol:acetic acid:water (4:1:5).The procedure in reference  was foll2,3-Dihydro-4′-O-methylamentoflavone (1). Amorphous yellow powder; UV (MeOH) λmax: 230, 294 and 331 nm see  δ (ppm) 7.50 , 7.48 , 7.38 , 7.10 , 6.68 , 6.51 , 6.24 , 5.80 , 5.77 , 5.37 , 3.66 , 3.07 , 2.68  see  δ (ppm) 196.2 (C-4), 182.8 (C-4″), 167.0 (C-7), 164.5 (C-5), 164.1 (C-2″), 163.4 (C-9), 161.9 (C-5″), 161.3 (C-7″), 160.8 (C-4′′′), 158.1 (C-4′), 154.8 (C-9″), 131.1 (C-2′), 130.7 (C-1′), 127.9 , 127.8 (C-6′), 121.7 (C-1′′′), 121.1 (C-3′), 115.4 , 110.7 (C-5′), 104.8 (C-8″), 103.8 (C-10″), 101.9 (C-3″), 101.7 (C-10), 98.3 (C-6″), 95.7 (C-6), 94.8 (C-8), 79.0 (C-2), 54.7 (4′-OCH3), 43.0 (C-3) see (3OD) see  see ; 13C-NMRC-3) see ; HSQC an3OD) see ; ESIMS: -H]− see .2,3-Dihydrohinokiflavone (2). Amorphous yellowish white powder; UV (MeOH) λmax: 223, 274 and 333 nm see  δ (ppm) 7.91 , 7.44 , 6.97 , 6.96 , 6.69 , 6.66 , 5.93 , 5.90 , 5.43 , 3.14 , 2.76  see  δ (ppm) 196.1 (C-4), 182.7 (C-4″), 167.0 (C-7), 165.1 (C-2″), 163.3 (C-5), 162.5 (C-9), 161.4 (C-4′′′), 158.2 (C-4′), 158.0 (C-7″), 154.4 (C-9″), 154.3 (C-5″), 132.3 (C-1′), 128.1 , 127.3 , 125.9 (C-6″), 121.7 (C-1′′′), 115.6 , 114.7 , 104.2 (C-10″), 102.1 (C-3″), 101.9 (C-10), 95.7 (C-6), 94.7 (C-8), 94.3 (C-8″), 78.8 (C-2), 42.7 (C-3) see  see ; 13C-NMRC-3) see ; ESIMS: -H]− see .Methyl gallate (3). White needle crystals; m.p: 189 °C; UV (MeOH) λmax: 234 and 275 nm see  δ (ppm) 7.05 , 3.83  see  δ (ppm) 167.6 (C-7), 145.1 , 138.3 (C-4), 119.9 (C-1), 108.5 , 50.8 (7-OCH3) see  see ; 13C-NMRCH3) see ; ESIMS: -H]− see .Amentoflavone 4′-O-methyl ether (4). Amorphous yellow powder; UV (MeOH) λmax: 234, 270 and 330 nm see  δ (ppm) 8.02 , 7.96 , 7.44 , 7.26 , 6.72 , 6.64 , 6.60 , 6.43 , 6.35 , 6.19 , 3.81  see  δ (ppm) 182.7 (C-4″), 182.3 (C-4), 164.7 (C-2″), 164.4 (C-7), 164.3 (C-2), 161.9 (C-7″), 161.8 (C-5), 161.2 (C-4′′′), 161.0 , 158.0 (C-9), 154.8 (C-9″), 130.8 (C-2′), 127.8 , 127.7 (C-6′), 122.8 (C-1′), 122.0 (C-3′), 121.6 (C-1′′′), 115.4 , 111.0 (C-5′), 103.9 (C-8″), 103.8 , 103.1 (C-3), 101.8 (C-3″), 98.8 (C-6″), 98.4 (C-6), 93.8 (C-8), 55.0 (4′-OCH3) see  see ; 13C-NMRCH3) see ; ESIMS: -H]− see .Amentoflavone (5). Amorphous yellow powder; UV (MeOH) λmax: 232, 274 and 329 nm see  δ (ppm) 7.95 , 7.77 , 7.46 , 7.05 , 6.69 , 6.52 , 6.52 , 6.40 , 6.32 , 6.15  see  δ (ppm) 182.7 (C-4″), 182.2 (C-4), 164.7 (C-2″), 164.5 (C-2), 164.4 (C-7), 162.5 (C-7″), 161.7 (C-5), 161.0 , 159.6 (C-4′), 157.8 (C-9), 154.9 (C-9″), 131.3 (C-2′), 127.8 , 127.4 (C-6′), 121.7 (C-1′′′), 121.6 (C-1′), 120.2 (C-3′), 116.0 (C-5′), 115.4 , 103.9 (C-8″), 103.8 , 102.4 (C-3), 101.8 (C-3″), 98.8 (C-6″), 98.7 (C-6), 93.7 (C-8) see  see ; 13C-NMRC-8) see ; ESIMS: -H]− see .Naringenin 7-O- β-D glucoside (6). Amorphous light brown powder; UV (MeOH) λmax: 243, 294 and 326 nm see  δ (ppm) 7.21 , 6.71 , 6.10 , 6.08 , 5.27 , 3.06 , 2.64 , 4.86 , 3.75 . 3.58 , 3.28-3.36  see  δ (ppm) 197.1 (C-4), 165.6 (C-7), 163.5 (C-5), 163.2 (C-9), 157.7 (C-4′), 129.4 (C-1′), 127.7 , 114.9 , 103.4 (C-10), 96.5 (C-6), 95.4 (C-8), 79.3 (C-2), 42.7 (C-3), 99.8 (Glu C-1), 76.8 (Glu C-5), 76.3 (Glu C-3), 73.2 (Glu C-2), 69.6 (Glu C-4), 60.8 (Glu C-6) see (3OD) see  see ; DEPTQ-NC-6) see ; HSQC an3OD) see ; ESIMS: -H]− see .4′,4′′′Biapigenin-8″-C-β-D-glucopyranosyl [1′′′′′′→3′′′′′]β-D-glucopyranoside-8-C-β-D-glucopyranoside) (7). Amorphous light brown powder; max: 238, 268 and 324 nm see  δ (ppm) 7.99 , 7.85 , 6.95 , 6.62 , 6.27 , 5.14 , 3.83-3.98 , 3.59 , 3.48-3.56 , 3.01 , 2.93 , , 5.07 , 4.41 , 4.34 , 3.37-3.42 , 3.17 , 3.10-3.17 , 4.21 , 3.76 , 3.71 , 3.30 . 3.24 , 2.73  see  δ (ppm) 182.8 (C-4″), 182.7 (C-4), 165.1 (C-2″), 164.8 (C-2), 163.5 (C-7″), 163.2 (C-7), 161.5 (C-5″), 161.4 , 161.2 (C-5), 156.8 (C-9″), 156.2 (C-9), 128.7 , 128.2 , 122.1 , 115.7 , 115.6 , 104.1 (C-10″), 104.0 (C-10), 103.6 (C-8″), 103.0 (C-8), 102.5 (C-3″), 102.3 (C-3), 99.3 (C-6″), 98.1 (C-6), 81.4 (Glu-I-C-5), 76.1 (Glu-I-C-3), 74.3 (Glu-I-C-2), 73.3 (Glu-I-C-1), 69.8 (Glu-I-C-4), 61.5 (Glu-I-C-6), 81.0 (Glu-II-C-5), 80.2 (Glu-II-C-3), 76.2 (Glu-II-C-2), 72.2 (Glu-II-C-1), 69.6 (Glu-II-C-4), 61.1 (Glu-II-C-6), 104.3 (Glu-III-C-1), 78.7 (Glu-III-C-5), 76.4 (Glu-III-C-3), 75.6 (Glu-III-C-2), 70.6 (Glu-III-C-4), 60.8 (Glu-III-C-6) see (3OD) see  see ; DEPTQ-NC-6) see ; HSQC an3OD) see ; HRMS  in μg/mL was calculated and compared to that of cotrimoxazole as a reference drug.The assay was carried out using virulent RH orted by . The mea.Hepatocellular carcinoma (HEPG-2), mammary gland breast carcinoma (MCF-7), colorectal carcinoma (HCT-116), prostate carcinoma (PC-3), cervical carcinoma (HELA) and the normal (WISH) amniotic cell lines were obtained from the American Type Culture Collection (ATCC) via Holding Company for Biological Production and Vaccines (VACSERA), Cairo-EgyptC. rumphii in DMSO against the tested cancer cell lines and one normal cell line (WISH) to ensure the safety of the plant extract on normal cells. The most affected cancer cell lines were tested against the extracts of petroleum ether, methylene chloride, ethyl acetate and n-butanol fractions and the constituents of the most active fraction were also tested using the same previous concentrations. Doxorubicin was used as a positive control. IC50 was calculated and the cytotoxic effect was assessed according to the classification of Hossan and Abu Melha, 2014 [The cytotoxicity assay was carried out according to the reported procedures using the MTT assay method ,33. The ha, 2014 .All experiments were carried out at least three times, and the data were expressed as the mean ± standard error of the mean (SEM).C. rumphii different fractions indicated that the ethyl acetate fraction has potent toxoplasmocidal and cytotoxic activities against various cell lines. Therefore, the phytochemical investigation of the most active fraction of the C. rumphii leaves was carried out and resulted in the isolation of a previously undescribed biflavonoid C-glycoside in addition to six known compounds, three of them were isolated for the first time from C. rumphii leaves. These compounds were identified using different spectroscopic techniques and compared to the data reported in the literature. In addition, the isolated compounds were evaluated for cytotoxic activity using the MTT assay method. The results showed that compounds  have significant cytotoxic activity. Future studies for in vivo assessment of these activities are necessary.In conclusion, the biological screening for


D-value of 5.9 µM as well as IC50 values of 4.0 µM in PBS and 10.2 µM in saliva. We thus introduce a rapid test for kynurenine as a potential biomarker for kidney transplant failure.Kynurenine is a tryptophan metabolite linked to several inflammatory processes including transplant failure, a significant challenge in transplant medicine. The detection of small molecules such as kynurenine, however, is often complex and time consuming. Herein, we report the successful synthesis of a fluorescently labelled kynurenine derivative, showing proper fluorescence and anti-kynurenine antibody binding behavior in a magnetic bead immunoassay (MIA). The fluorescent kynurenine–rhodamine B conjugate shows a K Kynurenine is a metabolite of the tryptophan degradation pathway linked to several inflammatory, metabolic, oncogenic  as well The link between inflammatory response and elevated kynurenine levels in serum via upregulation of IDO by pro-inflammatory factors renders kynurenine an interesting biomarker for clinically relevant inflammatory processes. Upregulated IDO and, therefore, elevated kynurenine levels of 3.9 ± 2.1 µM in serum have been found in the case of chronic kidney disease (CKD) in patients in the pre-dialysis stage. Values increase along with CKD severity up to 5.6 ± 2.3 µM because of chronic inflammation during CKD progression ,20,21,22Apart from transplant failure, elevated kynurenine levels have also been linked to titanium dental implants and to bone-osseointegration processes , as wellMost approaches in clinical trials to quantifying kynurenine from different tissues are focused on LC-MS ,34,35 orTo date and due to the small size of the kynurenine molecule, no fluorescently labelled kynurenine derivatives bound to an anti-kynurenine antibody are described in the literature. Likewise, there is no functional bioassay based on antibody binding of kynurenine described in the literature. Herein, we report the synthesis of a fluorescent, rhodamine B-labelled kynurenine derivative and demonstrate its binding ability to an anti-kynurenine antibody. We demonstrate that this denotes a promising approach for the development of a competitive kynurenine antibody-binding assay and ultimately a kynurenine rapid test from saliva and possibly other body fluids.4 in 1N NaOH. Flash column chromatography (FC) was performed on M&N Silica Gel 60 (0.063–0.200 mm). 1H NMR and 13C NMR spectra were recorded on a Bruker Avance I 200 (200 MHz), Bruker Avance II 400 , or Varian Unity 500 (500 MHz) spectrometer . Chemical shifts are reported in parts per million relative to solvent signals . Signals were assigned by first-order analysis and assignments were supported where feasible by 2-dimensional 1H, 1H and 1H, 13C correlation spectroscopy. Coupling constants are reported in hertz. UV/vis spectra were recorded on a PerkinElmer Lambda XLS+ UV/vis spectrometer  in a 10.00 mm QS quartz cuvette. Fluorescence was recorded on a Tecan Infinite M200 multiplate reader in commercially available 96-well multitier plates. Chemicals and reagents were purchased from Acros Organics, Alfa Aesar, Sigma-Aldrich, Carl Roth, Carbolution or ABCR and were used without further purification.TLC was carried out on Silica Gel 60 F254  with detection by UV light (254 nm) or by charring with 1% KMnO2Cl2 (250 mL) under N2 atmosphere. EDC  and DMAP  were added, and the mixture obtained was stirred for 15 min at room temperature. After the addition of propargyl alcohol  and after stirring overnight at room temperature, the mixture was washed 2x with 1M HCl (250 mL) and 1x with brine (250 mL). Drying the organic layer over Na2SO4 and evaporating the solvent yielded the crude product. Purification via flash column chromatography (CH2Cl2: MeOH 9:1) gave the final product: violet powder .Rhodamine B  was dissolved in dry CH1H-NMR : 8.66 , 7.88 , 7.78 , 7.38 , 7.09 , 6.95 , 6.88 , 4.65 , 3.68 , 2.46 , 1.36 .13C-NMR : 164.4 (C(=O)O), 157.6 (Ar-C), 155.6 (Ar-C), 134.0 (Ar-C), 133.8 (Ar-C), 131.3 (Ar-C), 131.3 (Ar-C), 131.0 (Ar-C), 129.3 (Ar-C), 115.1 (Ar-C), 113.4 (Ar-C), 107.4 (Ar-C), 96.4 (Ar-C), 78.5 (C), 77.8 (CH), 53.3 (CH2), 45.8 (CH2), 12.9 (CH3).2 protection of kynurenine was carried out by Boc-chemistry following Schotten–Baumann conditions: kynurenine  was dissolved in a water/THF 1:1 mixture containing NaOH . After the solution turned clear, Boc2O  was added dropwise at 0 °C, and the reaction was monitored by TLC. Complete conversion was observed after 90 min. Acidification using 10% HCl, followed by 3 extractions with ethyl acetate, drying of the organic layer and evaporation of the solvent yielded the crude product: a yellowish oil. Column chromatography (ethyl acetate) yielded the pure product: a yellowish powder .NH1H-NMR : 7.73 , 7.34 , 6.83–6.72 , 5.64 , 4.67 , 3.80–3.69 , 3.53 , 1.45 .13C-NMR : 199.54 (Ar-C(=O)), 172.43 (C(C=O)OH), 155.68 (Boc-C(=O)), 149.45 (Ar-C), 134.96 (Ar-C), 131.08 (Ar-C), 125.83 (Ar-C), 118.10 (Ar-C), 116.73 (Ar-C), 69.98 (Boc-tert-C), 52.60 (CH), 41.47 (CH2), 28.34 (CH3).1  and the corresponding azido-linker  were dissolved in CH2Cl2 (50 mL). EDC  and DMAP  were added, and the mixture was stirred at room temperature for 48 h. Washing 3× with 2 M NaOH (80 mL) followed by washing with brine (80 mL), drying over Na2SO4 and evaporation yielded the crude product. Column chromatography (ethyl acetate) yielded the pure product: an orange solid, which was used directly in the next step without further analysis .Boc-protected kynurenine 3  and propargyl rhodamine B 1  were dissolved in 30 mL of a mixture of CH2Cl2/MeOH/H2O 10:10:3. After adding an aqueous CuSO4 solution , TBTA  and Na ascorbate , the mixture was heated to 60 °C for 16 h. After cooling down, 20 mL of ddH2O was added and the mixture was extracted 3 times with 50 mL CH2Cl2, followed by drying over Na2SO4 and evaporation of the solvent. The pure product was obtained after column chromatography (CH2Cl2/MeOH 3:1) as a pink powder .Azido functionalized Boc-L-Kynurenine 1H-NMR : 8.20 , 7.91–7.85 , 7.83–7.79 , 7.47 , 7.08–7.00 , 6.97–6.90 , 5.04 , 4.65 , 4.44 , 3.74 , 3.63 , 3.48–3.45 , 3.44–3.40 , 3.30 , 3.00 , 1.33 , 1.20 .13C-NMR : 198.9 (Ar-C(=O), 172.1 (C(=O)-NH), 164.9 (C(=O)-O), 157.8 (Ar-H), 157.5 (Boc-C(=O)), 156.0 (Ar-C), 155.6 (Ar-C), 155.5 (Ar-C), 141.04 (Triazol-Ar-C), 134.6 (Ar-C), 133.9 (Ar-C), 133.8 (Ar-C), 133.7 (Ar-C), 131.3 (Ar-C), 131.2 (Ar-C), 131.0 (Ar-C), 130.9 (Ar-C), 129.6 (Ar-C), 129.2 (Ar-C), 125.3 (Triazol-Ar-C), 115.0 (Ar-C), 114.9 (Ar-C), 114.8 (Ar-C), 113.3 (Ar-C), 96.3 (Ar-C), 78.4 (tert.-C), 78.0 (NH(Boc)-CH), 77.8 (O-CH2), 70.1 (O-CH2), 70.0 (O-CH2), 69.9 (O-CH2), 69.6 (O-CH2), 69.3 (O-CH2), 68.0 (O-CH2), 58.6 (Ar-CH2), 53.2 (RhB-CH2), 49.8 (Ar-CH2), 45.8 (N-CH2), 28.7 (Boc-CH3), 12.9 (RhB-CH3).4 was carried out by dissolving conjugate 4 in 6 mL of CH2Cl2 containing 25% trifluoroacetic acid. After stirring at room temperature for 1 h, the solution was precipitated in 50 mL ice cold Et2O and centrifuged for 5 min at 4 °C and max speed. The precipitate was dissolved in ddH2O and lyophilized. Purification by HPLC yielded the pure product: a pink solid .Boc-deprotection of rhodamine B–kynurenin conjugate 1H-NMR : 8.20 , 8.11 NH), 7.93–7.84 , 7.85–7.78 , 7.47 , 7.05–6.99 , 6.97–6.89 , 5.05 , 4.47–4.41 , 3.75 , 3.63 , 3.54 , 3.50–3.42 , 2.92 , 1.20 .13C-NMR : 197.3 (Ar-C(=O)), 168.8 (C(=O)-NH), 164.9 (C(=O)-O), 158.2 (Ar-C), 157.9 (Ar-C), 157.5 (Ar-C), 155.5 (Ar-C), 141.1 (Triazol-Ar-C), 133.2 (Ar-C), 133.7 (Ar-C), 131.2 (Ar-C), 130.9 (Ar-C), 129.6 (Ar-C), 125.3 (Triazol-Ar-C), 117.5 (Ar-C), 114.9 (Ar-C), 113.3 (Ar-C), 96.3 (Ar-C), 70.1 (O-CH2), 70.0 (O-CH2), 69.9 (O-CH2), 69.0 (O-CH2), 67.1 (O-CH2), 58.6 (Ar-CH2), 49.8 (Ar-CH2), 45.7 N-CH2), 12.87 (CH3).1  and azidotetraethylene glycol  was added, TBTA  and Na ascorbate  were added, and the mixture was heated to 60 °C. The reaction was monitored by TLC. After 16 h, the mixture was left to cool down, followed by the addition of 25 mL of ddH2O. Extraction with 50 mL of CH2Cl2 3 times, followed by drying over Na2SO4, gave the crude product. Column chromatography (CH2Cl2/MeOH 3:1) gave the pure product: a pink oil .Propargyl rhodamine B terature ) were di1H-NMR : 8.22 , 7.89 , 7.82 , 7.48 , 7.04 , 6.96 , 6.95–6.92 , 5.06 , 4.56 , 4.45 , 3.76 , 3.65 , 3.49 , 3.46–3.41 , 3.37–3.34 , 1.22 .13C-NMR : 164.88 (C(=O)), 157.88 (Ar-H), 157.53 (Ar-H), 155.53 (Ar-H), 141.05 (Ar-H), 133.71 (Ar-H), 131.24 (Ar-H), 130.93 (Ar-H), 129.67 (Ar-H), 125.36 (Ar-H), 114.94 (Ar-H), 113.32 (Ar-H), 96.29 (Ar-H), 72.75 (CH2), 70.21 (CH2), 70.17 (CH2), 70.05 (CH2), 69.97 (CH2), 69.01 (CH2), 66.81 (CH2), 60.63 (CH2), 58.63 (CH2), 49.77 (CH2), 45.76 (CH2), 12.89 (CH3).®12-biotin, Sigma Aldrich, Taufkirchen, Germany) was added to 100 µL of an anti-kynurenine antibody . The mixture was incubated for 50 min at room temperature with gentle shaking. Excess Biotin-NHS was removed using a VivaSpin 500 centrifugal concentrator with 10 kDa MWCO (Sigma Aldrich).A total of 1 µL of a 6 mg/mL Biotin-NHS solution  was diluted to 1 mg/mL with 450 µL of PBS and pelleted on a magnetic rack for 2 min. The supernatant was discarded, and the beads were washed three times with 500 µL PBS. After the last washing step, 20 µL of the supernatant was replaced with 20 µL biotinylated antibody (0.5 mg/mL). The mixture was incubated for 30 min at RT under gentle shaking. The reaction was blocked by washing the beads three times with PBS containing 1.5% BSA and 0.5% Tween-20. The final concentration was 20 µg of antibody per 1 mg of beads.5 or rhodamine B-PEG-Linker 6 was incubated with 50 µL of antibody-conjugated magnetic beads and 50 µL of PBS (1:3 dilution). The beads were pelleted on a magnetic rack, and the unbound rhodamine B in the supernatant was quantified in a 96-well-plate with a fluorescence measurement of 100 µL of supernatant at 561 nm excitation and 592 nm emission, using a Tecan Infinite M200 multiplate reader. A standard curve of rhodamine B fluorescence intensity between 0 and 100 µM diluted 1:3 in PBS was used for the calculation of bead-bound conjugate 5 or 6.For 3 h at RT, 0–100 µM rhodamine B–kynurenine conjugate 5 and 50 µL of spiked PBS or artificial saliva  containing 0–250 µM native L-kynurenine for 3 h at RT on a hula shaker (1:3 dilution). Beads were pelleted on a magnet and fluorescence intensity of unbound rhodamine B–kynurenine conjugate 5 in 100 µL supernatant was measured in a Tecan Infinite M200 multiplate reader at 561 nm excitation and 592 nm emission. Bead-bound L-kynurenine was indirectly calculated by calculating the amount of displaced rhodamine B–kynurenine conjugate 5. Therefore, the fluorescence intensity of bound rhodamine B–kynurenine conjugate 5 without L-kynurenine was subtracted from the fluorescence intensity of samples with different L-kynurenine concentrations.Fifty microliters of antibody coupled beads was incubated with 50 µL of 12 µM rhodamine B-kynurenine conjugate 1 using Steglich esterification by EDC and DMAP, which was used as the fluorescent probe for click-conjugates 5 and 6.Chemical synthesis of the fluorescent L-kynurenine–rhodamine B conjugate was carried out as shown in 3, Boc-protection of the primary amine of kynurenine was carried out to obtain product 2, followed by attaching an amino-azido-tetraethylene glycol linker, synthesized according to a published protocol [3. Click reaction of 3 with 1 gave product 4, followed by Boc-deprotection to the fluorescent kynurenine probe 5. To study the effect of the linker as well as the rhodamine B moiety on antibody binding, rhodamine B clickamer 6 containing only a tetraethylene glycol linker [4.For the azido tetraethylene glycol L-kynurenine derivative protocol  yieldingl linker  was prep2O (cf. 5 and 6, containing a benzoic ester instead of a free benzoic acid at the rhodamine’s benzoic acid residue, is slightly shifted to 560 nm. Emission maxima were determined to be at 586 nm for the L-Kyn-4EG-RhB probe 5, while the 4EG-RhB clickamer 6 shows an emission maximum of 584 nm, indicating suitable absorption and emission properties for immunoassays.To determine emission and absorption maxima for the synthesized rhodamine B click-conjugates, UV/vis spectra of the compounds were recorded in ddH2O cf. . The abs5 and 6 as well as their competitive binding capability by using magnetic bead-bound anti-kynurenine antibodies, followed by incubation with the fluorescent conjugates and the subsequent fluorescence measurements of unbound conjugate in the supernatant. The tetraethylene glycol rhodamine B clickamer 6 was used as a negative control to exclude unspecific interactions between the rhodamine B or the tetraethylene glycol linker with the antibody. Antibody binding data are shown in 5 and native L-kynurenine.To determine the antibody binding capabilities of the synthesized fluorescent L-kynurenine conjugate, we elucidated the antibody binding capability of fluorescent conjugates 5, followed by a pelleting of the beads and the measurement of the fluorescence in the supernatant. The binding curves of 5 and 6 as well as the competitive binding curve are shown in To this end, antibodies were bound to magnetic beads in 1 µm diameter, followed by incubation with the target compounds, pelleted via magnet and the fluorescence measured in the supernatant. In the competitive assay, 0–83 µM L-kynurenine was incubated together with either 4 µM RhB-4EG-L-Kyn conjugate 5, the measured fluorescence in the supernatant decreases since more fluorescent conjugates bind to the antibody. 5 and 4EG-RhB clickamer 6 with and without incubation with the bead-bound antibodies. In higher concentrations, the 4EG-RhB clickamer 6 binding affinity also increases, probably due to unspecific interactions between either the linker or the rhodamine B residue. Apart from this, the difference in measured fluorescence for the L-kynurenine conjugate 5 is significantly stronger, showing a KD-value of 5.9 µM, and therefore, unspecific interactions between the 4EG-RhB clickamer 6 and the magnetic bead-bound antibody can be neglected.With increasing concentration of the fluorescent L-kynurenine conjugate 50 values of this competition were calculated to be 4.0 µM in PBS and 10.2 µM in saliva. As expected, the IC50 value in saliva is higher than in PBS due to interfering components such as enzymes and proteins in saliva samples.As shown in D-value of 5.9 µM for the L-kynurenine conjugate 5 and IC50 values of 4.0 µM in PBS and 10.2 µM in saliva for the competitive assay. Since an increase in kynurenine levels in saliva to 4.6 ± 1.6 µM under pathological conditions, compared to 0.7 ± 0.4 µM in the healthy subject, are observed [We successfully synthesized a fluorescent kynurenine conjugate based on rhodamine B with unique antibody binding properties, a promising component of a future rapid diagnostic test for kynurenine, a metabolite with a clinically relevant marker for the diagnosis of distinct diseases. Spectral properties of the products were elucidated, showing only a slight shift of 6 nm in the fluorescence emission maximum when compared to native rhodamine B. The antibody binding was investigated, and the magnetic bead assay showed a good sensitivity with a Kobserved , it thusobserved , even th


Their structures were elucidated by extensive spectroscopic analysis and electronic circular dichroism calculation (ECD). Compound 1 featured an unprecedented 5,8-spiroketal subunit as well as the loss of C-2′ carbonyl in the phloroglucinol ring. In addition, compounds 1 and 4 showed weak hepatoprotective activity against paracetamol-induced HepG2 cell damage at 10 μm. The plausible biosynthetic pathway of 1 was proposed via a retro-Clasisen reaction and decarboxylation.Two previously undescribed polycyclic polyprenylated acylphloroglucinols, hyperacmosins R-S ( Hypericum, are a group of structurally fascinating and synthetically challenging natural products, which possess highly oxygenated acylphloroglucinol-derived cores decorated with isoprenyl, geranyl, or other substituted side chains 20D -230.5 . HR-ESI-MS spectrum  revealed its molecular formula to be C34H52O7, indicating nine degrees of unsaturation, and its IR data implied the existence of hydroxyl (3533 cm−1) and carbonyl (1716 cm−1). The 1H NMR spectrum of 1 , 1.07 , 1.41  ], one hydroxyl [δH 2.66 ], two olefinic protons [δH 4.87  and 5.31 ], and nine singlet methyls . Combined with its HSQC and HMBC spectra, the 13C NMR data of 1  and four olefinic carbons , accounting for five indices of hydrogen deficiency. Apart from the aforementioned 7 carbons, the remaining 27 carbon signals were assigned to two oxygenated tertiary carbons (δC 85.4 and 90.1), three oxygenated secondary carbons , three quaternary carbons , five methylenes , three methines , and 11 methyls . Combined with the remaining four indices of hydrogen deficiency, the above data indicated that 1 might be a tetracyclic PPAP analogue.Hyperacmosin R  to C-2/C-3/C-5/C-6, from H2-5 (δH 1.25/2.45) to C-1/C-3/C-4/C-6, from H-17 (δH 3.04) to C-1/C-2/C-3/C-15/C-16, and from H2-15 (δH 1.34/1.89) to C-2/C-3/C-4/C-16/C-17, along with 1H-1H COSY correlations of H-4/H2-5, H2-15/H2-16, and H2-16/H-17, established the cyclohexanone (B-ring) and cyclopentane moieties (A-ring), respectively. Moreover, the HMBC correlations (δH 4.00) to C-8/C-26/C-28/C-29/C-30, from H3-29 (δH 1.22)/H3-30 (δH 1.33) to C-27/C-28, and from H2-26 (δH 2.37) to C-7/C-8/C-27/C-28, combined with the presence of two oxygen-bearing carbons C-8 (δC 113.1) and C-28 (δC 90.1), implied the presence of the 2,2-dimethyl-3-hydroxy-furan unit (D-ring). Considering one remaining unsaturation as well as the diagnostic ketal carbon C-8 (δC 113.1), the fourth circle (C-ring) was formed via an oxygen connecting C-8 and C-18. Moreover, an isobutyryl group and two isoprenyl groups were attached to C-2, C-4, and C-6, respectively, supported by the HMBC cross-peaks from H-11 (δH 2.90) to C-2, from H2-21 (δH 1.70/2.08) to C-3/C-4/C-5, and from H-31 (δH 3.20) to C-1/C-5/C-6/C-7. It is noteworthy that the abnormal chemical shift of C-31 (δC 36.9) was much lower than the normal value. By viewing the 3D model of compound 1, it might be attributed to the shielding effect of the C-1 carbonyl group. The polycyclic core structure of elations  from H-4elations  from H-21 was confirmed on the basis of the ROESY spectrum  calculation, using the time-dependent density functional theory (TD-DFT). A pair of enantiomers, -1a and -1b, were calculated for the ECD spectra based on the known relative configuration of 1. The ECD spectrum  was obtained as a colorless oil. The molecular formula was established as C31H46O5 according to its HRESIMS data , indicating nine degrees of unsaturation. The 1H NMR spectrum  and 4.94 ], a sec-butyl group , and eight singlet methyls . The 13C NMR spectrum  and four olefinic carbon . Detailed analysis of the 1D and 2D NMR ; garsubellin B: [α]24D -36 ] ..2) was o3)  nonane-2,4,9 trione core, is formed via a series of prenylation and cyclization reactions 20D -230.5 ; UV (MeOH) λmax (log ε) 204 (4.05) nm; ECD (MeOH) λmax (∆ε) 212 (−9.02), 320 (9.18) nm; IR υmax 3533, 2977, 2929, 1716, 1687, 1450, 1380 cm−1; 1H and 13C NMR data, see m/z 573.3786 [M + H]+ .Hyperacmosin R (2): colorless oil; [α]20D +92.3 ; UV (MeOH) λmax (log ε) 203 (4.45), 271 (4.43) nm; ECD (MeOH) λmax (∆ε) 224 (17.32), 250 (−5.31), 274 (19.29), 304 (−10.79), 339 (0.93) nm; IR υmax 3446, 2975, 2930, 1730, 1626, 1452, 1369 cm−1; 1H and 13C NMR data, see m/z 499.3417 [M + H]+ .Hyperacmosin S (1–11 were determined by a (MTT) colorimetric assay in HepG2 cells. Each cell suspension of 2 × 104 cells in 200 µL of RPMI 1640 containing fetal calf serum (10%), penicillin (100 U/mL), and streptomycin (100 µg/mL) was placed in a 96-well microplate and precultured for 24 h at 37 ℃ under 5% CO2 atmosphere. Fresh medium (100 µL) containing bicyclol and test samples was added, respectively, and the cells were cultured for 1 h. The cultured cells were exposed to 16 mM paracetamol for 24 h. Then, 100 µL of 0.5 mg/mL MTT was added to each well, after the withdrawal of the culture medium, and incubated for additional 4 h. The resulting formazan was dissolved in 150 µL DMSO after aspiration of the culture medium. The optical density (OD) of the formazan solution was measured on a microplate reader at 570 nm. Percentage inhibition was calculated as: inhibition (%) = [OD (sample) − OD (control)]/[OD  − OD (control)] × 100%. The hepatoprotective effects of compounds H.acmosepalum led to the identification of 11 PPAPs, including 2 previously undescribed ones, hyperacmosins R-S (1–2). Especially, hyperacmosin R (1) possesses a rare 5,8-spiroketal subunit, together with the loss of C-2′ carbonyl in the phloroglucinol ring. All the isolates were evaluated for their hepatoprotective activities. Among them, hyperacmosin R (1) and furoadhyperforin isomer B (4) exhibited weak capabilities against paracetamol-induced HepG2 cell damage at 10 μm. Furthermore, the plausible biosynthetic pathway of hyperacmosins R (1) was proposed. This study enriched the members and the structural diversity of PPAPs from H.acmosepalum.In summary, a detailed chemistry investigation of


A sulforhodamine B assay was used to evaluate the cytotoxicity effect against two human cancer cell lines, SKLU-1 and HeLa. Electronic descriptors and redox potential were obtained by DFT calculations. Structure–activity relationships are strongly determined by the redox potential (E1/2) of copper(II) and molar volume (V) of the complexes. These compounds can be used as a template to open a wide field of research both experimentally and theoretically.Casiopeinas are a family of copper(II) coordination compounds that have shown an important antineoplastic effect and low toxicity in normal cells. These compounds induce death cells by apoptosis through a catalytic redox process with endogenous reducing agents. Further studies included a structural variation, improving the activity and selectivity in cancer cells or other targets. In the present work we report the third generation, which contains a bioactive monocharged secondary ligand, as well as the design, synthesis, characterization and antiproliferative activity, of sixteen new copper(II) coordination compounds with curcumin or dimethoxycurcumin as secondary ligands. All compounds were characterized by elemental analysis, FTIR, UV-Vis, magnetic susceptibility, mass spectra with MALDI-flight time, cyclic voltammetry, electron paramagnetic resonance (EPR) spectroscopy and X-ray diffraction. Crystallization of two complexes was achieved in dimethylsulfoxide (DMSO) with polar solvent, and crystal data demonstrated that a square-based or square-base pyramid geometry are possible. A 1:1:1 stoichiometry (diimine: copper: curcuminoid) ratio and the possibility of a nitrate ion as a counterion were supported.  Historically, design of coordination compounds has led to the synthesis, characterization and biological evaluation of new molecules. In this sense, rare earth  and tranAccording to the World Health Organization (WHO) , in 20203 and [Cu(N-N)(N-O)]NO3, where N-N is 2,2′-bipyridines or 1,10-phenanthrolines with or without substituents (primary ligands), O-O is acetylacetonate or salycilaldehydates and N-O can be aminoacidates or peptidates (secondary ligands). Casiopeinas have shown good cytotoxic activity against several cancer cell lines and antineoplastic activity in vitro and in vivo +) (3). On the other hand, the results confirm that the stoichiometry of the copper(II) coordination compounds is 1:1:1 (diimine: Cu: secondary ligand), as determined by elemental analysis.For many molecules, it was observed that the molecular ion corresponds to the base peak; these data suggest that the most predominant species is the complex cation of copper(II) ([DnCuLn]+) , which im/z data, which represent the complex fragments without one or two –OH or –OMe groups over the aromatic rings of curcumin. The same behavior was observed for all complexes with dimethoxycurcumin with the loss of 2 or 4 –OMe groups. m/z of 638 is assigned to molecular ion [D4CuL2]+, and the peaks at m/z 582 and 522 are assigned to the fragment of the molecule that loses 2 or 4 –OMe groups, respectively.In addition, all coordination compounds with curcumin as secondary ligand exhibited + + 2e− → R-COHCHCOH-R) NO golden solid (98% yield); C31H27N3O9Cu (649.1 g/mol); elemental analysis, %calculated (%founded): C, 57.36 (57.13); H, 4.19 (4.17); N, 6.47 (6.46). m.p.: 248.0 °C; FT-IR (cm−1): 3423 ν(O-H), 1619 ν(C=N), 1598 ν(C-O), 1508 ν(C=C), 2969 ν(C-H), 1280 ν(Ar-O), 1384.7 ν (NO3), 480 ν (M-O); μeff = 1.05 B.M. Molar conductance Λ (DMSO) = 37.3 μS. MALDI MS M+ (m/z): 586.11 (586.9).3:[Cu(D2)(L1)]NO orange solid (94% yield); C33H31N3O9Cu.H2O (695.17 g/mol); elemental analysis, %calculated (%founded): C, 57.01 (56.77); H, 4.78 (4.76); N, 6.04 (6.07). Decomposition temperature: 222.5 °C; FT-IR (cm−1): 3423 ν(O-H), 1621 ν(C=N), 1589 ν(C-O), 1508 ν(C=C), 2939 ν(C-H), 1280 ν(Ar-O), 1384.7 ν (NO3), 470 ν (M-O); μeff = 1.79 B.M. Molar conductance Λ (DMSO) = 33.03 μS. MALDI MS M+ (m/z): 614.47 (615.03).3:[Cu(D3)(L1)]NO reddish solid (96% yield); C33H31N3O9Cu.H2O (695.17 g/mol); elemental analysis, %calculated (%founded): C, 57.01 (57.29); H, 4.78 (4.79); N, 6.04 (6.01). Decomposition temperature: 208.6 °C; FT-IR (cm−1): 3425 ν(O-H), 1600 ν(C=N), 1596 ν(C-O), 1510 ν(C=C), 2935 ν(C-H), 1280 ν(Ar-O), 1384.7 ν (NO3), 480 ν (M-O); μeff = 1.13 B.M. Molar conductance Λ (DMSO) = 32.9 μS. MALDI MS M+ (m/z): 614.47 (614.08).3:[Cu(D4)(L1)]NO orange solid (94% yield); C33H27N3O9Cu (673.12 g/mol); elemental analysis, %calculated (%founded): C, 58.88 (58.63); H, 4.04 (4.02); N, 6.24 (6.28). Decomposition temperature: 249.2 °C; FT-IR (cm−1): 3436 ν(O-H), 1618 ν(C=N), 1589 ν(C-O), 1510 ν(C=C), 2944 ν(C-H), 1278 ν(Ar-O), 1384.7 ν(NO3), 476 ν(M-O); μeff = 1.10 B.M. Molar conductance Λ (DMSO) = 32.6 μS. MALDI MS M+ (m/z): 610.11 (610.98).3:[Cu(D5)(L1)]NO reddish solid (80% yield); C35H31N3O9Cu.H2O (719.19 g/mol); elemental analysis, %calculated (%founded): C, 58.45 (58.70); H, 4.62 (4.59); N, 5.84 (5.82). Decomposition temperature: 200.5 °C; FT-IR (cm−1): 3419 ν(O-H), 1621 ν(C=N), 1596 ν(C-O), 1510 ν(C=C), 2977 ν(C-H), 1282 ν(Ar-O), 1384.7 ν(NO3), 478 ν(M-O); μeff = 1.35 B.M. Molar conductance Λ (DMSO) = 34.0 μS. MALDI MS M+ (m/z): 638.14 (638.04).3:[Cu(D6)(L1)]NO orange solid (95% yield); C35H31N3O9Cu (701.18 g/mol); elemental analysis, %calculated (%founded): C, 59.95 (60.20); H, 4.45 (4.47); N, 5.99 (5.97). Decomposition temperature: 205.4 °C; FT-IR (cm−1): 3432 ν(O-H), 1621 ν(C=N), 1600 ν(C-O), 1511 ν(C=C), 2936 ν(C-H), 1278 ν(Ar-O), 1384.7 ν (NO3), 478 ν (M-O); μeff = 1.10 B.M. Molar conductance Λ (DMSO) = 59.1 μS. MALDI MS M+ (m/z): 638.14 (638.04).3:[Cu(D7)(L1)]NO orange solid (95% yield); C37H35N3O9Cu.H2O (747.24 g/mol); elemental analysis, %calculated (%founded): C, 59.47 (59.20); H, 4.99 (4.97); N, 5.62 (5.59). Decomposition temperature: 243.2 °C; FT-IR (cm−1): 3446 ν(O-H), 1619 ν(C=N), 1594 ν(C-O), 1510 ν(C=C), 2925 ν(C-H), 1282 ν(Ar-O); 1384.7 ν(NO3), 478 ν(M-O); μeff = 1.92 B.M. Molar conductance Λ (DMSO) = 31.9 μS. MALDI MS M+ (m/z): 666.17 (667.18).3:[Cu(D8)(L1)]NO orange solid (80% yield); C45H35N3O9Cu (825.32 g/mol); elemental analysis, %calculated (%founded): C, 65.48 (65.51); H, 4.27 (4.25); N, 5.09 (5.12). Decomposition temperature: 246.0 °C; FT-IR (cm−1): 3417 ν(O-H), 1618 ν(C=N), 1589 ν(C-O), 1510 ν(C=C), 2942 ν(C-H), 1280 ν(Ar-O); 1384 ν(NO3), 474 ν(M-O); μeff = 2.16 B.M. Molar conductance Λ (DMSO) = 32.8 μS. MALDI MS M+ (m/z): 762.17 (762.10).3:[Cu(D1)(L2)]NO orange solid (88% yield); C33H31N3O9Cu (677.16 g/mol); elemental analysis, %calculated (%founded): C, 58.53 (58.24); H, 4.61 (4.59); N, 6.2 (6.18). m.p.: 236.9 °C; FT-IR (cm−1): 3427 ν(O-H), 1616 ν(C=N), 1579 ν(C-O), 1510 ν(C=C), 2948 ν(C-H), 1265 ν(Ar-O); 1384.7 ν(NO3), 486 ν(M-O); μeff = 1.91 B.M. Molar conductance Λ (DMSO) = 37.4 μS. MALDI MS M+ (m/z): 614.14 (615.30).3:[Cu(D2)(L2)]NO orange solid (92% yield); C35H35N3O9Cu.H2O (723.22 g/mol); elemental analysis, %calculated (%founded): C, 58.12 (57.86); H, 5.15 (5.12); N, 5.81 (5.83). m.p.: 244.9 °C; FT-IR (cm−1): 3471 ν(O-H), 1616 ν(C=N), 1581 ν(C-O), 1510 ν(C=C), 2939 ν(C-H), 1265 ν(Ar-O); 1384.7 ν(NO3), 480 ν(M-O); μeff = 1.86 B.M. Molar conductance Λ (DMSO) = 23.2 μS. MALDI MS M+ (m/z): 642.17 (643.29).3:[Cu(D3)(L2)]NO light green solid (84% yield); C35H35N3O9Cu.H2O (723.22 g/mol); elemental analysis, %calculated (%founded): C, 58.12 (58.39); H, 5.15 (5.18); N, 5.81 (5.84). m.p.: 262.7 °C; FT-IR (cm−1): 3452 ν(O-H), 1619 ν(C=N), 1581 ν(C-O), 1508 ν(C=C), 2939 ν(C-H), 1263 ν(Ar-O); 1384.7 ν(NO3), 484 ν(M-O); μeff = 1.9 B.M. Molar conductance Λ (DMSO) = 24.1 μS. MALDI MS M+ (m/z): 642.17 (642.10).3:[Cu(D4)(L2)]NO light green solid (80% yield); C35H31N3O9Cu.2H2O (737.21 g/mol); elemental analysis, %calculated (%founded): C, 57.02 (56.75); H, 4.78 (4.76); N, 5.69 (5.66). m.p.: 254.3 °C; FT-IR (cm−1): 3436 ν(O-H), 1619 ν(C=N), 1581 ν(C-O), 1510 ν(C=C), 2939 ν(C-H), 1261 ν(Ar-O); 1384.7 ν(NO3), 462 ν(M-O); μeff = 1.9 B.M. Molar conductance Λ (DMSO) = 23.7 μS. MALDI MS M+ (m/z): 638.14 (638.03).3:[Cu(D5)(L2)]NO solid (% yield); C37H35N3O9Cu (729.23 g/mol); elemental analysis, %calculated (%founded): C, 60.94 (60.68); H, 4.83 (4.82); N, 5.76 (5.74). Decomposition temperature: 244.5 °C; FT-IR (cm−1): 3428 ν(O-H), 1621 ν(C=N), 1579 ν(C-O), 1508 ν(C=C), 2935 ν(C-H), 1259 ν(Ar-O); 1384.7 ν(NO3), 472 ν(M-O); μeff = 2.26 B.M. Molar conductance Λ (DMSO) = 33.8 μS. MALDI MS M+ (m/z): 666.17 (668.24).3:[Cu(D6)(L2)]NO solid (% yield); C37H35N3O9Cu (729.23 g/mol); elemental analysis, %calculated (%founded): C, 60.94 (61.19); H, 4.83 (4.85); N, 5.76 (5.79). Decomposition temperature: 237.0 °C; FT-IR (cm−1): 3444 ν(O-H), 1619 ν(C=N), 1581 ν(C-O), 1510 ν(C=C), 2935 ν(C-H), 1261 ν(Ar-O); 1384.7 ν(NO3), 472 ν(M-O); μeff = 1.96 B.M. Molar conductance Λ (DMSO) = 28.7 μS. MALDI MS M+ (m/z): 666.17 (668.24).3:[Cu(D7)(L2)]NO solid (% yield); C39H39N3O9Cu (757.28 g/mol); elemental analysis, %calculated (%founded): C, 61.85 (61.56); H, 5.19 (5.21); N, 5.54 (5.51). Decomposition temperature: 241.0 °C; FT-IR (cm−1): 3446 ν(O-H), 1612 ν(C=N), 1579 ν(C-O), 1504 ν(C=C), 2931 ν(C-H), 1265 ν(Ar-O); 1384.7 ν(NO3), 484 ν(M-O); μeff = 2.10 B.M. Molar conductance Λ (DMSO) = 28.8 μS. MALDI MS M+ (m/z): 694.21 (696.14).3:[Cu(D8)(L2)]NO solid (% yield); C47H39N3O9Cu (853.37 g/mol); elemental analysis, %calculated (%founded): C, 66.14 (65.86); H, 4.60 (4.63); N, 4.92 (4.94). Decomposition temperature: 238.8 °C; FT-IR (cm−1): 3413 ν(O-H), 1625 ν(C=N), 1562 ν(C-O), 1511 ν(C=C), 2933 ν(C-H), 1263 ν(Ar-O); 1384.7 ν(NO3), 480 ν(M-O); μeff = 2.30 B.M. Molar conductance Λ (DMSO) = 27.2 μS. MALDI MS M+ (m/z): 790.21 (791.56).4][PF6] as supporting electrolyte. Before to each experiment, the working electrode was cleaned and polished with diamond powder (3 μm). All of the solutions were degassed with nitrogen prior to measurement. Cyclic voltammetry was initiated from open-circuit potential to the negative direction; the scan rate employed was 100 mVs−1 in both cathodic and anodic directions. On the other hand, by varying the scanning speed, the kinetics of the reactions can be studied, or the appearance of intermediate species can be detected accordingly in redox processes. We performed cyclic voltammetry with a variable scan rate between 20 and 1000 mV/s in the reductive direction. All potentials were reported versus the coupled ferrocene as internal standard (E°Fc+/Fc) according to IUPAC.Cyclic voltammetry was performed using a typical three-electrode cell comprising a pseudo-reference Ag, working glassy carbon electrodes and platinum wire as counter electrode. All electrochemical experiments were carried out using 1mM solutions of each coordination compound in DMSO in the presence of 0.1 M tetrabutylammonium hexafluorophosphate 0/+ value of 5.3 eV at the same level of theory.We calculated the redox potential of a half reaction for a one-electron exchange using the Nernst equation (Equation (4)), where F is the Faraday constant. Here, experimental redox potentials are reported with respect to ferrocene/ferrocenium  coordination compounds were prepared (third generation of Casiopeinas) and characterized, showing a 1:1:1 stereochemistry (diimine:Cu:curcuminoid). These complexes can have a square-based pyramid or square planar geometry, depending on whether the water molecules are coordinated or not to the metal center according to X-ray diffraction. The synthesis of copper(II) complexes with curcumin and dimethoxycucumin can be used as a template for the generation of new molecules that present novel properties.The measured cytotoxic activity of the coordination compounds against HeLa and SKLU-1 showed a improved biological activity compared to free ligands and that including a bioactive molecule as a secondary ligand in the Casiopeinas enhances their biological activity. One hypothesis to be resolved is that the toxicity of Casiopeinas may decrease due to the presence of these curcuminoids, given their low toxicity both in normal cells and in vivo, making them a potential alternative for the treatment of cancer.1/2) is adequate to describe the antiproliferative activity in the SKLU-1 tumor cell line for copper(II) coordination compounds with curcumin, which was validated with theoretical models. Increased activity of coordination compounds is important in pharmaceuticals. Our results will allow us to generate important research around the most active molecules to guide them towards pharmaceutical applications.It was found that the experimental redox potential (E


Glycosmis pentaphylla (Rutaceae), collected in Vietnam, yielded three new compounds named glyfuran (1), glyphyllamide (2), and glyphyllazole (3), along with twenty-five known compounds (4–28). The structures of isolates were determined by IR, MS, NMR, and UV data analyses. In the anti-diabetic activity screening, (+)-isoaltholacton (4), glycoborinine (17), 2′,4′-dihydroxy-4,6′-dimethoxychalcone (24), and flavokawain A (25) simultaneously exhibited inhibition of dipeptidyl peptidase-4 (DPP4) and stimulation of the glucagon-like peptide-1 (GLP-1) secretion on the murine intestinal secretin tumor cell line (STC-1).A phytochemical investigation of the leaves and twigs of  Diabetes mellitus is a metabolic disease characterized by excessive amounts of blood sugar, leading to a family of diseases that includes hypertension, osteoporosis, retinopathy, and urethritis. Along with the increasing popularity of a sedentary lifestyle, diabetes has gradually become a global epidemic. According to the World Health Organization, approximately 422 million people worldwide suffer from this disease, with up to 1.5 million dying yearly ,4.Gastrodia elata water extract can increase the quality of islet β cells and reduce cell apoptosis , two olefinic protons , four oxymethines , and one methyl group [δH 0.95 ]. The 13C NMR and DEPT spectra (δC 167.2), one olefinic quaternary carbon (δC 140.2), seven olefinic methines , four oxymethines , one oxymethylene (δC 64.8), two methylenes (δC 19.1 and 30.6), and one methyl group (δC 13.6).Compound NMR data  of 1 cle spectra  exhibite1, three proton sequences of H-2/H-3/H-4/H-5/H-6, H-7/H-8/H-9/H-10/H-11/H-12, and H2-1′ (δH 4.15)/H2-2′ (δH 1.66)/H2-3′ (δH 1.40)/H3-4′ (δH 0.95) were observed. The proton spin-spin coupling systems of H-2/H-3/H-4/H-5/H-6 and the HMBC correlations (δC 140.2) and H-2/H-6 to C-7 (δC 84.0) revealed the presence of a monosubstituted benzene ring (ring A), while the sequences of H-7/H-8/H-9/H-10 and the HMBC correlation from H-10 to C-7 (δC 84.0) suggested 1 contained a tetrahydrofuran ring (ring B). The n-butyl ester attached at C-12 was constructed by the COSY consequences H2-1′/H2-2′/H2-3′/H3-4′ and the HMBC correlation from H-11, H-12, and H-1′ to C-13 (δC 167.2). On the basis of these definitive 2D NMR analyses, the planar structure of 1 was established.In the COSY spectrum  of 1, thelations  of H-7 t1, the presence of NOESY correlations , suggesting compound 1 might be derived from 4 . The high-resolution ESIMS data showed a protonated molecule peak at [M + H]+ at m/z 336.18067, which indicated the molecular formula of C18H25NO5 and seven degrees of unsaturation. The IR spectrum of 2 showed absorption bands at 1736 and 1671 cm−1, revealing the presence of ester and amide functionality. The 1H NMR data , 7.31 , and 7.37 ], two methylenes [δH 4.02  and δH 4.19 ], and two methyl groups [δH 0.93  and δH 1.24 ]. The 13C NMR and DEPT spectra exhibited eighteen carbons, including two ester carbonyls (δC 170.5 and 170.8), one amide carbonyl (δC 170.7), one olefinic quaternary carbon (δC 134.4), five olefinic methines , two oxymethylenes (δC 61.8 and 64.9), one N-bearing methine (δC 48.6), four methylenes , and two methyl groups (δC 13.6 and 14.0). The planar structure of 2 was elucidated by dividing it into three partial structures, X, Y, and Z (δH 7.37)/H-3 (δH 7.29)/H-4 (δH 7.31)/H-5 (δH 7.29)/H-6 (δH 7.37) was observed from the COSY spectrum. In the HMBC spectrum, H2-7 (δH 3.62) showed correlations to C-1 (δC 134.4) and C-8 (δC 170.7), and the correlation from H-2/H-6 to C-1 revealed the presence of partial structure X. For the partial structure Y, a butyl group attached to an ester group was identified by the COSY correlations of H-1‴ (δH 4.02)/H-2‴ (δH 1.54)/H-3‴ (δH 1.33)/H-4‴ (δH 0.93) and the HMBC correlations from H2-3′ (δH 2.82 and 3.00) and H-1‴ to C-4′ (δC 170.8). Additionally, the HMBC correlations from H-1″ (δH 4.19) to C-1′ (δC 170.5), along with the proton spin system between H-1″ (δH 4.19) and H-2″ (δH 1.24) established the partial structure Z. These three parts were connected by the HMBC correlations from H-2′ (δH 4.83) to C-8, C-1′, and C-4′. According to the above 2D NMR analyses, the planar structure of 2 was established (2 showed similar 1H and 13C NMR data to a synthetic compound N-PhAc-D-Asp(OEt)OEt +), accounting for nine degrees of unsaturation. The IR spectrum showed the absorption bands due to hydroxy (3412 cm−1), amide (1625 cm−1), and phenyl (1512 and 1443 cm−1) functional groups. The UV spectrum displayed absorption maxima at 307 and 259 nm; therefore, 3 was defined to have a carbazole skeleton . The 1H NMR spectrum also displayed a methyl at δH 2.39 , along with two methoxy groups at δH 3.96  and δH 4.00 . The 13C NMR exhibited the signals of fifteen carbons, being twelve aromatic carbons , one methyl carbon (δC 16.1), and two methoxy carbon groups (δC 56.9 and 60.9). The COSY correlation showed one fragment of H-5/H-6. In the HMBC experiment, 3J correlations from δH 3.96 to δC 149.4 (C-7) and from δH 4.00 to δC 133.7 (C-8) indicated the positions of two methoxy groups. In addition, the correlations from 3-Me to C-2 (δC 152.5), C-3 (δC 116.2), and C-4 (δC 121.4) revealed the position of a methyl group. The HMBC correlations from H-4 to C-5a (δC 119.5)/C-1a (δC 139.6) and from H-5 to C-4a (δC 117.8) were used to construct the pyrrole moiety. Therefore, compound 3 was determined to be 7,8-dimethoxy-3-methyl-9H-carbazol-2-ol, and the trivial name glyphyllazole was given.Compound hydroxy 32 cm−1, aspectrum  of 3 sho1, 4, 6, 7, 17, 24, and 25 exhibited stimulatory effects on GLP-1 secretion from murine intestinal secretin tumor cell line (STC-1) at a concentration of 100 μM. In the acute toxicity evaluation  using solvent peaks as the internal standard on Bruker AVIII HD 700X NMR and Varian VNMRS 600 MHz spectrometers. HRESIMS data were obtained by using a Bruker 7T solariX spectrometer. Column chromatography was performed using Merck silica gel 60 (0.040–0.063 mm), C18 silica gel (0.040–0.075 mm), and Sephadex LH-20 material. NP-HPLC was composed of a Hitachi L-6000 pump and a Hitachi L-4000 UV detector with Phenomenex Luna silica  and CN  columns. RP-HPLC was carried out on a Shimadzu chromatography system consisting of a LC-20AT pump, a SPD-M20A PDA detector, and a CBM-20A system controller with Phenomenex Luna C18 , phenyl-hexyl , and Kinetex biphenyl  columns.The optical rotations, UV, and IR spectra were recorded on a Jasco P-2000 digital polarimeter, a Jasco V-530 UV/VIS spectrophotometer, and a Jasco FT-IR 4600 spectrometer, respectively. NMR spectra data were corrected in CDClG. pentaphylla were purchased in July 2018 from Cu Lao Cham Island  by Associate Professor Quang Vinh Nguyen  and Associate Professor Chia-Hung Yen . A voucher specimen (code No. KMU-VN016) was deposited at Kaohsiung Medical University.The leaves and twigs of G. pentaphylla (0.7 kg) were extracted by 95% EtOH  at room temperature to give an ethanolic extract, which was then partitioned between EtOAc and H2O (1:1) to provide an EtOAc layer. The EtOAc layer was further partitioned by hexanes/MeOH/H2O (4:3:1) to obtain a 75% MeOH(aq) layer (13.3 g). This methanol extract was subjected to a silica gel flash column  to afford subfractions GP-1 to GP-6. GP-3 (5.2 g) was separated by a silica gel open column stepwise-eluted with hexanes/EtOAc (10/1 to 0/1) and EtOAc/MeOH (5/1 to 0/1) to yield 18 subfractions (GP-3-1 to GP-3-18). GP-3-7 (80.5 mg) was subjected to a Sephadex LH-20 open column eluting with CH2Cl2/MeOH (1/1) to obtain four fractions (GP-3-7-1 to GP-3-7-4). Compound 25 (2.1 mg) was purified from GP-3-7-4 (17.8 mg) by semi-preparative RP-HPLC , isocratic, 360 nm). GP-3-10 (342.6 mg) was applied to chromatography on a silica gel open column  to give 6 (32.5 mg) and fractions GP-3-10-2 to GP-3-10-4. GP-3-10-3 (220.9 mg) was further separated on a Sephadex LH-20 column  to afford seven fractions (GP-3-10-3-1 to GP-3-10-3-7). GP-3-10-3-5 (12.4 mg) was isolated by semi-preparative NP-HPLC  to obtain 2 (3.1 mg). Compounds 1 (2.5 mg), 15 (1.1 mg), and 23 (38.1 mg) were purified from GP-3-10-3-6 (108.4 mg) by repeated semi-preparative NP-HPLC . GP-3-11 (1.8 g) was fractionated by a Sephadex LH-20 column eluted with CH2Cl2/MeOH (1/1) to give five fractions (GP-3-11-1 to GP-3-11-5). GP-3-11-3 (167.3 mg) was subjected to semi-preparative RP-HPLC , isocratic, 280 nm) to afford 4 (38.6 mg), 5 (4.0 mg), 7 (12.9 mg), 8 (1.5 mg), and 18 (1.4 mg). GP-3-11-4 (237.2 mg) was isolated by semi-preparative RP-HPLC , isocratic, 280 nm) to yield 21 (1.4 mg) and 24 (43.2 mg). GP-3-11-5 (23.1 mg) was purified by semi-preparative RP-HPLC , isocratic, 280 nm) to obtain 3 (3.5 mg), 16 (1.9 mg), and 17 (2.9 mg). GP-3-12 (315.4 mg) was subjected to Sephadex LH-20 column eluted with CH2Cl2/MeOH (1/1) to obtain five fractions (GP-3-12-1 to GP-3-12-5). Compound 22 (6.2 mg) was purified from GP-3-12-3 (55.8 mg) by semi-preparative RP-HPLC , isocratic, 280 nm). Compound 28 (1.7 mg) was isolated from GP-3-12-4 (84.1 mg) by semi-preparative RP-HPLC , isocratic, 210 and 280 nm). GP-3-13 (218.5 mg) was processed using a Sephadex LH-20 open column eluted with CH2Cl2/MeOH (1/1). GP-3-13-2 (153.3 mg) was fractionated over C18 open column  to give GP-3-13-2-1 to GP-3-13-2-5. Compounds 11 (15.3 mg) and 13 (4.0 mg) were obtained from GP-3-13-2-1 (24.1 mg) by semi-preparative RP-HPLC , isocratic, 254 nm). Compounds 10 (12.7 mg) and 14 (13.5 mg) were isolated from GP-3-13-2-2 (65.2 mg) by repeated semi-preparative RP-HPLC , isocratic, 254 nm). GP-3-13-2-3 (17.5 mg) was purified by semi-preparative RP-HPLC , isocratic, 254 nm) to yield 9 (4.7 mg). GP-3-13-2-4 (13.7 mg) was isolated by semi-preparative RP-HPLC , isocratic, 280 nm) to afford 20 (3.2 mg). GP-3-14 (282.1 mg) was subjected to Sephadex LH-20  and ODS  open columns, respectively. Compound 12 (2.9 mg) was purified from GP-3-14-2-2 (18.8 mg) by semi-preparative RP-HPLC , isocratic, 254 nm). GP-3-15 (364.1 mg) was chromatographed by a Sephadex LH-20 open column eluted with CH2Cl2/MeOH (1/1) to obtain three fractions (GP-3-15-1 to GP-3-15-3). GP-3-15-3 (163.5 mg) was applied to a C18 gel open column  to obtain fractions GP-3-15-3-1 to GP-3-15-3-5. Compounds 26 (18.8 mg) and 27 (44.8 mg) were purified by semi-preparative RP-HPLC , isocratic, 280 nm). GP-3-15-3-3 (18.6 mg) was isolated by semi-preparative RP-HPLC , isocratic, 280 nm) to give 19 (8.8 mg).The dried leaves and twigs of Glyfuran (1): White amorphous powder; c 0.05, MeOH); UV (MeOH) λmax (log ε) 276 (3.23), 226 (3.20) nm; IR (KBr) vmax 3380, 2923, 2853, 1684, 1456, 1259, 1192, 1026 cm−1; 1H NMR and 13C NMR data, see m/z 329.13605 [M + Na]+ .Glyphyllamide (2): White amorphous powder; c 0.05, MeOH); UV (MeOH) λmax (log ε) 285 (2.83), 213 (3.53) nm; IR (KBr) vmax 2925, 2854, 1736, 1671, 1457, 1179 cm−1; 1H NMR and 13C NMR data, see m/z 336.18067 [M + H]+ .Glyphyllazole (3): Brown-yellow solid; UV (MeOH) λmax (log ε) 307 (3.80), 259 (3.96), 237 (2.23), 212 (4.0) nm; IR (KBr) vmax 3142 (OH), 2925, 1626, 1513, 1443, 1296, 1229, 1140, 1068 cm−1; 1H NMR and 13C NMR data, see m/z 280.09468 [M + Na]+ .v/v) horse serum (HS) and 5% (v/v) fetal bovine serum (FBS).Murine enteroendocrine cell line (STC-1) purchased from American Type Culture Collection (ATCC) was maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 15%  with/without VN016 or testing compound preparations for 1 h. At the end of treatment, the supernatant was collected and measured for GLP-1 using an active GLP-1 assay kit (Cisbio). The viability of cells after extract or compound treatment was measured by neutral red assay according to the previous description [Cells were seeded into a 24-well plate at 1.5 × 10cription .According to the manufacturer’s instructions, the measurement of the activity and potential inhibition of DPP-IV, a type II membrane glycoprotein, was performed using the DPP-IV GloTM Protease Assay (Promega).G. pentaphylla has been carried out, resulting in the purification of three new (1−3) and twenty-five known (4–28) compounds. This research led to the isolation of eleven styryl-lactones (4–14) and five alkaloids (15–19), which proved the genus Glycosmis is a rich source of both alkaloid and styryl-lactone. In anti-diabetic evaluation, compounds 4, 17, 24, and 25 displayed dual activities by stimulating GLP-1 secretion while inhibiting DPP-4 (which can rapidly degrade GLP-1) without cytotoxicity in STC-1 cells. Consequently, our pharmacological data supports G. pentaphylla as being a folk medicine with a hypoglycemic effect.As part of an ongoing program to search for bioactive compounds from Vietnam’s medicinal plants, a chemical study on the leaves and twigs of