Helietta parvifolia stems. We identified eight compounds, consisting of two coumarins and six alkaloids. Among these, a new alkaloid, 2-hydroxy-3,6,7-trimethoxyquinoline-4-carbaldehyde (6), was elucidated, along with seven known compounds. The phytotoxicity of purified compounds was evaluated, and chalepin (4) was active against Agrostis stolonifera at 1 mM with 50% inhibition of seed germination and it reduced Lemna pausicotata (duckweed) growth by 50% (IC50) at 168 μM. Additionally, we evaluated the antifungal activity against the fungal plant pathogen Colletotrichum fragariae using a thin-layer chromatography bioautography assay, which revealed that three isolated furoquinoline alkaloids (flindersiamine (3), kokusagenine (7), and maculine (8)) among the isolated compounds had the strongest inhibitory effects on the growth of C. fragariae at all tested concentrations. Our results indicate that these active natural compounds, i.e., (3), (4), (7), and (8), could be scaffolds for the production of more active pesticides with better physicochemical properties.The identification of natural and environmentally friendly pesticides is a key area of interest for the agrochemical industry, with many potentially active compounds being sourced from numerous plant species. In this study, we report the bioassay-guided isolation and identification of phytotoxic and antifungal compounds from the ethyl acetate extract of  Lolium rigidum) in Australia in 1996 posed an unexpected challenge for weed scientists +, calculated for C16H19O4 275.1283. 1H NMR  δ 7.53 , 6.87 , 6.83 , 6.37–6.04 , 5.13 , 5.09 , 3.94 , 3.93 , 1.50 . 13C NMR  δ 160.1 (C-2), 152.0 (C-7), 149.0 (C-9), 146.1 (C-6), 145.5 (C-2′), 137.6 (C-4), 132.1 (C-3), 112.1 (C-10), 111.7 (C-3′), 108.0 (C-5), 99.3 (C-8), 56.3 (OCH3), 56.3 (OCH3), 40.4 (C-1′), 26.0  [2 (pellitorine) as a white solid. High-resolution DART positive m/z 224.2007 [M + H]+, calculated for C14H26NO 224.2014. 1H NMR  δ 7.18 , 6.18 , 5.75 , 5.58 , 3.15 , 2.13 , 1.78 , 1.41 , 1.29 , 0.92 , 0.91 , 0.88 . 13C NMR  δ 166.53 (C-2), 143.33 (C-4), 141.41 (C-6), 128.32 (C-5), 121.87 (C-3), 47.05 (C-1′), 33.05 (C-7), 31.50 (C-9), 28.76 (C-8), 28.62 (C-2′), 22.61 (C-10), 20.26 , 14.14 (C-11) [3 (flindersiamine) as yellow powder. High-resolution DART positive m/z 274.0829 [M + H]+, calculated for C14H12NO5 274.0715. 1H NMR  δ 7.59 , 7.28 , 7.03 , 6.07 , 4.41 , 4.28 . 13C NMR  δ 162.62 (C-2), 156.08 (C-6), 146.74 (C-4), 143.03 (C-9), 138.03 (C-8), 137.74 (C-2′), 135.98 (C-7), 114.99 (C-10), 104.34 (C-1′), 102.93 (C-3), 101.52 (C-3′), 92.42 (C-5), 60.63 (OCH3), 58.96 (OCH3) +, calculated for C19H23O4 315.1596. 1H NMR  δ 7.50 , 7.22 , 6.73 , 6.19 , 5.15–5.05 , 4.74 , 3.22 , 1.49 , 1.28 . 13C NMR  δ 162.23 (C-7), 160.18 (C-2), 154.65 (C-9), 145.62 (C-2′), 138.05 (C-4), 130.90 (C-3), 124.55 (C-6), 123.24 (C-5), 113.15 (C-10), 112.08 (C-3′), 97.16 (C-8), 94.03 (C-7′), 71.71 (C-8′), 40.31 (C-1′), 29.71 (C-6′), 26.12 (2CH3), 24.19 (2CH3) [5 (1-hydroxy-3-methoxy-10-methylacridone) as yellow powder. High-resolution DART positive m/z 256.1063 [M + H]+, calculated for C15H14NO3 256.0973. 1H NMR  δ 14.82 , 8.43 , 7.70 , 7.46 , 7.30–7.27 , 6.31–6.25 , 3.89 , 3.77 . 13C NMR  δ 180.76 (C-4), 166.03 (C-2′), 165.95 (C-4′), 144.70 (C-2), 142.38 (C-9), 134.05 (C-7), 126.73 (C-5), 121.41 (C-10), 121.03 (C-6), 114.49 (C-8), 105.26 (C-3), 94.05 (C-3′), 90.01 (C-1′), 55.58 (OCH3), 34.07 (NCH3) [Fraction 8 was subjected to reversed-phase HPLC preparative chromatography with a RP-C9 (2CH3) . Compoun6  as yellow powder. High-resolution DART positive m/z 264.0892 [M + H]+, calculated for C13H14NO5 264.0872  δ 10.22 , 7.25 , 6.85 , 4.04 , 3.85 , 3.82   δ 189.94 (C-2′), 168.37 (C-2), 164.05 (C-3), 155.53 (C-6), 146.02 (C-7), 137.84 (C-9), 111.23 (C-10), 108.97 (C-4), 105.05 (C-8), 98.12 (C-5), 64.30 (OCH3), 56.36 (OCH3), 56.23 (OCH3) (7 (kokusagenine) as yellow crystals. High-resolution DART positive m/z 260.0992 [M + H]+, calculated for C14H14NO4 260.0922. 1H NMR  δ 7.58 , 7.49 , 7.35 , 7.05 , 4.45 , 4.04 , 4.04 . 13C NMR  δ 163.09 (C-2), 155.58 (C-4), 152.61 (C-7), 147.79 (C-6), 142.56 (C-9), 142.45 (C-2′), 112.96 (C-10), 106.70 (C-8), 104.62 (C-1′), 102.22 (C-3), 100.23 (C-5), 58.87 (OCH3), 56.05 (OCH3), 56.01 (OCH3) [8 (maculine) as yellow crystals. High-resolution DART positive m/z 244.0598 [M + H]+, calculated for C13H10NO4 244.0609. 1H NMR  δ 7.57 , 7.51 , 7.31 , 7.03 , 6.10 , 4.41 . 13C NMR  δ 163.14 (C-2), 155.98 (C-4), 150.76 (C-7), 146.09 (C-6), 143.85 (C-2′), 142.60 (C-9), 114.32 (C-10), 104.51 (C-8), 104.49 (C-1′), 102.50 (C-5), 101.60 (C-3), 98.03 (C-3′), 58.93 (OCH3) [Fraction 14 was chromatographed using reversed-phase preparative HPLC with a gradient mode of methanol (solvent B) in water (solvent A)  to yield Compound 264.0872 . 1H NMR H, OCH3) . 13C NMR3 (OCH3) . CompounLactuca sativa) and bentgrass (Agrostis stolonifera), following the protocol described by Dayan et al. [L. sativa (Iceberg A Crisphead from Burpee Seeds) and A. stolonifera  were first subjected to surface sterilization by immersing them in a 2.5 % sodium hypochlorite for 10 min. Afterward, the seeds were thoroughly rinsed with sterile deionized water and air-dried in a sterile environment.Plant extracts, fractions obtained through silica gel column chromatography, and isolated pure compounds were assessed for their phytotoxic effects on seeds of lettuce  or L. sativa (5 seeds) separately positioned on a filter paper (Whatman no. 1). The test compounds and fractions were dissolved in a mixture of acetone and DI water, ensuring a final acetone concentration of 10%. Subsequently, 200 μL of the test solution was added to each well containing seeds, while the control wells received only 200 μL of acetone and DI water. A 1 mM atrazine  solution served as the positive control. The plate was covered and sealed using Parafilm and placed in a Percival Scientific CU-36L5 incubator, with continuous light conditions at 24 °C and an average photosynthetically active radiation (PAR) of 120 μmol s−1 m−2.In a 24-well multiwell plate , each well was filled with L. sativa and after 10 days for A. stolonifera, using a rating scale ranging from 0 to 5. A rating of 0 indicated no effect , while a rating of 5 indicated no seed germination [The phytotoxic activity was qualitatively assessed visually by comparing seed germination in each well after 7 days for mination . Each exLemna paucicostata cultures were cultivated from a single colony, comprising a mother and two daughter fronds, in a beaker filled with Hoagland’s No. 2 Basal Salt Mixture  at a concentration of 1.6 g/L, supplemented with iron (1 mL of 1000× FeEDTA solution per 1 L of Hoagland media). The pH of the medium was adjusted to 5.5 using 1 N NaOH and then filter-sterilized through a 0.2 μm filter. These L. paucicostata cultures were grown in approximately 100 mL of sterile jars with vented lids in a Percival Scientific CU-36L5 incubator, maintaining continuous light conditions at 24 °C and an average 120 μmol s−1 m−2 PAR. The doubling time for the plants was approximately 24 to 36 h. Nonpyrogenic polystyrene sterile six-well plates  were used for assays. Each well contained 4950 μL of Hoagland’s media and 50 μL of water, solvent, or the compound dissolved in the appropriate solvent, resulting in a final solvent concentration of 1% by volume. Atrazine was used as the positive control. Two three-frond plants of the same age (4 to 5 days old) and approximate size were inoculated into each well. As mentioned earlier, all six-well plates were placed in the Percival incubator, maintaining conditions at 24 °C and a 120 μmol s−1 m−2 average PAR. The LabScanalyzer , an image analyzer was used to measure the frond surface area. The measurements were recorded on day 0 and day 7. The dose-response analysis and calculation of half-maximal inhibitory concentration (IC50) was performed with R 4.2.1 software with support of the drc package. Colletotrichum fragariae (isolate cf63) that infects strawberries and many other vegetables and fruits. Pure compounds (10 μL) were dissolved in methanol and then applied at concentrations of 10, 20, 50, and 100 μM into silica gel TLC plates . After solvent evaporation, these plates were then sprayed with spore suspensions of C. fragariae, adjusted to a final concentration of 3.0 × 105 conidia/mL using potato dextrose broth  and 0.1% Tween-80. The TLC plate was sprayed until it appeared damp with the prepared conidial suspension (approx. 1 mL/plate). The inoculated TLC plates, placed in moisture chamber (30 × 13 × 7.5 cm) boxes to maintain a 100% relative humidity, were incubated in a growth chamber for 4 days, maintained at 27 ± 1 °C, with a 12 h photoperiod under photon flux conditions of 60 ± 5 μmol s·m−2 s−1. To determine the sensitivity of each tested compound against fungal species, inhibitory zone areas were compared. Bioautography experiments were conducted in triplicate, including both dose-response and non-dose-response assessments. A technical grade fungicide standard, captan , served as the positive control.The assessment of antifungal activity against fungal plant pathogens followed a published TLC bioautography method . We sele


A rapid and sensitive method that combined ultra-high-performance liquid chromatography combined with Fourier transform ion cyclotron resonance mass spectrometry (UHPLC-FT-ICR-MS) was used to identify the chemical constituents in Lanqin oral liquid. On the basis of UHPLC-FT-ICR-MS analysis, systematic characterization of the chemical profile of Lanqin oral liquid was carried out, and a total of 441 compounds were identified or tentatively characterized including alkaloids, flavonoids, terpenoids, organic acids, phenylpropanoids, and other types. The results provide a reference for improving quality control, contribute to establishing higher quality standards, provide a scientific basis for further research on the pharmacodynamic material basis, and help illustrate the relationship between the complicated constituents and therapeutic effects of Lanqin oral liquid. Lanqin oral liquid is the classic traditional Chinese medicine antipyretic prescription. Lanqin oral liquid has the functions of clearing heat, detoxification, liyan, and detumescence effects. The recipe is composed of five valuable herbal medicines, including banlangen-Isatidis Radix, guangqin-Scutellariae Radix, zhizi-Gardeniae Fructus, pangdahai-Sterculiae Lychnophorae Semen, and huangbai-Phellodendri Chinensis Cortex, and has been widely employed in treatments as an antipyretic and anti-inflammatory preparation. Radix Scutellariae possesses anti-inflammatory and antipyretic activities ,2. IsatiTraditional Chinese medicine (TCM) prescriptions collectively exert therapeutic effects and display a synergistic action via the multicomponents-to-multitargets mechanism guided by the compatibility rules of TCM theory. Given the highly complicated and diverse components in these systems, it is inevitably difficult to identify compounds because these ingredients have rich structural types, wide polarity ranges, and significant differences in content; this always makes the separation and identification challenging and, accordingly, creates difficulties in quality control and the elucidation of pharmacodynamic substances, which hinders modernization. Hence, to ensure the safety and effectiveness of TCM, it is unquestionable that systematic and comprehensive chemical characterization using analytical instruments with high resolution, sensitivity, and accuracy are needed. Recently, the chemical compositions in TCM are increasingly being studied systematically through the use of high-performance liquid chromatography (UHPLC) combined with high-resolution mass spectrometry (HRMS) ,8. The oIn this study, a rapid, sensitive, and systematic chemical identification and characterization method with UHPLC-FT-ICR-MS was applied to identify and characterize the chemical components of Lanqin oral liquid. A total of 441 compounds including alkaloids, flavonoids, terpenoids, organic acids, phenylpropanoids, and other types were detected, and their structures were tentatively identified based on the retention time, accurate molecular weight measurement, and characteristic mass fragment information with correlative reference standards or reference data. Results of this investigation lay a foundation for the quality control of Lanqin oral liquid; provide a reference and promising experimental data support for further study of the relationship between the effective substances and their pharmacology; illustrate the relationship between the complicated constituents and the therapeutic effects; and, thus, promote the development of Lanqin oral liquid.As shown in Flavonoid compounds are natural compounds containing the basic skeleton C6-C3-C6. A total of 134 flavonoids were tentatively identified in Lanqin oral liquid. The chemical structures of major flavonoid compounds are shown in − of compound F12 was at m/z 461.07229 and was observed at 8.22 min. The molecular formula was C21H18O12, as predicted by the precursor ion. The fragment ion at m/z 285.04039 was observed, indicating the elimination of a glucuronosyl unit in its structure. The compound was identified as scutellarin − peak at m/z 521.12952. The characteristic fragments were m/z 359.07727, m/z 344.05343, and m/z 329.02992, corresponding to an excimer ion [M + H]+ successively removing a fragment ion of glucose, one and two methyl groups. It was tentatively characterized as 5,2′,6′-trihydroxy-6,7,8-trimethoxyflavone-2′-O-glucoside + successively removing a fragment ion of glucose, one and two methyl groups. It was tentatively characterized as 5,7-dihydroxy-6,8-dimethoxyflavone-7-O-glucuronide [O-glucuronide are shown in O-glycoside.The precursor ion [M-H]+ of compound F26 and compound F46 were at m/z 417.11859 and m/z 417.11836 and were observed at 9.40 and 11.24 min; the molecular formula was C21H20O9, as predicted by the precursor ion. In the MS/MS spectrum, the characteristic fragment ions of compound F26 at m/z 399.10614, m/z 381.09575, m/z 363.08537, m/z 351.08537, m/z 339.08545, m/z 335.09070, m/z 327.08588, m/z 321.07497, m/z 307.09563, m/z 297.07507, m/z 279.06454, and m/z 267.06457 were detected, which corresponded to [M + H-H2O]+, [M + H-2H2O]+, [M + H-3H2O]+, [M + H-CH2O-2H2O]+, [M + H-C2H4O2-H2O]+, [M + H-3H2O-CO]+, [M + H-C3H6O3]+, [M + H-C2H4O2-2H2O]+, [M + H-3H2O-2CO]+, [M + H-C4H8O4]+, [M + H-C4H8O4-H2O]+, and [M + H-C5H10O5]+, respectively. The characteristic fragment ions of compound F46 at m/z 381.09610, m/z 363.08528, m/z 351.08569, m/z 345.07486, m/z 335.09070, m/z 333.07491, m/z 321.07497, m/z 307.09570, m/z 297.07508, m/z 293.08047, m/z 279.06454, and m/z 267.06455 were detected, which corresponded to [M + H-2H2O]+, [M + H-3H2O]+, [M + H-CH2O-2H2O]+, [M + H-4H2O]+, [M + H-3H2O-CO]+, [M + H-CH2O-3H2O]+, [M + H-C2H4O2-2H2O]+, [M + H-3H2O-2CO]+, [M + H-C4H8O4]+, [M+H-C2H4O2-2H2O-CO]+, [M + H-C4H8O4-H2O]+, and [M + H-C5H10O5]+, respectively. Compound F26 and compound F46 were tentatively characterized as chrysin 8-C-glucoside and chrysin 6-C-glucoside lucoside . The ion− at m/z 343.08212 and m/z 343.08199 in the negative ion mode, and the molecular formula was predicted as C18H15O7. In the MS/MS experiments of compound F118, fragment ions at m/z 328.05879, m/z 313.03510, m/z 298.01173, m/z 285.04033, and m/z 270.01684 corresponded to [M-H-CH3]−, [M-H-2CH3]−, [M-H-3CH3]−, [M-H-2CH3-CO]−, and [M-H-3CH3-CO]−. In the MS/MS experiments of compound F130, fragment ions at m/z 328.05887, m/z 313.03516, m/z 298.01176, m/z 295.02482, and m/z 270.01718 corresponded to [M-H-CH3]−, [M-H-2CH3]−, [M-H-3CH3]−, [M-H-2CH3-H2O]−, and [M-H-3CH3-CO]−. Compounds F118 and F130 were tentatively characterized as rivularin and 5,2′-dihydroxy-6,7,8-trimethoxyflavone [Compounds F118 and F130 with a retention time of 20.12 min and 22.60 min generated the precursor ion of [M-H]N-methylhigenamine 7-glucopyranoside, protoberberines such as palmatine, and other types such as indirubin. The chemical structures of the major alkaloid compounds are shown in Alkaloids are a class of basic organic compounds containing nitrogen that exist in nature. A total of 88 alkaloids were tentatively identified in Lanqin oral liquid, mainly containing tetrahydroperoberberines such as phellodendrine, aporphines such as magnoflorine, benzylisoquinoline such as m/z 342.16909 was observed at 6.05 min. We speculated that the molecular formula was C20H24NO4. In the MS/MS spectrum, the characteristic fragment ion produced was at m/z 192.10132, corresponding to fragment ion [M-C9H10O2]+. The compound was tentatively characterized as phellodendrine +, [M-NH(CH3)2-CH3]+, [M-NH(CH3)2-CH3OH]+, and [M-NH(CH3)2-CH3OH-CO]+. The compound was tentatively characterized as magnoflorine + and [M + H-glucose-NH2CH3]+. The compound was tentatively characterized as N-methylhigenamine 7-glucopyranoside +, [M-CH2O]+, and [M-CH4-CO]+. The compound was chemically defined as palmatine +, [M+H-CO]+, and [M + H-CO2]+. The compound was chemically defined as indirubin. The precursor ion [M + H]+ of compound A88 was at m/z 263.08146 and was observed at 21.69 min. The MS spectra of the two compounds were highly similar to each other. The compound was chemically defined as indigo [The precursor ion [M + H]s indigo .Terpenoid compounds are compounds and derivatives derived from methylglutaric acid, and their basic structural unit is a prenyl unit (C5 unit). A total of 108 terpenoids were tentatively identified in Lanqin oral liquid, mainly contain iridoids such as geniposidic acid, monoterpenoids such as jasminoside B, diterpenoids such as crocin I, and triterpenoids such as obacunone. The chemical structures of major terpenoid compounds are shown in − of compound T5 was at m/z 373.11374 and was observed at 2.34 min, indicating the formation of C16H22O10. The characteristic fragments were m/z 211.06098, m/z 193.05035, m/z 167.07122, m/z 149.06069, and m/z 123.04507, corresponding to fragment ions [M-H-Glc]−, [M-H-Glc-H2O]−, [M-H-Glc-CO2]−, [M-H-Glc-H2O-CO2]−, and [M-H-Glc-H2O-CO2-C2H2]−. The compound was tentatively characterized as geniposidic acid − were observed in the MS/MS spectrum. The compound was tentatively characterized as genipin − peak at m/z 595.18640 and the [M-H]− peak at m/z 549.18166. Daughter ions at m/z 225.07659 [M-H-2C6H10O5]−, m/z 207.06613 [M-H-2C6H10O5-H2O]−, m/z 147.04499 [M-H-2C6H10O5-C2H6O3]−, m/z 123.04505 [M-H-2C6H10O5-H2O-C4H4O2]−, and m/z 101.02435 [M-H-2C6H10O5-C7H8O2]− were observed in the MS/MS spectrum. Compound T22 was tentatively characterized as genipin-1-O-β-D-gentiobioside −, m/z 367.10302 [M-H-C15H20O8]−, m/z 349.09286 [M-H-C15H20O8-H2O]−, m/z 307.08244 [M-H-C11H14O5-C6H10O5]−, m/z 265.07151 [M-H-C19H26O11]−, m/z 235.06085 [M-H-C20H28O12]−, m/z 207.06596 [M-H-C21H28O13]−, m/z 163.03996 [M-H-C11H12O4-2C6H10O5]−, m/z 145.02945 [M-H-C11H12O4-2C6H10O5-H2O]−, and m/z 123.04507 [M-H-C9H6O2-2C6H10O5-H2O-C4H4O2]− were observed in the MS/MS spectrum. The compound was tentatively characterized as 6″-O-trans-p-coumaroylgenipin gentiobioside [O-trans-p-coumaroylgenipin gentiobioside are shown in The precursor ion [M-H]dic acid . The pre genipin ,19,20. C− and [M-H]− of compound T18 were at m/z 391.16044 and m/z 345.15548 and were observed at 4.96 min, indicating the formation of C16H26O8. The characteristic fragment ions at m/z 179.05598 [M-H-C10H14O2]−, m/z 165.09202 [M-H-C6H12O6]−, and m/z 161.04543 [M-H-C10H16O3]− were observed in the MS/MS spectrum. The compound was tentatively characterized as jasminoside B [The precursor ions [M + COOH]598 M-H-CH14O2−, m− of compound T60 was at m/z 975.37046 observed at 10.34 min, indicating the formation of C44H63O24. Daughter ions at m/z 651.26536 [M-H-2glucose]− were observed in the MS/MS spectrum. The compound was tentatively characterized as crocin I [The precursor ion [M-H]+ of compound T96 was at m/z 455.20596 and was observed at 24.02 min, indicating the formation of C26H30O7. Daughter ions at m/z 437.19518 [M + H-H2O]+, m/z 419.18444 [M + H-2H2O]+, m/z 409.20033 [M + H-CH2O2]+, m/z 391.1899 [M + H-CH2O2-H2O]+, 359.12727 [M + H-C3H12O3]+, 349.14242 [M + H-C4H10O3]+, 331.13297 [M + H-C4H12O4]+, and 315.13702 [M + H-C4H12O5]+ were observed in the MS/MS spectrum. The compound was tentatively characterized as obacunone [The precursor ion [M + H]bacunone . The ext− at m/z 191.05582 in negative ion mode, and the molecular formula was predicted as C7H12O6. In the MS/MS spectrum, the characteristic fragment ions produced were at m/z 173.04533 and m/z 127.03983, corresponding to fragment ions [M-H-H2O]− and [M-H-H2O-H2CO2]−. It was tentatively characterized as quinic acid −. It was identified as caffeic acid −, m/z 364.07967 [M-H-C10H11O4]−, m/z 353.08759 [M-H-C11H10O4]−, m/z 335.07676 [M-H-C11H12O5]−, m/z 223.06100 [M-H-C16H16O8]−, m/z 205.05048 [M-H-C16H18O9]−, m/z 191.05598 [M-H-C20H16O7]−, m/z 179.03487 [M-H-C18H20O9]−, m/z 173.04543 [M-H-C20H18O8]−, m/z 164.04778 [M-H-C18H19O10]−, m/z 161.02423 [M-H-C18H22O10]−, m/z 155.03476 [M-H-C20H20O9]−, m/z 149.02429 [M-H-C19H22O10]−, m/z 137.02427 [M-H-C20H22O10]−, and m/z 135.04506 [M-H-C19H20O11]− were observed in the MS/MS spectrum. Fragment ions at m/z 379.10338 and m/z 364.07967 corresponded to successive losses of caffeoyl and methyl groups. Fragment ions at m/z 353.08759 and m/z 335.07680 corresponded to successive losses of a sinapoyl group and H2O. Fragment ions at m/z 223.06100, m/z 205.05048, and m/z 164.04778 corresponded to successive losses of caffeoyl + quinic acid, H2O, and CO2, and also can be regarded as [M-H]−, [M-H-H2O]−, and [M-H-H2O-CO2]− ions of sinapinic acid. Fragment ions at m/z 191.05598, m/z 173.04543, m/z 155.03476, and m/z 137.02427 corresponded to successive losses of a caffeoyl + sinapoyl group, H2O, H2O, and CO2. Fragment ions at m/z 179.03487, m/z 161.02423, and m/z 135.04506 corresponded to losses of sinapoyl + quinic acid, H2O, and CO2, and also can be regarded as [M-H]−, [M-H-H2O]−, and [M-H-H2O-CO2]− ions of caffeic acid. The compound was tentatively characterized as 3-O-caffeoyl-4-O-sinapoylquinic acid +. The compound was tentatively characterized as esculin − and m/z 179.07122 [M-H-glucose]− were observed. The compound was tentatively characterized as coniferin −. The compound was tentatively characterized as (−)-secoisolariciresinol 4-O-β-D-glucopyranoside [O-β-D-glucopyranoside are shown in Phenylpropanoid compounds are natural compounds with one or more C6-C3 as the basic skeleton. A total of 29 phenylpropanoids were tentatively identified in Lanqin oral liquid. The chemical structures of major phenylpropanoid compounds are shown in  esculin . The preoniferin . The preAccording to the above analysis results, chemical identification of the compounds was performed based on an integrated consideration of accurate MS data, characteristic fragment ions, and retention time using a reference substance or the reference literature. A total of 441 compounds in Lanqin oral liquid were identified in this study. In the previous literature, a total of 170 compounds were identified or putatively characterized by ultra-high-performance liquid chromatography coupled with ion mobility-quadrupole time-of-flight mass spectrometry , and 175O-β-D-gentiobioside, geniposide, obaculactone, obacunone, and chlorogenic acid, were determined in Lanqin oral liquid [O-glucoside shows antioxidant activity [O-β-D-glucopyranoside shows anti-inflammatory and antiviral effects [Compounds including flavonoids, alkaloids, terpenoids, organic acids, and phenylpropanoids were identified in Lanqin oral liquid. The content of compounds, such as baicalin, wogonoside, baicalein, wogonin, phellodendrine, magnoflorine, berberine, palmatine, indirubin, genipin-1-l liquid . Based ol liquid ,30, and l liquid . Alkaloil liquid ,33, and l liquid . Terpenol liquid , and genl liquid ,37,38. Ol liquid . Organicl liquid ,41. Phenl liquid ; pinoresactivity ; and lar effects . It can Lanqin oral liquid (lot no. 19122821) was purchased from Yangtze River Pharmaceutical Group . Reference compounds (purity > 98%) of chlorogenic acid, caffeic acid, scutellarin, baicalin, palmatine, wogonoside, apigenin, baicalein, wogonin, indirubin, and indigo were purchased from Chengdu Must Bio-Technology Co., Ltd. . Acetonitrile and formic acid of HPLC grade were obtained from Fisher Scientific . Purified water was purchased from Hangzhou Wahaha Corporation . All other chemical reagents used in this study were of analytical grade.1 mL of Lanqin oral liquid was accurately measured and diluted with 1 mL of water. The mixture was vortexed for 3 min, and then the solution was prepared by diluting 5-fold with ultrapure water. The mixture was vortexed for 3 min, then centrifuged at 12,000 rpm for 10 min. The supernatant was collected and filtered through a 0.22 µm filter membrane before UHPLC-FT-ICR-MS/analysis.An Agilent 1260 UHPLC system  was used for the analysis. Chromatographic separation was performed on an ACQUITY UPLC HSS T3 column  with an ACQUITY UPLC HSS T3 VanGuard Pre-Column  kept at 35 °C. With a flow rate of 0.30 mL/min, the mobile phase was a gradient system consisting of water infused with 0.1% formic acid (A) and acetonitrile (B), with the gradient as follows: 0–2 min, 8–20% B; 2–20 min, 20–50% B; 20–26 min, 50–98% B; 26–33 min, an isocratic elution of B. The injection volume was set at 10 µL.2), respectively. Full-scan MS data were acquired over an m/z range of 100–1200 Da. And the collision energy were set from 10 to 40 eV for fragmentation information. Bruker Compass Hystar  and Fourier Transform Mass Spectrometer Control  were used for instrument control and data acquisition. Data Analysis Software  was used for data analysis.Mass spectrometric detection was carried out on a Solarix 7.0T FT-ICR mass spectrometer  coupled with an electrospray ionization (ESI) interface. The full-scan MS data acquired in both positive and negative modes scanned from 100 to 1200 Da. The capillary voltage was 4500 V, the endplate offset was 500 V, dry gas flow was 8 L/min, dry gas temperature was 200 °C, and nebulizer gas pressure was 4 bar. The collision gas and nebulizing gas were high-purity argon (Ar) and high-purity nitrogen (NIn our study, a rapid, sensitive, and specific analytical method using UHPLC-FT-ICR-MS/MS was successfully applied to separate and identify chemical constituents of Lanqin oral liquid. A total of 441 compounds were identified or tentatively characterized in Lanqin oral liquid, revealing that flavonoids, alkaloids, terpenoids, organic acids, and phenylpropanoids were the main components. The applied UHPLC-FT-ICR-MS method with the advantages of good separation, high sensitivity, high resolution, and high accuracy for the detection of chemical constituents in Lanqin oral liquid provided comprehensive chemical information on Lanqin oral liquid and could aid in quality control and an understanding of the pharmacodynamic material basis and thus promote the development of Lanqin oral liquid.


The new compounds were designed based on the oxadiazole-linked aryl core of tubulin inhibitors of IMC-038525 and IMC-094332, prepared in five steps and further characterized via spectral analyses. The anticancer activity of the compounds was assessed against several cancer cell lines belonging to nine different panels as per National Cancer Institute (NCI US) protocol. 4-Chloro-2--1,3,4-oxadiazol-2-yl)amino)phenol (6h) demonstrated significant anticancer activity against SNB-19 (PGI = 65.12), NCI-H460 (PGI = 55.61), and SNB-75 (PGI = 54.68) at 10 µM. The compounds were subjected to molecular docking studies against the active site of the tubulin–combretastatin A4 complex (PDB ID: 5LYJ); they displayed efficient binding and ligand 4h  lay within the hydrophobic cavity surrounded by important residues Leu252, Ala250, Leu248, Leu242, Cys241, Val238, Ile318, Ala317, and Ala316. Furthermore, the antibacterial activity of some of the compounds was found to be promising. 4-Chloro-2-((5-(4-nitrophenyl)-1,3,4-oxadiazol-2-yl)amino)phenol (6c) displayed the most promising antibacterial activity against both Gram-negative as well as Gram-positive bacteria with MICs of 8 µg/mL and a zone of inhibition ranging from 17.0 ± 0.40 to 17.0 ± 0.15 mm at 200 µg/mL; however, the standard drug ciprofloxacin exhibited antibacterial activity with MIC values of 4 µg/mL.We report herein the synthesis, docking studies and biological evaluation of a series of new 4-chloro-2-amino)phenol analogues ( A), furamizole  (B), tidazosin (α1-adrenergic receptor antagonist) (C), fenadiazole (hypnotic) (D), zibotentan (anticancer) (E), setileuton  (F), nesapidil  (G), and AZD1979 (melanin concentration receptor 1 antagonist) (H) contain a 1,3,4-oxodiazole ring in their structure , IMC-094332 (tubulin inhibitor), and FABT , 3.9 ± 1.1 (SW707) and 4.2 ± 1.2 (T47D) µg/mL, respectively) was used in the design strategy for the newer oxadiazole analogues shown in 6a-h). Microtubules are highly dynamic structures made up of α- and β-tubulin heterodimers that are indispensable for cell division (mitosis) and growth + was observed at 306.01, while the isotopic peak [M + 3]+ was observed at 308.00. The HPLC chromatogram of one of the compounds (6h) was recorded at λ210 nm and a retention time of 4.19 min demonstrated a purity level of >99%  ,35. In ts (6a-h) . The rea6b, 6e, and 6g showed maximum sensitivity against PC-3 (PGI = 14.13), NCI-H460 (PGI = 19.55), and NCI-H522 (PGI = 11.4), respectively, while compound 6a was found to be sensitive against NCI-H522 (PGI = 23.32). Compound 6d displayed maximum sensitivity against breast cancer cell line, MCF7 (PGI = 24.79), and MDA-MB-468 (PGI = 26.01), while compound 6f demonstrated maximum sensitivity against UACC-62 (PGI = 21.25), NCI-H522 (PGI = 20.32), and HCT-116 (PGI = 20.15). Compound 6h showed maximum anticancer activity among the series and was found to be sensitive against SNB-19 (PGI = 65.12), NCI-H460 (PGI = 55.61), and SNB-75 (PGI = 54.68). Compound 6h demonstrated <50 to >40 PGI against eight cancer cell lines including NCI/ADR-RES (PGI = 48.28), NCI-H226 (PGI = 42.59), HOP-62 (PGI = 40.28), SF-295 (PGI = 45.26), SF-539 (PGI = 43.79), OVCAR-4 (PGI = 40.03), MDA-MB-231 (PGI = 42.58), and HS 578T (PGI = 47.22), while it demonstrated <40 to >20 PGI against nine cancer cell lines including HCT-116 (PGI = 35.59), H322M (PGI = 33.58), A459 (PGI = 34.85), SF-268 (PGI = 29.96), NCI- ACNH (PGI = 27.99), U251 (PGI = 26.04), SN 12C (PGI = 23.24), NCI-H23 (PGI = 20.82), and 786-O (PGI = 20.42) [6h exhibited far better anticancer activity against non-small cancer, colon, CNS, melanoma, ovarian, renal, and breast cancer cell lines. The data for imatinib were retrieved from the NCI website with NSC Code 759854 [6h) substitution of the phenyl ring exhibited promising anticancer activity followed by 2-hydroxy (6f) substitution. The overall structure–activity relationship (SAR) can be represented as 3,4,5-trimethoxy > 2-hydroxy > 4-nitro > 4-hydroxy-3-methoxy substitution in the phenyl ring directly attached to the oxadiazole ring.All the compounds were designed based on the pharmacophore of tubulin inhibitors, and their anticancer activity was accessed using the National Cancer Institute protocol (NCI US) reported elsewhere ,38,39,40= 20.42) . The avee 759854 . The str6a-h) ranged from −7.617 to −8.389 kcal/mol and they displayed two types of interactions, including H-bonding and halogen bonding. Ligand 6d displayed maximum binding affinity with a docking score of −8.389 kcal/mol and exhibited a halogen bond interaction with the residue Ala317, while ligand 6e displayed a docking score of −7.617 kcal/mol with no significant interaction. Ligands 6d and 6f displayed a similar type of interaction with the residue Ala317 through a halogen bond interaction. The 2D and 3D interactions of ligands 6d and 6f are shown in 6a and 6b displayed a H-bond interaction with the residue Asn258, while they showed a halogen bond interaction with the residues Ala317 and Asn349, respectively. Ligand 6h displayed no significant electrostatic interaction; however, the trimethoxy phenyl ring lies with the hydrophobic cavity of containing residues Leu252, Ala250, Leu248, Leu242, Cys241, Val238, Ile318, Ala317, and Ala316, as shown in Some of the oxadiazole analogues displayed good anticancer activity against some of the cancer cell lines and the compounds were also designed based on the pharmacophore of tubulin inhibitors, hence a molecular docking study of tubulin was explored against the active site of tubulin–combretastatin A4 (PDB ID: 5LYJ). The molecular docking scores and interactions are given in 6a-h against Gram-negative  as well as Gram-positive  bacterial strains was assessed via a disc diffusion method and broth dilution method as per the reported protocol [6d, 6f, 6g, and 6h displayed less antibacterial activity with zones of inhibition ranging from 4.8 ± 0.75 to 6.7 ± 0.20 mm at 200 µg/mL drug concentrations and exhibited MICs ranging from 128 to 512 µg/mL against various bacterial strains. Compounds 6a and 6d displayed moderate antibacterial activity with MICs ranging between 16 and 64 µg/mL and zones of inhibition ranging between 13.0 ± 0.21 and 9.8 ± 0.25 mm at 200 µg/mL. Compound 6e displayed good antibacterial activity against S. aureus, E. coli, and P. aeruginosa with MICs of 8 µg/mL, while it inhibited B. subtilis with an MIC of 8 µg/mL and a zone of inhibition ranging between 14.0 ± 0.45 and 16.0 ± 0.26 mm at 200 µg/mL. Compound 6c displayed the most promising antibacterial activity against both the Gram-negative as well as Gram-positive bacteria with MICs of 8 µg/mL and zones of inhibition ranging from 17.0 ± 0.40 to 17.0 ± 0.15 mm at 200 µg/mL; however, the standard drug ciprofloxacin exhibited MICs of 8 µg/mL. The antibacterial activity in terms of zones of inhibition and MICs is given in 6c and 6e are given in 6c and 6e) that had the maximum docking score  demonstrated promising antibacterial activity.The antibacterial activity of compounds protocol ,42,43. C6h demonstrated significant anticancer activity against SNB-19 (CNS cancer), NCI-H460 , and SNB-75 (CNS cancer) with PGIs of 65.12, 55.61, and 54.68 percent, respectively at 10 µM. Compound 6h also displayed efficient binding against the tubulin–combretastatin A4 binding site of the D-chain with a docking score of −8.030 kcal/mol and a trimethoxy phenyl ring lying within the hydrophobic cavity surrounded by important residues Leu252, Ala250, Leu248, Leu242, Cys241, Val238, Ile318, Ala317, and Ala316. The antibacterial activities of the oxadiazoles were also assessed against Gram-negative (E. coli and P. aeruginosa) as well as Gram-positive (S. aureus and B. subtilis) bacterial strains via a disc diffusion method and broth dilution method as per the reported protocol. Compounds 6c and 6e demonstrated good bacterial inhibition against both the Gram-positive as well as Gram-negative bacterial strains. A molecular docking study was conducted for the title compounds (6a-h) against DNA gyrase, one of the attractive molecular targets for many antibacterial drugs. All the compounds exhibited docking scores ranging between −5.883 and −6.424 kcal/mol. The binding affinities of compounds 6c  and 6e  were found to be the highest among the series, and these compounds also possessed promising antibacterial activity.The 1,3,4-oxadiazoles were designed using the pharmacophore found in some tubulin inhibitors (IMC-094322 and IMC-038525) and the anticancer agent FTAB. The current research aimed to create scaffolds with the basic pharmacophore of tubulin inhibitors (IMC-094322 and IMC-038525) and a low-molecular-weight small compound. Molecular docking studies were performed for the tubulin–combretastatin A4 binding site of the D-chain (PDB ID: 5LYJ). We discovered that the benzene ring substitution produced higher docking scores than IMC-094322 and IMC-038525; hence, we performed modifications in the benzene ring rather than the dioxane ring of IMC-094322 and IMC-038525. The oxadiazole analogues were prepared in satisfactory yields and characterized using spectroscopic techniques followed by their anticancer evaluation at 10 µM against nine panel of 56 cancer cell lines as per the NCI US protocol. Some of the compounds displayed promising anticancer activity against a few cancer cell lines. Compound 2SO4 and 15 mL of concentrated HNO3 was mixed in a 250 mL round-bottom flask with a few smaller pieces of porcelain. 4-Chlorophenol  was added in portions of 2–3 g to the acidic mixture, shaking the reaction mixture after each addition. The temperature was controlled to no more than 55 °C by immersing the flask into the cold-water bath. The reaction mixture was then heated to 45 °C for 45 min with continuous shaking on magnetic stirrer [2). Colour, light yellow; yield, 82%; Mp 86–88 °C.An amount of 21 mL of concentrated H stirrer . The con2)  was dissolved in concentrated HCl (50 mL) and 45 g granulated tin was added, and the reaction mixture was left to heat up in a boiling water bath (100 °C) for 60 min [3). Colour, brown; yield, 78%; Mp 142–144 °C.4-Chloro-2-nitrophenol (r 60 min . The rea3)  was dissolved in 20 mL glacial acetic acid with heating and 40 mL hot water was added and 40 mL of NaCNO  was added with continuous stirring at room temperature (25 °C) for 30 min. It was then allowed to stand for another 30 min and then dipped into cold-water bath to obtain precipitate, which was then filtered, dried, and re-crystallized with hot water to obtain light brown coloured precipitate of 1-(5-chloro-2-hydroxyphenyl)urea (4) [2-Amino-4-chlorophenol (urea (4) ,35. Colo4)  was refluxed at 80 °C with hydrazine hydrate  in absolute ethanol for 24 h to obtain N-(5-chloro-2-hydroxyphenyl)hydrazinecarboxamide (5) [1-(5-chloro-2-hydroxyphenyl)urea (mide (5) ,35. ColoN-(5-chloro-2-hydroxyphenyl)hydrazinecarboxamide (5)  and aromatic aldehyde (1 mmol) was dissolved in 5 mL ethanol, then 5 mL water was added and the reaction mixture was allowed to reflux at 100 °C for 8–10 h with an addition of 10% mol NaHSO3 (5 mL). The reaction mixture was then concentrated and poured into the crushed ice, filtered, dried, and recrystallized with ethanol to obtain the final product, 4-chloro-2-amino)phenol analogues (6a-h) [An equimolar mixture of s (6a-h) .4-Chloro-2-((5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)amino)phenol (6a): yield: 78%; Mp 102–104 °C; 1H NMR : δ ppm: 6.85 , 7.01 , 7.13–7.31 , 7.70–7.75 , 7.89 , 8.76 , 11.05 ; 13C NMR : δ ppm: 152.60, 148.66, 148.11, 144.54, 141.39, 128.64, 125.66, 122.81, 122.41, 117.17, 115.51, 108.54; Anal. Calc. for C14H9ClFN3O2: C, 55.01; H, 2.97; N, 13.75, found: C, 54.98; H, 2.99; N, 13.70%. ESI-MS m/z = 306.01 (M + 1)+, 308.00 (M + 3)+.4-Chloro-2-((5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)amino)phenol (6b): yield: 72%; Mp 154–156 °C; 1H NMR : δ ppm: 6.91 , 7.03 , 7.40 , 7.57 , 8.11 , 8.75 , 11.00 ; 13C NMR : δ ppm: 152.59, 148.53, 143.51, 135.69, 134.52, 129.97, 128.99, 127.79, 124.49, 124.21, 118.19, 116.89; Anal. Calc. for C14H9Cl2N3O2: C, 52.20; H, 2.82; N, 13.04, found: C, 52.15; H, 2.80; N, 13.01%. ESI-MS m/z = 322.14 (M + 1)+, 324.12 (M + 3)+.4-Chloro-2-((5-(4-nitrophenyl)-1,3,4-oxadiaol-2-yl)amino)phenol (6c): yield: 83%; Mp 196–198 °C; 1H NMR : δ ppm: 6.86 , 6.91 , 7.92 , 8.08 , 8.27 , 8.76 , 11.37 ; 13C NMR : δ ppm: 152.12, 147.57, 145.01, 140.47, 139.02, 128.04, 127.37, 124.12, 122.62, 122.06, 118.04, 115.63; Anal. Calc. for C14H9ClFN4O4: C, 50.54; H, 2.73; N, 16.84, found: C, 50.49; H, 2.75; N, 16.81%. ESI-MS m/z = 333.23 (M + 1)+, 335.25 (M + 3)+.4-Chloro-2-((5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)amino)phenol (6d): yield: 81%; Mp 162–164 °C; 1H NMR : δ ppm: 3.83 , 6.72 , 6.99 , 7.13 , 7.32 , 8.09 , 9.81 , 10.42 ; 13C NMR : 152.65, 148.52, 147.66, 142.57, 135.39, 127.24, 124.45, 118.13, 116.99, 115.97, 114.83, 56.18; Anal. Calc. for C15H12ClN3O3: C, 56.70; H, 3.81; N, 13.23, found: C, 56.67; H, 3.79; N, 13.21%. ESI-MS m/z = 318.11 (M + 1)+, 320.10 (M + 3)+.4-Chloro-2-((5-(2-chlorophenyl)-1,3,4-oxadiazol-2-yl)amino)phenol (6e): yield: 71%; Mp 182–184 °C; 1H NMR : δ ppm: 6.84 , 6.86 , 7.41–7.45 , 7.51–7.54 , 7.91–7.94 , 8.10 , 8.78 , 11.26 ; 13C NMR : 152.63, 148.59, 142.51, 136.92, 132.22, 130.13, 129.31, 128.92, 127.71, 127.34, 124.41, 118.11, 116.91; Anal. Calc. for C14H9Cl2N3O2: C, 52.20; H, 2.82; N, 13.04, found: C, 52.17; H, 2.80; N, 13.00%. ESI-MS m/z = 322.13 (M + 1)+, 324.19 (M + 3)+.4-Chloro-2-((5-(2-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)amino)phenol (6f): yield: 82%; Mp 168–170 °C; 1H NMR : δ ppm: 6.85 , 6.93 , 7.14–7.69 , 8.14 , 9.00 , 10.25 , 10.97 ; 13C NMR : 152.63, 148.95, 147.14, 142.51, 135.61, 130.11, 127.72, 126.30, 124.42, 121.81, 118.13, 117.82, 116.91, 108.11; Anal. Calc. for C14H10ClN3O3: C, 55.37; H, 3.32; N, 13.84, found: C, 55.34; H, 3.30; N, 13.81%. ESI-MS m/z = 304.23 (M + 1)+, 306.23 (M + 3)+.4-Chloro-2-((5-(4-hydroxy-3-methoxyphenyl)-1,3,4-oxadiazol-2-yl)amino)phenol (6g): yield: 88%; Mp 112–114 °C; 1H NMR : δ ppm: 3.83 , 6.82 , 6.85 , 7.31 , 7.86 , 8.15 , 9.80 , 10.48 , 10.88 ; 13C NMR : 152.59, 148.65, 148.10, 144.53, 141.39, 128.64, 125.67, 122.80, 121.40, 121.23, 117.17, 115.51, 115.47, 108.55, 55.40; Anal. Calc. for C15H12ClN3O4: C, 53.99; H, 3.62; N, 12.59, found: C, 53.87; H, 3.60; N, 12.57%. ESI-MS m/z = 334.09 (M + 1)+, 336.06 (M + 3)+.4-Chloro-2--1,3,4-oxadiazol-2-yl)amino)phenol (6h): yield: 89%; Mp 180–182 °C; 1H NMR : δ ppm: 3.69 , 3.83 , 6.87 , 7.03 , 7.47 , 7.88 , 8.94 , 11.10 ; 13C NMR : 153.20, 152.42, 144.54, 138.79, 129.72, 128.48, 122.72, 121.46, 117.05, 115.43, 104.61, 103.62, 60.15, 55.82; Anal. Calc. for C17H16ClN3O5: C, 54.05; H, 4.27; N, 11.12, found: C, 54.02; H, 4.25; N, 11.10%. ESI-MS m/z = 378.27 (M + 1)+, 378.28 (M + 3)+. HPLC purity: 99.129%.The anticancer activity of the compounds was assessed using the standard NCI US protocol at 10 µM drug concentration ,38,39,40The tubulin–combretastatin A4 complex’s X-ray crystallographic structure, with a resolution of 2.40 Å and an R-value of 0.192 (observed), was retrieved from protein data bank (PDB) . The comE. coli DNA gyrase complex with 2-oxo-1,2-dihydroquinoline derivative’s X-ray crystal structure with a resolution of 2.40 Å, R-value 0.199 (obs.), was retrieved from protein data bank (https://www.rcsb.org/structure/6KZV accessed on 16 March 2023) [The ch 2023) . The stach 2023) .B. subtilis, E. coli, S. aureus, and P. aeruginosa bacterial strains via disc diffusion method and broth dilution method [The antibacterial activities of the compounds were tested against n method ,42,43 (p6a-h) in the current investigation. The compounds were designed based on the pharmacophoric features of tubulin inhibitors (IMC-094322 and IMC-038525). Therefore, the compounds were further subjected to molecular docking studies against the active site of the tubulin–combretastatin A4 complex (PDB ID: 5LYJ); they displayed efficient binding with docking scores ranging between −7.617 and −8.389 kcal/mol and demonstrated two types of binding interactions: H-bond and halogen bond interactions. The compounds’ binding affinities against the active site of the tubulin–combretastatin A4 complex were found to be higher than those of the tubulin inhibitors IMC-094322 and IMC-038525 in the molecular docking studies, with the exception of compound 6e. The compounds were prepared efficiently in five steps in good yields. The anticancer activity of the compounds was evaluated at 10 µM drug concentration as per the standard protocol. 4-Chloro-2--1,3,4-oxadiazol-2-yl)amino)phenol (6h) demonstrated significant anticancer activity against some of the cancer cell lines, including SNB-19, NCI-H460, and SNB-75 at 10 µM. Compound 6h was discovered to be highly sensitive to the CNS cancer cell line SNB-19 (PGI = 65.12%), but less sensitive to the renal cancer cell line A498 (PGI = −30.65%). The average anticancer activity of compound 6h against nine panels of cancer cell lines was compared with that of the reference drug imatinib at 10 µM. It was discovered that compound 6h displayed significantly greater anticancer activity against non-small cancer, colon, CNS, melanoma, ovarian, renal, and breast cancer cell lines than imatinib. Based on anticancer activity, the SAR was concluded. It was discovered that the electron-donating group, such as 1,3,4-trimethoxy  and 2-hydroxy  substitution, as well as the electron-withdrawing 4-nitro  substitution on the phenyl ring demonstrated significant anticancer activity compared to other substitutions. The 4-chloro  substitution showed slightly more anticancer activity than 2-chloro  and 4-fluoro  substitution on the phenyl ring. Additionally, the 1,3,4-oxadiazoles’ antibacterial activities are well documented in the literature. The antibacterial activity of the compounds was studied against Gram-negative (E. coli and P. aeruginosa) as well as Gram-positive (S. aureus and B. subtilis) bacterial strains via the disc diffusion method and broth dilution method. The compounds 4-chloro-2-((5-(4-nitrophenyl)-1,3,4-oxadiazol-2-yl)amino)phenol (6c) and 4-chloro-2-((5-(2-chlorophenyl)-1,3,4-oxadiazol-2-yl)amino)phenol (6e) displayed the most promising antibacterial activities against both the Gram-negative as well as Gram-positive bacteria. Compound 6c displayed the most significant antibacterial activities with MICs of 8 µg/mL and a zone of inhibition ranging from 17.0 ± 0.40 to 17.0 ± 0.15 mm at 200 µg/mL; however, the standard drug ciprofloxacin exhibited MICs of 4 µg/mL. The electron-withdrawing group, such as 4-nitro and 2-chloro substitutions on the phenyl ring, demonstrated the most promising antibacterial activity among the series; 4-chloro and 4-fluoro substitutions exhibited moderate antibacterial activity, while the electron-releasing group, such as 4-methoxy, 2-hydroxy, 4-hydroxy-3-methoxy, and 1,3,4-trimethoxy substitutions on the phenyl ring, displayed less antibacterial activity. A molecular docking study was performed for the title compounds (6a-h) against DNA gyrase (PDB ID: 6KZV), one of the most appealing targets for many antibacterial agents, and it was discovered that both compounds 6c and 6e had efficient binding affinity with docking scores of −6.200 and −6.424 kcal/mol, respectively. The results of the molecular docking studies revealed a correlation between docking scores and antibacterial activity. We have reported the anticancer and antibacterial activities of 1,3,4-oxadiazoles in the present investigation. The bioactivities of the compounds were found to be significant and some of the in vitro experimental results were found to be in good agreement with in silico studies. The current discovery could pave the way for future breakthroughs in cancer and bacterial disease treatment. The biological activities of these oxadiazoles can be increased through structural modification. In our laboratory, we are currently conducting additional research into the anticancer and antibacterial potential of modified oxadiazoles.We discussed the design, synthesis, and anticancer activity of new 4-chloro-2-amino)phenol analogues (


Eutypella sp. D-1 based on the OSMAC (one strain many compounds) approach resulted in the isolation of five cytosporin polyketides (compounds 1–3 and 11–12) from rice medium and eight cytosporins (compounds 2 and 4–11) from solid defined medium. The structures of the seven new compounds, eutypelleudesmane A (1), cytosporin Y (2), cytosporin Z (3), cytosporin Y1 (4), cytosporin Y2 (5), cytosporin Y3 (6), and cytosporin E1 (7), were elucidated by analyzing their detailed spectroscopic data. Structurally, cytosporin Y1 (4) may be a key intermediate in the biosynthesis of the isolated cytosporins, rather than an end product. Compound 1 contained a unique skeleton formed by the ester linkage of two moieties, cytosporin F (12) and the eudesmane-type sesquiterpene dihydroalanto glycol. Additionally, the occurrence of cyclic carbonate moieties in compounds 6 and 7 was found to be rare in nature. The antibacterial, immunosuppressive, and cytotoxic activities of all compounds derived from Eutypella sp. D-1 were evaluated. Unfortunately, only compounds 3, 6, 8, and 10–11 displayed immunosuppressive activity, with inhibitory rates of 62.9%, 59.5%, 67.8%, 55.8%, and 68.7%, respectively, at a concentration of 5 μg/mL.A chemical investigation of the Arctic-derived fungus  Cytospora sp. in 1996, cytosporins were recognized as inhibitors of angiotensin II binding inhibitors , whi, whi1 wa2) was obtained as a light-brown oil and determined to have a molecular formula of C19H30O4 based on HRESIMS and NMR data, corresponding to an unsaturation index of 5. The presence of hydroxy functionality was indicated by IR absorption bands at 3359 cm−1. The 13C NMR  and five oxygenated carbon signals . The COSY spectrum of 2 showed three distinct spin systems: C-2/C-3, C-7/C-8/C-9/C-10/C-11/C-12/C-13, and C-15/C-16  and C-2 (δC 57.5) to those of cytosporins D and F −, consistent with the molecular formula of C19H28O4. The similarity of the 1H and 13C NMR data between 3 and 11 indicated that compound 3 was the derivative of 11. The presence of a pentasubstituted benzene moiety ; δC 115.1 (CH), 118.8 (C), 123.6 (CH), 126.8 (C), 144.8 (C), and 146.7 (C)) instead of the oxirane-fused cyclohexene moiety in 11 was suggested by the 1H and 13C NMR spectra. This was further confirmed by the further HMBC correlations from H-4β to C-5, C-6, and C-10; from H-6 to C-7, C-8, and C-10; from H2-13 to C-8, C-9, and C-10; and from H-14 to C-7, C-8, and C-9 (C 146.7) and the molecular formula. The conjugated C-14/C-15 double bond in 3 was assigned as trans based on the similar 1H NMR chemical shift values and coupling constants (16.5 Hz) observed in 3 and 2. To determine the absolute configuration at C-3 in compound 3, the specific rotation  was measured. The configuration of C-3 could be assigned as S by comparison to the literature data for synthetic (R)-2,2-dimethylchromane-3,7-diol (c 0.03) +), which is larger than that of cytosporin Y (2) by 18 amu. The NMR data of 4  and one methine (δH/δC 3.29/57.5) in 2 to δC 74.3 and δH/δC 3.76/75.0 in 4, respectively, suggested that 4 was the oxirane ring-opening product of 2. This hypothesis was further supported by further COSY and key HMBC correlations, as shown in E-geometry of the Δ7,8 double bond was deduced from a NOESY correlation between H-7 and H2-9, as well as the coupling constants (16.5 Hz). Additional NOESY correlations of H-2/H2-15, H-2/H-6, H-3/H-6, and H-6/H2-15 indicated the same orientation of these protons. Furthermore, the comparison of the calculated and the experimental ECD spectra confirmed the absolute configurations of 4 as 1S,2R,3R,6S  was obtained as a light-yellow oil. Extensive NMR analyses and HRESIMS data (m/z 389.1928 [M + Na]+) led to the determination of its molecular formula as C20H30O6. The overall NMR data of 5 indicated a structure similar to 4, with the notable difference of an additional quaternary carbon. This carbon was identified as a carbonate moiety based on the strong IR absorption at 1647 cm−1 and the diagnostic 13C NMR signal at δC 154.8 +, consistent with the molecular formula C20H32O7 with five degrees of unsaturation. The 1H and 13C NMR data of 7 (9), except for two additional methylenes (δC/δH 31.0/2.26 and 30.0/1.49) in 7 and the absence of two olefinic methines (δC/δH 123.5/6.39 and 137.1/6.03) in 9. These observations indicated that C-8 in 7 was substituted by a heptane subunit instead of the 1-heptene part in 9, which was also confirmed by COSY correlations of H2-14 (δH 2.26)/H2-15 (δH 1.49), H2-15/H2-16 (δH 2.25), H2-16/H2-17 (δH 1.35), H2-17/H2-18 (δH 1.31), H2-18/H2-19 (δH 1.32), and H2-19/H3-20 (δH 0.90), as well as HMBC correlations from H2-14 to C-7 (δC 78.5) and C-8 (δC 133.3). The relative configuration of 7 was inferred to be different from that of compound 9 through a comparison of the 13C NMR data between 7 (C-6 δC 81.3 and C-7 δC 78.5) and 9 (C-6 δC 81.0 and C-7 δC 75.3), as well as the analysis of NOESY correlations of H-3/H3-11, H-4α/H-6, H-4α/H-7, H-4β/H-10, H-4β/H3-12, and H-10/H3-12 in MeOD-d4 and 3-OH/H-10 and 5-OH/H-10 in DMSO-d6  + .Eutypelleudesmane A (2): light brown oil; c 0.10, MeOH); UV (MeOH) (log ε) λmax 241 (3.89) nm; CD (MeOH) (Δε) 238 (+9.1); IR (KBr) νmax 3359, 2956, 2927, 2857, 1454, 1376, 1261, 1014, 842, 725 cm−1; 1H and 13C NMR data, see m/z 367.2125 [M + COOH]− .Cytosporin Y (3): light-brown oil; c 0.10, MeOH), c 0.1, CDCl3); UV (MeOH) (log ε) λmax 210 (5.37), 312 (3.13) nm; IR (KBr) νmax 3378, 2954, 2927, 2856, 1708, 1614, 1513, 1434, 1380, 1369, 1255, 1218, 1184, 1143, 1064, 1029, 977, 852 cm−1; 1H and 13C NMR data, see m/z 319.1912 [M − H]− .Cytosporin Z (1 (4): light-yellow oil; c 0.1, MeOH); UV (MeOH) λmax (log ε) 221 (3.71), 240 (3.77) nm; IR νmax 3367, 2955, 2926, 2857, 1743, 1601, 1378, 1072, 1023 cm−1; CD (MeOH) (Δε) 208 (–6.4); 1H NMR and 13C NMR, see m/z 363.2139 [M + Na]+ .Cytosporin Y2 (5): light-yellow oil; c 0.1, MeOH); UV (MeOH) λmax (log ε) 214 (3.96), 244 (4.28) nm; IR νmax 3381, 2956, 2926, 2857, 1786, 1647, 1344, 1219, 1049, 1023, 1001, 822, 760 cm−1; CD (MeOH) (Δε) 222 (–13.2); 1H NMR and 13C NMR, see m/z 389.1928 [M + Na]+ .Cytosporin Y3 (6): light-yellow oil, c 0.1, MeOH); UV (MeOH) λmax (log ε) 217 (3.64), 241 (3.81) nm; IR νmax 3383, 2956, 2928, 2858, 1783, 1595, 1361, 1270, 1182, 1068, 907, 770, 737 cm−1; CD (MeOH) (Δε) 241 (+4.87); 1H NMR and 13C NMR, see m/z 389.1928 [M + Na]+ .Cytosporin Y1 (7): light-yellow oil, c 0.1, MeOH), UV (MeOH) λmax (log ε) 200 (4.10) nm; IR νmax 3393, 2926, 2856, 1783, 1464, 1361, 1184, 1158, 1086, 1058, 1023, 917, 772, 629 cm−1; CD (MeOH) (Δε) 210 (+14.2) nm; 1H NMR and 13C NMR, see m/z 363.2145 407.2034 [M + Na]+ .Cytosporin E1–12 against Staphylococcus aureus, Escherichia coli, and Bacillus subtilis were evaluated using a previous method [1–12 against DU145, SW1990, Huh7, and PANC-1 human cancer cell lines were determined using the CCK-8 method [1–12 against ConA-induced T cell proliferation were performed as previously described [The antimicrobial activities of compounds s method , and lev8 method , with ciescribed , with cyEutypella sp. D-1 when cultivated in different media. This approach resulted in the production of five cytosporin polyketides (compounds 1–3 and 11–12) from a rice medium and eight cytosporins (compounds 2 and 4–11) from a solid defined medium. Remarkably, compound 1 contained a unique skeleton formed by the ester linkage of two moieties: cytosporin F (12) and the eudesmane-type sesquiterpene dihydroalanto glycol. Compounds 6 and 7, characterized by a cyclic carbonate-fused cytosporin skeleton, were found to be rare in nature. However, these metabolites only exhibited weak immunosuppressive inhibitory activity against ConA-induced T cell proliferation in the antimicrobial, cytotoxic, and immunosuppressive evaluation. Collectively, this work showcased that changing the fermentation medium could be an effective strategy to trigger the production of secondary metabolites from fungi derived from the polar extreme environment.In summary, the utilization of the OSMAC (one strain many compounds) culture strategy effectively modified the chemical profile of the Arctic-derived fungus


Their structures were elucidated by extensive spectroscopic data . The absolute configuration of 1–3 was elucidated based on ECD calculation. Among them, stellerterpenoid A was a rare 13, 14-seco nortigliane diterpenoid and stellerterpenoid B was a guaiacane-type sesquiterpenoid with an unusual 1, 2-diketone moiety. The known stelleraguaianone B (5) exhibited moderate activity for suppressing NO production in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages cells with an IC50 value of 24.76 ± 0.4 μM. None of the compounds showed anti-influenza virus or anti-tumor activity in vitro.An undescribed diterpene, stellerterpenoid A ( Stellera is comprised of 10-12 species, two of which are distributed in China (Stellera chamaejasme Linn. and Stellera formosana Hayata ex Li) +  in the HR-ESI-MS , 1.78 , and 1.18 , an oxymethines at δH 3.85 , a suite of methines including an olefinic proton at δH 7.48 , and four methines at δH 4.31 , 3.57 , 3.24 , and 2.74 . Its 13C NMR and DEPT spectra (δc 30.2 (C-16), 18.0 (C-17), and 10.0 (C-18)], three methylenes , six methines , and seven quaternary carbons consisting of two carbonyl groups [δC 209.8 (C-3) and 209.2 (C-15)], one ester carbonyl group at δC 177.9 (C-13), one olefinic group at δC 137.7 (C-2), and three oxygenated ones at δC 91.8 (C-9), 76.2 (C-4), and 66.0 (C-6). The 5/7 carbocyclic ring system was determined via HMBC analysis. The HMBC correlations from H-18 to C-1, C-2, and C-3 indicated that C-3 and C-1 were connected through C-2  and a ketone carbonyl  in 1 instead of the ∆14,15 double bond  in crotonianoid A. The phenomena suggested that the ∆14,15 double bond in crotonianoid A was oxidized to the methylene and ketone carbonyl in 1. In addition, the methylene of C-5  in crotonianoid A was replaced by one oxy-methine , and the double bond was replaced with one epoxy structure at C-6 and C-7. The C-9 and C-13 are similar to crotonianoid A, and this was demonstrated by signal offset via an oxygen atom generating a five-membered lactone ring [H 3.57) to C-14 (δC 42.6) and C-15 (δC 209.2) and from H-16 (δH 2.19) to C-14 (δC 42.6) and C-15 (δC 209.2) supported the location of the acetone unit at C-8  was obtained as a yellow oil with a molecular formula of C15H16O3 according to its HR-ESI-MS at m/z 267.0981 [M + Na]+   and 4.80  and three methyl peaks at δH 1.95 , 1.85 , and 1.80 . The 13C NMR spectrum (δc 20.4 (C-13), 17.3(C-14), and 8.8 (C-15), an olefinic carbon at δc 111.3 (C-12), two methylenes at δc 50.6 (C-7) and 36.3 (C-5), one methine at δc 40.3 (C-6), and eight quaternary carbons at δc 204.3 (C-2), 202.6 (C-1 and C-8), 165.1 (C-4), 148.9 (C-11), 146.1 (C-3), 145.9 (C-10), and 132.6 (C-9). The NMR data indicated that 2 was a guaiacane sesquiterpene.Stellerterpenoid B (67.0992) , suggestspectrum  of 2 exhspectrum , in comb2 were very similar to those of oleodaphnone [2 by one methylene in oleodaphnone. This observation suggested that one methylene in oleodaphnone was oxygenated into one ketone carbonyl in 2. The HMBC correlation from H-14 (δH 1.95) to C-1 (δC 202.6) verified that the ketone was located at C-1 -2 -2 b. Theref2 was proposed based on oleodaphnone  was obtained as a colorless oil. Its HR-ESI-MS exhibited a quasi-molecular ion peak at m/z 273.1454 [M + Na]+  , 1.64 , and 0.85 , six methylenes at δH 4.79 , 4.76 , 2.57 , 2.34 , 2.20 , and 1.75 , and three methines at δH 3.69 , 3.00 , and 2.29 . The 13C NMR spectrum showed 15 signals which were ascribed to three methyls at δC 19.9 (C-13), 15.6 (C-14), and 7.9 (C-15), four methylenes at δC 112.8 (C-12), 50.3 (C-1), 32.2 (C-5), and 40.8 (C-8), three methylates at δC 70.5 (C-7), 51.3 (C-6), and 37.9 (C-9), and five quaternary carbons at δC 208.3 (C-2), 138.9 (C-3), 173.8 (C-4), 82.2 (C-10), and 149.0 (C-11) (Stellerterpenoid C (73.1461) , suggestpic data  show the0 (C-11) . 3 were extremely similar to those of known wikstronone C + .Yellow oil; + .Yellow oil; + .Colorless oil; [S,5S,6R,7S,8R,10R,11R)-1, (6S)-2, and -3 were generated within a 10 kcal/mol energy window under MMFF94S via the software CONFLEX [1–3, respectively, were further optimized by the density functional theory method at the B3LYP/6-31G (d) level. The ECD calculations were performed using level TD-DFT-B3LYP/6-311G  of the theory on optimized geometries through the CPCM model (in MeOH) [Based on the conformation determined from ROESY spectra and Chem 3D modeling, the low-energy conformers of model compounds (4 CONFLEX . The selin MeOH) . The calin MeOH)  and all 2. The RAW 264.7 (8 × 104/well) cells were left to acclimate for 24 h before any treatments and were seeded onto 96-well plates and pretreated by compounds 1–9 (3.125–50 µM) 1 h prior to treatment with 1 μg/mL LPS. Afterward, co-stimulation for 24 h at 37 °C was carried out in an incubator under 5% CO2. Then, Griess reagents I and II (100 µL) were mixed with cell culture medium (70 µL) and incubated at room temperature for 10 min with horizontal shaking, after which the absorbance was measured at 540 nm using a microplate reader . N(G)-monomethyl-Larginine, monoacetate salt (L-NMMA) and DMSO were used as positive and negative controls, respectively [The murine macrophage cell line RAW 264.7 was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin -streptomycin  at 37 °C in a humidified incubator containing 5% COectively .4/well) were inoculated in 96-well plates and cultured for 24 h before viral infection. The infection medium was MEM , and 100 U/mL penicillin/streptomycin). The cells were treated with various concentrations (from 3.125 μM to 50 μM) of tested compounds 1–9 and detected by Promega  CellTiter-Glo® reagent following the protocol provided by the supplier. The emitted luminescence (RLU) was quantified with a Promega Victor III plate reader. Oseltamivir was used as the positive control [Influenza strain A/Puerto Rico/8/1934 (H1N1) was used in this study. MDCK cells , and streptomycin  fluid, as well as 10% fetal bovine serum. The cells (1 × 105 per well) were seeded on 96-well microplates and allowed to adhere for 12 h before drug addition. Each cell line was treated with compounds 1–9 at different concentrations (3.125–50 μM) in triplicate for 48 h. Spectra-Max M2  was employed to record the optical density (λ = 490 nm). IC50 values were calculated based on the mean OD values measured three times versus the concentration curves of drugs. Adriamycin  and DMSO were used as the positive and negative control [HepG2, A549, and HeLa cell lines were used for cytotoxic activity. Cells were cultured in DMEM medium at 37 °C in 5% CO control .All biological experiments were repeated at least three times and the results were obtained using Statistical Package for Social Sciences (SPSS Version 21.0) software . 1), two guaiacane sesquiterpenoids (2–3), and six known ones (4–9) were isolated from the roots of Stellera chamaejasme. Their structures were elucidated by extensive spectroscopic data . The absolute configuration of compounds 1–3 were established by the experimental and calculated ECD. Compound 5 exhibited moderate NO inhibitory activity in LPS-induced RAW 264.7 macrophages (IC50 = 24.76 ± 0.4 μM). None of the compounds showed anti-influenza virus and anti-tumor activity in vitro.In summary, three previously unreported terpenoids including a diterpene (


Camellia taliensis (W. W. Smith) Melchior, belonging to the genus Camellia sect. Thea., is mainly distributed from northern Myanmar to western and southwestern Yunnan province of China, and its leaves have been used to make various teas by the locals of its growing regions. The chemical constituents of C. taliensis are significantly related to those of cultivated tea plants, C. sinensis and C. sinensis var. assamica. The HPLC-ESI-MS analysis of black tea prepared from the leaves of C. taliensis showed a rich existence of polyphenols. Further comprehensive chemical study led to the separation and recognition of 32 compounds (1–32), including one new hydrolyzable tannin, 1-O-galloyl-4,6-tetrahydroxydibenzofurandicarboxyl-β-D-glucopyranose (1), and one new natural product (24). The known compounds referred to seven hydrolyzable tannins (2–8), 10 flavonols and glycosides (9–18), and 14 simple phenolics (19–32). Their structures were elucidated by comprehensive spectroscopic analyses. Among them, 20 compounds  were isolated from black tea for the first time. Most isolates displayed obvious antioxidant activities on DPPH and ABTS+ assays, and the hydrolyzable tannins 1, 3–5, 7, and 8 exhibited stronger inhibitory activities on α-glycosidase than quercetin and acarbose , with IC50 values ranging from 0.67 to 2.01 μM. Camellia sinensis or C. sinensis var. assamica, through catalyzed oxidation by polyphenol oxidase (PPO) contained in the fresh tea leaves. Due to its fragrant aroma and various positive health effects, e.g., antioxidant, anti-hyperlipidemia, anti-hyperglycemic, and anti-microbial activities −; HRESI-MS: m/z 615.0625 [M − H]− ; UV λmax (MeOH) (log ε): 276 (4.35), 222 (4.51) nm; 1H NMR : δH 7.20 , 7.12 , 7.05 , 5.73 , 5.00 , 4.78 , 4.01 , 3.98 , 3.86 , 3.65 . 13C NMR : δC 171.5 (C-7‴), 170.0 (C-7″), 166.8 (C-7′), 147.7 (C-6‴), 147.6 (C-6″), 146.6 , 145.5 (C-4‴), 145.2 (C-4″), 140.6 (C-4′), 136.1 (C-5″), 134.0 (C-5‴), 120.4 (C-1′), 119.8 (C-2‴), 117.0 (C-2″), 116.2 (C-3″), 115.9 (C-1″), 115.4 (C-1‴), 112.1 (C-3‴), 110.6 , 95.7 (C-1), 76.2 (C-4), 74.9 (C-3), 74.2 (C-2), 71.7 (C-5), 67.6 (C-6).Yellow-brown amorphous powder; + were carried out using the previous method with some modifications −, 697 [M − H − gallic acid]−, and compared the tR (36.94 min) and UV absorptions  with those of the reference substance. In a similar way, peaks 32  and 34  were confirmed as theaflavin-3-gallate and theaflavin-3′-gallate, respectively.Peaks 32, 33, and 34 in m/z 331 [M − H]−, 169 [M − H − glucosyl]−) and tR (5.43 min), peak 2 was identified as β-glucogallin. Similarly, peaks 6 , 9  and 14  were recognized as 1,6-di-O-galloyl-β-D-glucopyranose (6), strictinin (4) and 1,4,6-tri-O-galloyl-β-D-glucopyranose (8), respectively, and these were obtained during further chemical isolation.Peaks 2, 6, 9, and 14 in m/z 125 [M − H]−; tR 6.82 min) and 5  were identified as pyrogallol and theogallin, respectively. Similarly, gallic acid (19), methyl gallate (23), chlorogenic acid (25), and 5-O-(E)-p-coumaroylquinic acid (26) were determined at peaks 3 , 7 , 10 , and 13 , respectively. All of them were obtained during further chemical isolation.Peaks 3, 4, 5, 7, 10, and 13 in 13) by the fragment ions , tR (23.79 min) and UV absorption . Likewise, peaks 18 , 21 , 22 , 23 , 24 , 25 , 27 , 31  and 35  were recognized as quercetin 3-O-β-D-glucopyranoside (11), kaempferol 3-O-rutinoside (12), quercetin 3-(2″-β-D-glucosy-l)-α-L-rhamnopyranoside (17), kaempferol 3-(2″-β-D-glucosyl)-α-L-rhamnopyranoside (18), kaempherol 3-O-β-D-glucopyranoside (9), kaempherol 3-O-β-D-galacopyranoside (14), kaempherol 3-O-α-L-rhamnopyranoside (15), kaempferol 3-O-(6″-trans-p-coumaroy-l)-β-D-glucopyranoside (10), and quercetin (16), respectively, and they were also isolated from further chemical study. In addition, peaks 20 , 28 , 30 , were speculated to be quercetin 3-O-β-D-galactopyranoside, quercetin-3-O-coumaroyl-rhamnosyl-glucosyl-rhamnopyranoside, and kaempferol-3-O-coumaroyl-rhamnosyl-glucosyl-rhamnopyranoside, respectively.Peaks 16, 18, 20, 21, 22, 23, 24, 25, 27, 28, 30, 31, and 35 in m/z 173 [M − H]− and fragment ions  with those of the standard sample, peak 1 was inferred to be theanine. Similarly, peak 15  was identified as 5,7-dihydroxycoumarin.In addition, two other types of compounds were also identified. Compared the quasi-molecular ion of C. taliensis using 60% aqueous acetone was dissolved with water and successively redistributed with CHCl3, EtOAc, and n-BuOH. The EtOAc extract was further isolated by various CC on Diaion HP20SS, RP-18, Toyopearl HW-40F, MCI-gel CHP20P, and Sephadex LH-20, to yield 32 compounds, including one undescribed hydrolyzable tannin (1) and one new natural product (24). The compounds, which had were in advance, were recognized as seven hydrolyzable tannins (2−8) including 4-O--cis-p-coumaric acid (2) [O-galloyl-4,6-O-(S)-hexahydroxydiphenoyl-β-D-glucopyranose (3) [O-galloyl-4,6-O-(S)-hexahydroxydiphenoyl-β-D-glucopyranose (4) [O-galloyl-4,6-O-(S)-hexahydroxydiphenoyl-β-D-glucopyranose (5) [O-galloyl-β-D-glucopyranose (6) [O-galloy-β-D-glucopyranose (7) [O-galloyl-β-D-glucopyranose (8) [9−18) including kaempferol 3-O-β-D-glucopyranoside (9) [O-(6″-trans-p-coumaroyl)-β-D-glucopyranoside (10) [O-β-D-glucopyranoside (11) [O-rutinoside (12) [13) [O-β-D-galactopyranoside (14) [O-α-L-rhamnopyranoside (15) [16) [β-D-glucosyl)-α-L-rhamnopyranoside (17) [β-D-glucosyl)-α-L-rhamnopyranoside (18) [19−32) including gallic acid (19) [20) [p-hydroxybenzoic acid (21) [m-hydroxybenzoic acid (22) [23) [24) [25) [O-(E)-p-coumaroylquinic acid (26) [O-caffeoylshikimic acid (27) [28) [E-p-hydroxycinnamic acid (29) [30) [31) [R)-thunberginol C (32) [2, 3, 6, 7, 8, 15, 17, 18, 20–22, 24–32) were never been separated from black tea.The extract of black tea from acid (2) , 2-O-galnose (3) , 1-O-galnose (4) , 1,2-di- product . The comnose (6) , 1,2,6-tnose (7) , and 1,4nose (8) , 10 flavside (9) , kaempfeide (10) , quercetide (11) , kaempfeide (12) , rutin (12) [13) , kaempfeide (15) , quercet15) [16) , quercetide (17) , and kaeide (18) , and 14 cid (19) , 3,4-dih19) [20) , p-hydrocid (21) , m-hydrocid (22) , methyl 23) [24) , chlorog24) [25) , 5-O-(E)cid (26) , 5-O-cafcid (27) , caffeic27) [28) , E-p-hydcid (29) , 1- [30) , 3,4,8,930) [31) , and (3Rl C (32) , respect1 was obtained as a yellow-brown amorphous powder with c 0.10, MeOH)  (1 had a great semblable structure to that of strictinin (4)  with C1 form proved by the study on the conformation of ellagitannins (ETs) [J = 8.2 Hz) of anomeric proton (δH 5.73) [δC 146.6 (2C), 140.6, 120.4, 110.6 (2C), 166.8; δH 7.12, 2H, s), and one HHDP-related acyl  groups were deduced apparently from the 1H and 13C NMR spectra (δH 5.73) to galloyl carboxyl carbon (δC 166.8) clarified that the galloyl group was connected at glucosyl C-1. The obvious lower field shift of glucosyl C-6 (δC 67.6), H-4 (δH 5.00), and H-6  compared with those of 1-O-galloyl-β-D-glucopyranose [4. However, 1H and 13C NMR data assignable the HHDP-related acyl group in 1 were very different to those of the HHDP group in 4, and the molecular formula of 1 was differed from 4 by one H2O, indicating that 1 should be a dehydrated derivative of 4. The HHDP-related acyl group at glucosyl C-6 and C-4 in 1 was determined to be 1,1′- dibenzofurandicarboxyl group, compared with the NMR data with those in mallotusinin, the first compound with a 1,1′- dibenzofurandicarboxyl group isolated from Mallotus japonicus [1. Hence, compound 1 was confirmed to be 1-O-galloyl-4,6-tetrahydroxydibenzofurandicarboxyl-β-D-glucopyranose, as shown in Compound 0, MeOH) . Its mol15.0628) , demonstinin (4) . The exins (ETs)  and the δH 5.73) , one gal spectra . The HMBpyranose , suggestaponicus . Other 26–8 were classified as GTs, and 1, 3, 4, and 5 were classified as ETs. ETs are often present as an equilibrium mixture of two isomers with a different configuration at anomeric carbon [3 was isolated as a mixture, due to the chirality of anomeric carbon. It was reported that the original product of the oxidative coupling of two galloyl groups in ETs was the dehydrohexahydroxydiphenoyl (DHHDP) group, which was then reduced to the HHDP group [1 could be a reduction product, reduced through the two galloyl groups of 4 which were converted into DHHDP by the oxidative coupling reaction, followed by redox disproportionation. Hydrolyzable tannins are segmented into two major classes based on their structures: gallotannins (GTs) and ETs. GTs are esters with only a galloyl group bounded to glucose, while ETs are esters with hexahydroxydiphenoyl (HHDP) or its similar acyl groups commonly bounded to glucose . Isolatec carbon . For exaDP group , as showDP group . ConsequDP group . Thus, c31 (resembled a urolithin) demonstrated a possible occurrence of the ester hydrolysis of ET acyl groups and further decarboxylation during the fermentation of black tea made from the leaves of C. taliensis.Urolithins, as natural metabolites of ETs with better gastrointestinal absorption, were reported to have inhibitory effects on the proliferation of prostate and colon cancer cells as well as anti-inflammation activity . It is r+ assays for all of the hydrolyzable tannins (1–8), flavonol and its glycosides (9–18), and simple phenolic compounds  isolated from C. taliensis black tea, were assessed. The results, shown in The antioxidant activities through DPPH and ABTS1–8) showed a stronger free radical scavenging activities than ascorbic acid and trolox. The sequence of activity for inhibiting the DPPH radical was 8 > 6 > 5 > 4 > 1 > 2 > 7 > 3 > ascorbic acid, and the sequence of activity for inhibiting the ABTS+ radical was 5 > 8 > 4 > 6 > 3 > 1 > 7 > 2 > trolox. In hydrolyzable tannins, galloyl group at C-2 of glucose could weaken the radical scavenging effects from the weaker results of 3 and 7 than those of 4 and 8. All eight hydrolyzable tannins  showed equivalent effects to the positive control (ascorbic acid and trolox). The sequence of activity for inhibiting the DPPH radical was 13 > 16 > 11 > ascorbic acid > 17, and the sequence of activity for inhibiting the ABTS+ radical was 16 > 13 > 11 > trolox > 17. The p-coumaryl group has a little positive influence on the antioxidant effects of flavonol and its glycosides, which could be found from 10 (6.28%) and 9 (0.97%), respectively. Part of the flavonol and its glycosides  showed stronger activity than the positive control (ascorbic acid and trolox). The sequence of activity for the inhibiting DPPH radical was 19 > 23 > 31 > 25 > 28 > 30 > ascorbic acid, and the sequence of activity for inhibiting the ABTS+ radical was 31 > 23 > 28 > 19 > 25 > 30 > trolox. The p-coumaric acid derivatives showed little radical scavenging effects, weaker than caffeic acid derivatives from the order of activity (25 > 30 > 27 > 26 > 29), which was consistent with reports in the literature [Part of the simple phenolic compounds  on α-glucosidase were investigated. At a concentration of 50 μM, 1, 3–5, 7 and 8 with three or more galloyl groups showed a higher inhibition ratio (>50%) on α-glucosidase. Their IC50 values were further evaluated, and as shown in 50 = 5.75 and 223.30 μM, respectively), with IC50 values ranging from 0.67 to 2.01 μM. Their activity order was 5 > 3 > 1 > 4 > 7 > 8 > quercetin > acarbose. Compounds 2 and 6 with one or two galloyl groups showed equivalent inhibitory effects to acarbose on α-glucosidase at a concentration of 50 μM, while gallic acid (19) showed almost no inhibitory effect. The results revealed that the number of phenolic hydroxyl group plays a positive role on the α-glucosidase inhibitory activities of hydrolyzable tannins, probably due to their hydrophobic association with α-glucosidase [The inhibitory activities of hydrolyzable tannins cosidase .C. taliensis, with similar chemical constituents to the extensively cultivated tea plants, C. sinensis and its variety assamica, is the earliest, most used and widely distributed wild tea tree, whose leaves were utilized by the locals of its growing regions to manufacture various types of tea. A comprehensive chemical study of black tea from C. taliensis resulted in the separation and recognition of one new hydrolyzable tannin, 1-O-galloyl-4,6-tetrahydroxydibenzofurandicarboxyl-β-D-glucopyranose (1), together with 31 known compounds, comprising seven hydrolyzable tannins (2–8), 10 flavonols and their glycosides (9–18), and 14 simple phenolics (19–32) with 24 as a new natural product. It is noted that 15 compounds  were obtained from C. taliensis and 20 compounds  were isolated from black tea for the first time. The isolation of hydrolyzable tannins, especially new ones from fermented tea, is challenging, because hydrolyzable tannins are easily hydrolyzed during fermentation process. Nonetheless, one new hydrolyzable tannin was isolated and identified in the study. As far as we know, the hydrolyzable tannin with 1,1′- dibenzofurandicarboxyl group was discovered from tea for the first time. Most of the isolates showed significant antioxidant and α-glucosidase inhibitory activities, which were positively correlated with the amount of phenolic hydroxyl group. However, more antioxidant and hypoglycemic activity experiments could be carried out. In short, C. taliensis is also an ideal material to produce black tea with mild bitter and astringent flavor and rich polyphenols with significant antioxidant and α-glucosidase inhibitory activities, which indicated that the black tea from C. taliensis has the potential to be developed as health products.


Fungus-derived secondary metabolites are fascinating with biomedical potential and chemical diversity. Mining endophytic fungi for drug candidates is an ongoing process in the field of drug discovery and medicinal chemistry. Endophytic fungal symbionts from terrestrial plants, marine flora, and fauna tend to produce interesting types of secondary metabolites with biomedical importance of anticancer, antiviral, and anti-tuberculosis properties.Penicillium verruculosum sponge-derived endophytic fungi from Spongia officinalis yielded seven different secondary metabolites which are purified through HPLC. The isolated compounds are of averufin (1), aspergilol-A (2), sulochrin (3), monomethyl sulochrin (4), methyl emodin (5), citreorosein (6), and diorcinol (7). All the seven isolated compounds were characterized by high-resolution NMR spectral studies. All isolated compounds', such as anticancer, antimicrobial, anti-tuberculosis, and antiviral, were subjected to bioactivity screening.An organic ethyl acetate extract of 50 concentration of 1.00μm, which is nearly significant to that of the standard anticancer drug taxol. A virtual computational molecular docking approach of averufin with HL60 antigens revealed that averufin binds strongly with the protein target alpha, beta-tubulin (1JFF), with a −10.98 binding score. Consecutive OSIRIS and Lipinski ADME pharmacokinetic validation of averufin with HL60 antigens revealed that averufin has good pharmacokinetic properties such as drug score, solubility, and mutagenic nature. Furthermore, aspergilol-A (2) is the first report on the Penicillium verruculosum fungal strain.Out of seven tested compounds, compound (1) exhibits strong anticancer activity toward myeloid leukemia. HL60 cell lines have an ICPenicillium verruculosum can be taken for further preliminary clinical trials like animal model in-vivo studies and pharmacodynamic studies. A future prospect of in-vivo anticancer screening of averufin can be validated through the present experimental findings.We concluded that averufin (1) isolated from  Pullularia sp., isolated from a terrestrial plant Culophyllum sp., produces cytotoxic hexadepsipeptides named pullulans A-D, which exhibited cytotoxicity against human epidermoid cancer cells and human breast cancer cells.  derived from a marine sponge Spongia officinalis. The fungus was fermented on a large scale and extracted, and its bioactive compound isolation was conducted through chromatographic purification, like HPLC. Structural elucidation of isolated compounds was carried out through NMR spectroscopic studies. Herein, we have reported on the fermentation, extraction, and isolation strategies of those bioactive metabolites.2SO4 in H2O. Semi-preparative HPLC (RP-HPLC) was conducted on Agilent HPLC . All the positive control standard drugs used in the bioassay were procured from Sigma Aldrich (USA).NMR studies were recorded on a Bruker AC 500MHz NMR  spectrometer using TMS as an internal standard. Chemical shifts were expressed in “d” (ppm) and coupling constant ‘J' in Hz. HR-ESI-MS were measured using a Bruker micro TOF-QII mass spectrometer . Size exclusion chromatography was conducted using Sephadex LH-20 gel . Column chromatography was performed using a silica gel (200–300) at Qingdao Marine Chemical Factory . TLC spots were detected under UV light and by heating after spraying with 12% HS. officinalis were collected from the Xidao Island (18o23.18'N and 109o36.71'E), Hainan province of the South China Sea coast, in July 2014, during a marine cruise collection. Sponges were transported to the laboratory in ice-freeze conditions and stored at−20oC until further use. Sponges were identified based on morphological and spicule identification by the Department of Marine Bioresources and Ecological Sciences, SCISO, China. A voucher specimen (Voucher Number: SCISO 45874/2018) was deposited at SCSIO marine biological collections.Fresh sponges of S. officinalis. The strain was grown on a MactoBalt (MB) agar slant at 25°C. Two-week-old fungal hyphae were scraped for genomic DNA isolation using the Ultraclean Microbial DNA Kit (MoBio Laboratories). The internal transcribed spacer of ribosomal nucleotide sequence (ITS rDNA) primers ITS1 (5′-GCACAGGCAGCAGGAGCTG CCCCTCAGCTGTCTCCTCGTGCTCAAC-3′) and ITS (5′- AGAGCAAGCCGAGCAGGTCTATCGCCAAGTATCCTCAG AAGCTGTGCT-3′) were used  and sequenced using an ABI 3730 sequencer . Sequences were analyzed using the BLAST program . The phylogenetic tree was constructed based on the neighbor-joining (NJ) method using MEGA-5.0 by using 1,000 bootstrap replicates. The sequence was deposited in GenBank and assigned an accession number (Genbank: KU891245).The endophytic fungus XWSO1F60 was isolated from the sponge 0C for a week. The stock culture was inoculated in an optimized seed medium  and incubated at 250C for 72 h on a rotating shaker (180 rpm). The optimization of the growth medium was selected based on previous experimental validation with maximum fungal growth and UV-HPLC metabolite fingerprinting. The mass quantity of fermentation of fungal isolates XWSO1F60 was carried out using a solid rice medium in 1000 mL flasks , which were inoculated with 10 mL of seed solution. Flasks were incubated at 25oC under a day-night cycle. 60 days old of fungal cultures from 40 flasks were subjected to organic extraction using Acetone /Ethyl acetate (EtOAc). The combined acetone/EtOAc fungal culture extracts were partitioned between 90% aqueous MeOH and petroleum ether. The resultant MeOH yield was fractionated using a silica column, Sephadex LH-20, and finally, semi-preparative reversed-phase HPLC to yield compounds (1–7).A stock culture of the strain XWSO1F60 was grown on MB agar solid medium at 253-MeOH in a gradient eluent  to obtain fractions 1–8 based on the TLC spots. Fr. 4 (950 mg) was purified by Sephadex LH-20  to obtain three subfractions (fr. 4.1–4.3). Fr.4.1 (226 mg) was further purified by SP-RP HPLC eluting with CH3CN/H2O (55:45) to afford compound diorcinol (7) (16.7 mg) and compound methyl emodin (5) (9.6 mg). Fraction 3 (1.9 g) was purified using Sephadex LH-20  and SP-RP HPLC using a C-18 column  eluting with MeOH/H2O (10:90) to afford compound citreoresin (6) (13.2 mg). Fr. 7 (1.8 g) was subjected to Silica gel chromatography eluted with Acetone/MeOH in a step-wise eluent  to give three subfractions (fr. 7.1–7.3). Fr. 7.2 (159 mg) was purified using SP-RP HPLC eluting with CH3CN / H2O (79:21) to afford compound averufin (1) (5.6 mg) and compound apergilol-A (2) (12.2 mg). Fr. 8 (3.9 g) was subjected to silica gel column chromatography eluted with a CHCL3/MeOH in a gradient elution of  (v/v), which yielded six fractions (fr 8.1 – 8.3). Fr. 8.3 (700 mg) was subjected to ODS chromatography eluted with MeOH/H2O  to obtain five subfractions (fr. 8.3.1–8.3.3). Fr. 8.3.1 (96.1mg) was further purified using SP-RP HPLC eluting with CH3CN- H2O (50:50) to afford the compound monomethyl sulochrin (4) (14.9mg). Fr. 8.2.4 (23 mg) was purified using (SP-RP) HPLC eluting with CH3CN-H2O (67:33) to afford the compound sulochrin (3) (11.6mg).The EtOAc crude extracts were then purified by silica gel column chromatography eluted with CHCL1H and 13C-Nuclear Magnetic Resonance (NMR) spectroscopy . Trimethylsilane (TMS) was used as an internal standard. The NMR spectrum was analyzed using MestreNova spectral processing (Version. 14.2.0).All the isolated compounds were checked for purity using Thin-layer Chromatography (TLC). After ensuring the purity, the compounds were subjected to Penicillium sp. were dissolved in 100% DMSO with a maximum concentration of 200 μg/mL and serially diluted to a final 0.1% DMSO concentration to treat cells for 2 h. Taxol was used as a positive control, and DMSO was used as a negative control. Cytotoxicity screening assay experiments were conducted in triplicate to obtain the standard error ± mean value. The percentage of cytotoxicity (IC50) alongside the selectivity index was then calculated using the formula:Cytotoxicity was assessed using the Cell Counting Kit (CCK-8)  method adopted by Wang et al. . Cancer Mycobacterium tuberculosis (ATCC 25177) purchased from the American Type Culture Collection (ATCC) was used in the anti-TB bioassay. The anti-tuberculosis assay was based on the one used by Wang et al.  alongside the positive control isoniazid (1 to 417 μg/mL) was added to the well. The anti-tubercular assay was done in triplicate to obtain the SD± mean value.The H37Ra strain of g et al. . INH  and H3N2  were used in the present study. The antiviral activities against H1N1 and H3N2 were evaluated by the CPE inhibition assay based on the methodology  and Escherichia coli (CGMCC 1.2385) were evaluated by an agar dilution method based on the study by Wang et al. , averufin was docked with four major cancer antigens of HL60 cell lines, namely, human serum transferrin, CD-5 antigen, CD-20, and alpha-beta tubulin from zinc-induced sheet based upon the methodology derived by Notarte et al. . The anto. The calculation type was set to “Dock” mode, whereas “flexible mode” was selected for the ligand. The lowest energy represented the easy binding character of ligands and receptors. Molecular interactions between ligands and target proteins were visualized using Discovery Studio (Ver 3.1) software.For docking analysis, Argus Lab 4.0.1 software was used, followed in accordance to the methodology described by Duverna et al. . The actin-silico toxicity prediction to evaluate the mutagenicity, tumorigenicity, irritant effects, and reproductive toxicity efficacy of compound (1), i.e., averufin  averufin were predicted by using the Swiss ADME software  based on the methodology derived by Quimque et al. . Pharmac+ and 10 Cl−. The energy minimization was conducted and equilibrated by NVT at 300 K and 1 bar for 100 ns and then subjected to a molecular dynamics study using an NPT ensemble. A molecular dynamic (MD) prediction of drug and target was performed at 100 nanoseconds (ns) to assess the bonding stability and displacement of ligand with that of the target.A molecular dynamic (MD) analysis was performed using Schrödinger maestro based on the methodology of Wu et al. . TargetsP. verruculosum based upon the sequence obtained from the internal transcribed spacer (ITS) regions (Genbank accession number: KU891245), and it has 99% similarity with that of P.verruculosum strain C2-8 (JQ717338) , the fungal hyphae stained with lactophenol blue appears to be slender with dispersed conidiophores . The funQ717338) .P.verruculosum strain XWSO1F60 yielded seven different metabolites, which comprise polyketides, xanthones, and alkaloid derivatives. Structural determinations were carried out manually and referred to previously published NMR spectral data for their consistency : δH: 1.52, 1.62, 1.72 - 2.01, 1.82 - 1.99 , 5.28, 6.58,7.03, 7.11 , 12.109, 12.54; 13C-NMR : δC: 15.2 (C-4′), 26.8 (C-3′), 27.3 (C-6′), 35.1 (C-2′), 66.1(C-1′), 101.1 (C-5′), 107.1 (C-4), 108.0 (C-7), 108.4 (C-9a), 108.6 (C-8a), 108.9 (C-5), 115.9 (C-2), 133.1 (C-10a), 134.9 (C-4a), 158.1 (C-3), 159.8 (C-8), 164.2 (C-1),165.2 (C-6), 180.9 (C- 10), and 188.8 (C-9) : δH: 0.83 , 1.22 , 1.26 , 1.26 , 1.40 , 1.75 , 1.87 , 2.39 , 2.56 , 4.40 , 5.66 , 5.68 , 5.85 , 5.96 , 6.37 , 6.54 , 6.98 , 7.03 , 8.98 , 9.14 , 10.87 , 11.20 , 12.20 ,12.87 ; 13C-NMR : δC: 14.4 (C-16), 20.7 (C-7′), 21.2 (C-7″), 22.6 (C-15), 28.8 (C-13), 32.1 (C-14), 33.1 (C-12), 37.3 (C-11), 101.5 (C-4″), 105.8 (C-4′), 108.3 (C-2), 108.5 (C-5), 108.5 (C-8a), 108.7 (C-4), 108.8 (C-2″), 109.2 (C-1a), 109.6 (C-6″), 113.7 (C-2′), 122.9 (C-7), 126.4 (C-6′), 131.8 (C-5a), 135.2 (C-4a), 139.1 (C-1″), 139.7 (C-1′), 155.1 (C-5′), 155.9 (C-3′), 158.2 (C-3″), 158.8 (C- 5″), 164.1 (C-3), 164.2 (C-6), 164.4 (C-8), 165.1 (C-1), 181.6 (C-9), and 189.2 (C-10) : δH: 2.31 , 3.64 , 3.65 , 6.09 , 6.68 , 6.91 , 10.02 , 12.48 ; 13C NMR : δC: 21.5 (C-7′), 52.1 (C-9′), 55.9 , 103.3 (C-4), 107.1(C-6), 107.5 (C-5′ and3′), 109.1 (C-1′), 126.1 (C-2), 127.8 (C-1), 143.3 (C-4′), 156.7 (C-3), 158.1 (C-5), 161.6 (C-6′ and 2′), 165.6 (C-7), and 199.6 (C-8′) : δH: 2.25 , 3.33 , 3.62 , 3.63 , 6.26 , 6.38 , 6.69 ,6.90 , 10.19 , 12.95 ; 13C NMR : δC: 21.9 (C-7′), 52.1 (OCH3-7), 55.9 ,55.9 (OCH3-8), 103.1 (C-4),103.5 (C-5′), 107.1 (C-6), 110.1 (C-1′ and 3′), 125.8 (C-2), 127.9 (C-1), 147.8 (C-4′), 156.6 (C-3), 158.1 (C-5), 160.7 (C-6′),163.2 (C-6′),165.7 (C-7), and 199.3(C-8′) :δH: 2.38 , 3.86 , 6.68 , 7.08 , 7.10 , 7.39 , 13.58 ; 13C NMR : δC: 21.3 (3-CH3), 56.9 (1-OCH3), 105.1 (C-7), 108.7 (C-5), 110.6 (C-8a), 114.5 (C-9a), 118.7 (C-4), 123.9 (C-2), 132.1 (C- 10a), 136.6 (C-4a), 145.9 (C-3), 161.6 (C-6), 163.7 (C-1), 167.8 (C-8), 182.8 (C-10), and 185.3 (C-9) :δH: 4.60 ,6.59 ,7.12 , 7.24 , 7.63 , 12.06 ; 13C NMR : δC: 61.9 (CH2OH),107.9 (C-2),108.9 (C-4), 108.9 (C-8a), 114.1 (C-9a), 117.0 (C-7),120.7 (C-5), 132.9 (C-4a), 135.1 (C-10a),152.8 (C-6), 161.5 (C-3), 164.3 (C-8), 165.5 (C-1), 181.3 (C-10), and 189.6 (C-9) :δH: 2.23 , 6.23 , 6.29 , 6.39 ; 13C NMR : δC: 21.5 (CH3-5), 104.2 , 111.7 (C-4 and C-6), 112.1 (C-4′and C-6′), 141.6 (C-5), 159.5 (C-1 and C-3), and 159.5   and methyl emodin (5) exhibit a significant anticancer effect against cancer cell lines compared to the others. Compound (1) shows strong anticancer activity toward HL60 cells with an IC50 value of 1.005 μM concentration, and compound (5) shows moderate activity , with a MIC value of 3.98μM, which was recorded. For antiviral, Tamiflu was used as the positive control with ICin-vitro results.Averufin (1) showed a better docking score when compared to the standard drug Taxol against all the target proteins, as evidenced by the protein-ligand interaction . The intin-vivo experiments as this compound has a good drug score. The boiled egg simulation shows that the compound averufin (1) is hydrophilic, which is a good hallmark of its palatability in gastrointestinal digestion  analysis report states that the compound (1) averufin binds with the target protein (1A8E). At the arrival time of simulation, it displayed a steady state of around 0.15 ns to the completion until 100 ns, which is a hallmark for good drug target interaction stability without drug binding displacement from the 1A8E protein target domain . The rooP.verruculosum strain. Compounds (1-7) are of polyketide, quinone, and phenone derivatives. All seven isolated compounds were subjected to different biological assays, which include anticancer, antimicrobial, antiviral, and anti-tuberculosis screening. Compound (1) averufin exerts strong anticancer activity against HL60 (Human leukemic cell lines), with a strong IC50 value of 1.005μm concentration prominent at that of standard drug taxol. Reports have suggested that Averufin isolated from a marine-derived Penicillium flavidorsum SHK1-27 exerted a weak antitumor activity toward K562 cell lines with MIC values of 72.4 μmol/L , a recently reported new compound from Aspergillus versicolor  reported from marine isolates of fungi   can be found in the article/KK and LS equally contributed to the experimental work. YL and RK performed experimental supervision. SS and ZJ performed molecular docking studies, molecular dynamics, and data interpretations. SE and KS conducted manuscript drafting and reviewing. NMA, NA, and MS performed data interpretation. All authors reviewed and approved the final version of the manuscript.