Fungal Prenyltransferase AnaPT and Its F265 Mutants Catalyze the Dimethylallylation at the Nonaromatic Carbon of Phloretin.

Phloretin is widely found in fruit and shows various biological activities. Here, we demonstrate the dimethylallylation, geranylation, and farnesylation, particularly the first dimethylallylation at the nonaromatic carbon of phloretin (1) by the fungal prenyltransferase AnaPT and its mutants. F265 was identified as a key amino acid residue related to dimethylallylation at the nonaromatic carbon of phloretin. Mutants AnaPT_F265D, AnaPT_F265G, AnaPT_F265P, AnaPT_F265C, and AnaPT_F265Y were discovered to generally increase prenylation activity toward 1. AnaPT_F265G catalyzes the O-geranylation selectively at the C-2' hydroxyl group, which involves an intramolecular hydrogen bond with the carbonyl group of 1. Seven products, 1D5, 1D7-1D9, 1G2, 1G4, and 1F2, have not been reported prior to this study. Twelve compounds, 1D3-1D9, 1G1-1G3, and 1F1-1F2, exhibited potential inhibitory effects on α-glucosidase with IC50 values ranging from 11.45 ± 0.87 to 193.80 ± 6.52 µg/mL. Among them, 1G1 with an IC50 value of 11.45 ± 0.87 µg/mL was the most potential α-glucosidase inhibitor, which is about 30 times stronger than the positive control acarbose with an IC50 value of 346.63 ± 15.65 µg/mL.

Aloperine-Type Alkaloids with Antiviral and Antifungal Activities from the Seeds of Sophora alopecuroides L.

As a continuous flow investigation of novel pesticides from natural quinolizidine alkaloids, the chemical compositions of the seeds of Sophora alopecuroides were thoroughly researched. Fifteen new aloperine-type alkaloids (1-15) as well as six known aloperine-type alkaloids (16-21) were obtained from the extract of S. alopecuroides. The structures of 1-21 were confirmed via HRESIMS, NMR, UV, IR, ECD calculations, and X-ray diffraction. The antiviral activities of 1-21 against tobacco mosaic virus (TMV) were detected following the improved method of half-leaf. Compared with ningnanmycin (protective: 69.7% and curative: 64.3%), 15 exhibited excellent protective (71.7%) and curative (64.6%) activities against TMV. Further biological studies illustrated that 15 significantly inhibited the transcription of the TMV-CP gene and increased the activities of polyphenol oxidase (PPO), peroxidase (POD), superoxide dismutase (SOD), and phenylalanine ammonia-lyase (PAL). The antifungal activities of 1-21 against Phytophythora capsica, Botrytis cinerea, Alternaria alternata, and Gibberella zeae were screened according to a mycelial inhibition test. Compound 13 displayed excellent antifungal activity against B. cinerea (EC50: 7.38 µg/mL). Moreover, in vitro antifungal mechanism studies displayed that 13 causes accumulation of reactive oxygen species and finally leads to mycelia cell membrane damage and cell death in vitro.

Isolating Antipathogenic Fungal Coumarins from Coriaria nepalensis and Determining Their Primary Mechanism In Vitro.

Through bioassay-guided isolation, eight undescribed coumarins (1-8), along with six reported coumarins (9-14), were obtained from Coriaria nepalensis. The new structures were determined by using IR, UV, NMR, HRESIMS, and ECD calculations. The results of the biological activity assays showed that compound 9 exhibited broad spectrum antifungal activities against all tested fungi in vitro and a significant inhibitory effect on Phytophthora nicotianae with an EC50 value of 3.00 µg/mL. Notably, compound 9 demonstrated greater curative and protective effects against tobacco balack shank than those of osthol in vivo. Thus, 9 was structurally modified to obtain new promising antifungal agents, and the novel derivatives (17b, 17j, and 17k) exhibited better effects on Sclerotinia sclerotiorum than did lead compound 9. Preliminary mechanistic exploration illustrated that 9 could enhance cell membrane permeability, destroy the morphology and ultrastructure of cells, and reduce the exopolysaccharide content of P. nicotianae mycelia. Furthermore, the cytotoxicity results revealed that compound 9 exhibited relatively low cytotoxicity against HEK293 cell lines with an inhibition rate of 33.54% at 30 µg/mL. This research is promising for the discovery of new fungicides from natural coumarins with satisfactory ecological compatibility.

Quinolizidine Alkaloids and Isoflavones from the Herb of Thermopsis lupinoides and Their Antiviral, Antifungal, and Insecticidal Activities.

As part of our ongoing investigation of natural bioactive substances from the genus Thermopsis of the tribe Fabaceae for agricultural protection, the chemical constituents of the herb Thermopsis lupinoides were systematically investigated, which led to the isolation of 39 quinolizidine alkaloids (QAs) (1-39), including 14 new QAs (1-14) and 14 isoflavones (40-53). Their structures were elucidated through comprehensive spectroscopic data analysis (IR, UV, NMR, HRESIMS), ECD calculations, and X-ray crystallography. The antitomato spotted wilt virus (TSWV) and antifungal (against Botrytis cinerea, Gibberella zeae, Phytophythora capsica, and Alternaria alternata) and insecticidal (against Aphis fabae and Tetranychus urticae) activities of the isolated compounds were screened using the lesion counting method, mycelial inhibition assay, and spray method, respectively. The bioassay results showed that 34 exhibited excellent protective activity against TSWV, with an EC50 value of 36.04 µg/mL, which was better than that of the positive control, ningnanmycin (86.03 µg/mL). The preliminary mechanistic exploration illustrated that 34 induced systemic acquired resistance in the host plant by acting on the salicylic acid signaling pathway. Moreover, 1 showed significant antifungal activity against B. cinerea (EC50 value of 20.83 µg/mL), while 2 exhibited good insecticidal activity against A. fabae (LC50 value of 24.97 µg/mL). This research is promising for the invention of novel pesticides from QAs with high efficiency and satisfactory ecological compatibility.

Synergistic combination of isogarcinol isolated from edible fruits of Garcinia multiflora and dexamethasone to overcome leukemia glucocorticoid resistance.

Isogarcinol (ISO), a cytotoxic polycyclic polyprenylated acylphloroglucinol isolated from the edible fruits of Garcinia multiflora. However, synergistic combination of ISO and dexamethasone (DEX) to overcome leukemia glucocorticoid resistance has never been investigated. Therefore, in this study, the effects of ISO in combination with DEX was conducted on leukemia in vivo and glucocorticoid resistance in vitro. As a result, the combination of the two compounds could efficiently inhibit leukemia progression in mice and reverse DEX resistance in acute lymphoblastic leukemia (ALL) Jurkat cells. Significantly, our findings indicated that c-Myc may be a potential target of ISO, as it is involved in cell cycle arrest and apoptosis by the combination of ISO and DEX in Jurkat cells. Furthermore, western blot analysis revealed that ISO and DEX inhibits the PI3K/Akt/mTOR signaling pathway and promotes the nuclear translocation of glucocorticoid receptor (GR), which activates target genes NR3C1 and TSC22D3, leading to apoptosis in Jurkat cells. Hence, our results suggest that ISO, as a safe and effective food-derived agent, can enhance the anti-leukemia effects of DEX.

Four new pregnane C21 steroids isolated from the roots of Cynanchum auriculatum.

Four new steroids cynansteroid G-I (1-3) and cynansteroid K (4), a new natural product 5,6-deacidizingcaudatin (5), and a known compound glycocaudatin (6), were isolated from the roots of Cynanchum auriculatum. The structures of new compounds were identified by comprehensive spectroscopic analyses, including NMR, HRESI-MS, ECD, UV, and IR spectral data. The cytotoxic activities of all the isolates against two human tumour cell lines (COLO-205 and BGC-823) were screened, unfortunately, which were weaker than positive control.

UPLC-Q-Orbitrap-MS/MS-Guided Isolation of Bioactive Withanolides from the Fruits of Physalis angulata.

Physalis angulata Linn. is an exotic Amazonian fruit that is commonly recognized as wild tomato, winter cherry, and gooseberry. While its fruit is known to contain many nutrients, such as minerals, fibers, and vitamins, few papers have investigated withanolide derivatives from its fruits. UPLC-Q-Orbitrap-MS/MS, which produces fragmentation spectra, was applied for the first time to guide the isolation of bioactive withanolide derivatives from P. angulata fruits. As a result, twenty-six withanolide derivatives, including two novel 1,10-secowithanolides (1 and 2) and a new derivative (3), were obtained. Compounds 1 and 2 are rare rearranged 1,10-secowithanolides with a tetracyclic 7/6/6/5 ring system. All structures were assigned through various spectroscopic data and quantum chemical calculations. Nine withanolide derivatives exhibited significant inhibitory effects on three tumor cell lines with IC50 values of 0.51-13.79 µM. Moreover, three new compounds (1-3) exhibited potential nitric oxide inhibitory effects in lipopolysaccharide-stimulated RAW264.7 cells (IC50: 7.51-61.8 µM). This investigation indicated that fruits of P. angulata could be applied to treat and prevent cancer and inflammatory-related diseases due to their potent active withanolide derivatives.

Three new quinolizidine alkaloids from the roots of Sophora tonkinensis.

Three new quinolizidine alkaloids (1 - 3), including one new naturally isoflavone and cytisine polymer (3), along with 6 known ones were isolated from the ethanol extract of Sophora tonkinensis Gagnep. Their structures were elucidated by comprehensive spectroscopic data analysis (IR, UV, HRESIMS, 1D and 2D NMR), combined with ECD calculations. The antifungal activity against Phytophythora capsica, Botrytis cinerea, Gibberella zeae, and Alternaria alternata of the compounds was evaluated in a mycelial inhibition assay. Biological tests indicated that compound 3 exhibited strong antifungal activity against P. capsica with EC50 values of 17.7 µg/ml.

Synthesis and Antifungal Activity of Curcumol Derivatives.

To discover novel and effective antifungal candidates, a series of new curcumol derivatives were designed, synthesized, and evaluated their antifungal activity against five phytopathogenic fungi by the mycelium growth rate method. Derivatives c4, c22 and c23 exhibited excellent antifungal activity against Phomopsis sp. with EC50 values of 3.06, 3.07, and 3.16 µM, respectively. Specifically, compound c4 exhibited the strongest antifungal activity against Phomopsis sp., which was 44 times that of pyrimethanil (EC50 =134.37 µM). The results of scanning electron microscopy (SEM) and transmission electron microscopy (TEM) indicated that compound c4 could cause cell senescence and death of Phomopsis sp. by changing the normal hyphal morphology and disrupting the normal metabolism of hyphal cells. Moreover, compound c4 showed excellent curative effect against Phomopsis sp. on kiwifruit. These findings confirmed that compound c4 has great potential as a potent antifungal agent.

Chemical Constituents of Primulina eburnea (Gesneriaceae) and Their Cytotoxic Activities.

Two new ursane-type triterpenes, eburnealactones A and B (1 and 2), one new flavonoid, eburneatin A (6), and one new phenylethanoid glycoside, chiritoside D (7), along with 9 known compounds (3-5, 8-13) were isolated from the whole plant of Primulina eburnea. Their structures were elucidated by comprehensive spectroscopic data analysis (IR, UV, NMR, and HR-ESI-MS). All the compounds were evaluated for their cytotoxic activities. Compound 1 showed significant cytotoxic activities against MKN-45 cell lines and 5637 cell lines with the IC50 values of 9.57 µM and 8.30 µM, respectively. Compound 1 exhibited moderate cytotoxic activities against A549 and PATU8988T cell lines with the IC50 values of 30.70 µM and 38.22 µM, respectively. Compound 6 exhibited moderate cytotoxic activities against MKN-45, HCT116, PATU8988T, 5637 and A-673 cell lines with the IC50 values of 19.69 µM, 16.44 µM, 18.07 µM, 11.51 µM and 18.15 µM, respectively. Compound 5 showed moderate cytotoxic activities against A549 cell lines with the IC50 values of 24.06 µM.

Geranylation of Chalcones by a Fungal Aromatic Prenyltransferase.

Geranylated chalcones mainly exist in plants, and many of them have attracted attention because of their diverse pharmacological and biological activities. Herein, we report geranylation of eight chalcones by the Aspergillus terreus aromatic prenyltransferase AtaPT. Ten new mono-geranylated enzyme products (1G-5G, 6G1, 6G2, 7G, 8G1, and 8G2) were obtained. Most of the products are C-geranylated products with prenyl moieties at ring B. In comparison, plant aromatic prenyltransferases usually catalyze the geranylation at ring A. Therefore, AtaPT can be used complementarily for chalcone geranylation to increase the structural diversity of small molecules. In addition, seven compounds (1G, 3G, 4G, 6G1, 7G, 8G1, and 8G2) exhibited a potential inhibitory effect on α-glucosidase with the IC50 values ranging from 45.59 ± 3.48 to 82.85 ± 2.15 µg/mL. Among them, compound 7G (45.59 ± 3.48 µg/mL) was the most potential α-glucosidase inhibitor, which is about seven times stronger than the positive control acarbose (IC50 = 346.63 ± 15.65 µg/mL).

Five new limonoids isolated from Walsura robusta.

Five new toosendanin limonoids with highly oxidative furan ring walsurobustones A-D (1-4), and one new furan ring degraded limonoid walsurobustone E (5) together with one known compound toonapubesic acid B (6) were isolated from the leaves of Walsura robusta. Their structures were elucidated by NMR and MS data. Especially, the absolute configuration of toonapubesic acid B (6) was confirmed by X-ray diffraction study. Compounds 1-6 exhibited good cytotoxicity against the cancer cell lines HL-60, SMMC-7721, A-549, MCF-7, and SW480.

Matrine-Type Alkaloids with Anti-Tomato Spotted Wilt Virus Activity from the Root of Sophora tonkinensis Gagnep.

As a continuation of our research on the development of pesticide active quinolizidine alkaloids (QAs) from the family Fabaceae, the chemical constituents of the root of Sophora tonkinensis Gagnep. were systematically investigated. Seventeen new matrine-type alkaloids (1-17), including one new naturally occurring compound (17), along with 20 known ones were isolated from the EtOH extract of S. tonkinensis. Notably, compound 5 possessed an unprecedented 6/6/5/4/6/6 hexacyclic system. Their structures were confirmed via comprehensive spectroscopic data analysis (IR, UV, NMR, HRESIMS), ECD calculation, and X-ray crystallography. Biological tests indicated that compounds 1, 4, 10, 12, 13, and 30 displayed significant anti-tomato spotted wilt virus (TSWV) activities compared with the positive control ningnanmycin. Moreover, compound 12 strongly inhibited the expression of the TSWV N, NSs, and NSm genes and TSWV NSs protein in plant host. Furthermore, compounds 4, 10, 12, 20, and 22 exhibited moderate insecticidal activities against TSWV thrip vector (Frankliniella occidentalis).

Two new isoflavones from the roots of Sophora tonkinensis.

Two new isoflavones (1 and 2), as well as eight known ones were isolated from the roots of Sophora tonkinensis Gagnep. Compound 1 represents an unprecedented polymerization pattern constructed by isoflavone and cytisine. Their structures were elucidated by comprehensive spectroscopic data analysis, combined with ECD calculations. Compound 1 displayed significant anti-tobacco mosaic virus (TMV) activity compared with the positive control ningnanmycin. Moreover, compound 6 exhibited potent α-glucosidase inhibitory activity with IC50 value of 47.4 mg/L.

Quinoline Alkaloids from the Roots of Orixa japonica with the Anti-Pathogenic Fungi Activities.

A new quinoline alkaloid, 5-hydroxy-6-methoxy-N-methyl-2-phenylquinoline-4-one (1), and seventeen known quinoline alkaloids (2-18) were isolated from the roots of Orixa japonica. The structure of 1 was determined by analysis of spectroscopic data. Among them, compounds 2, 3, and 13 were isolated from this plant for the first time. All isolates were screened for the anti-pathogenic fungi activities, including Rhizoctonia solani, Magnaporthe oryzae, and Phomopsis sp. The results showed that five compounds (4, 8, 10, 11, and 12) exhibited significant anti-pathogenic fungi effects at 50.0 µg/mL. In special, compound 10 exhibited the best antifungal activities toward R. solani and M. oryzae with the IC50 values of 37.86 and 44.72 µM, respectively, better than that of the positive control, hymexazol (IC50 121.21 and 1518.18 µM, respectively). Moreover, eleven new quinoline alkaloids derivatives (12a-12k) were designed and synthesized to investigate the structure-activity relationships (SARs). The SARs analysis indicated that the furo[2,3-b]quinoline skeleton and the methoxy at C-7 (compounds 8, 11, and 12) played a key role for improving the antifungal activities.

Hyperpatone A, a polycyclic polyprenylated acylphloroglucinol with a rare 8/6/5/6/5 pentacyclic skeleton from Hypericum patulum.

Hyperpatone A (1), a highly oxidated polycyclic polyprenylated acylphloroglucinol (PPAP), along with a biosynthesized related PPAP (2) was isolated from Hypericum patulum under the guidance of LC-MS investigation. Architecturally, compound 1 represents the first PPAP with an unprecedented 8/6/5/6/5 pentacyclic skeleton and an intramolecular peroxy bridge, which might be derived from the [3.3.1]-type bicyclic polyprenylated acylphloroglucinol via the critical Baeyer-Villiger oxidation, decarboxylation, and intramolecular cyclization. The structures were established by extensive spectroscopic analysis, ACD software calculation, and quantum chemical computations. A plausible biogenetic pathway of 1 and 2 was also proposed. Importantly, both compounds exhibited moderate cytotoxic activities against the HEL cell line with the IC50 values ranging from 10.2 to 19.2 µM. Moreover, compound 1 showed an inhibitory effect on NO production in lipopolysaccharide-stimulated RAW264.7 cells at a lower concentration of 5 or 1 µM.

Maillard Reaction Products with Anti-Tobacco Mosaic Virus Activities Generated in Processed Thermopsis lanceolata R. Br. Seed Extract.

Six novel Maillard reaction products (MRPs) (1-6) were isolated from the processed Thermopsis lanceolata R. Br. seed extract, along with one biogenetically related intermediate (7). Compounds 1-4 possessed three rare dimerization patterns constructed by cytisine, whereas compounds 5 and 6 represented the first example of the addition products of cytisine and 5,6-dihydroxy-4-hexanolide. Their structures were elucidated by comprehensive spectroscopic data analysis and quantum chemistry calculations including GIAO 13C{1H} NMR and ECD calculation, combined with single-crystal X-ray diffraction analysis. Biologically, compound 3 displayed significant anti-tobacco mosaic virus activity compared with the positive control ningnanmycin.

Hypersampones A-C, Three Nor-Polycyclic Polyprenylated Acylphloroglucinols with Lipid-Lowering Activity from Hypericum sampsonii.

Hypersampones A-C (1-3), three unprecedented nor-polycyclic polyprenylated acylphloroglucinols (PPAPs), were isolated from Hypericum sampsonii. These compounds represent the first nor-PPAPs with an unexpected tetracyclic 6/5/5/6 ring system. Their structures were assigned through the analysis of detailed spectroscopic data, X-ray crystallography, and electronic circular dichroism calculations. Compound 1 significantly inhibited the accumulation of lipid in an oleic acid-treated HepG2 cell model by suppressing the protein expression of FAS and ACACA at 5 µM.

Quinolizidine Alkaloids with Antitomato Spotted Wilt Virus and Insecticidal Activities from the Seeds of Thermopsis lanceolata R. Br.

As part of our ongoing investigation of pesticide active quinolizidine alkaloids (QAs) from the family Fabaceae, the chemical constituents of the seeds of Thermopsis lanceolata R. Br. were systematically investigated. Bioassay-guided fractionation and purification of the crude extract led to the isolation of seventeen new QAs (1-17), including three new naturally occurring compounds (15-17), along with 15 known compounds (18-32). Their structures were elucidated by comprehensive spectroscopic data analysis (IR, UV, NMR, and HRESIMS) and quantum chemistry calculations (13C NMR and ECD). The antitomato spotted wilt virus activities and insecticidal activities against Aphis fabae, Nilaparvata lugens (Stal), and Tetranychus urticae of compounds 1-32 were screened using the lesion counting method, spray method, and rice-stem dipping method, respectively. Biological tests indicated that compounds 6, 9, 10, and 18 displayed significant anti-TSWV activities compared with the positive control ningnanmycin. Compounds 3, 4, and 5 showed better insecticidal activities against A. fabae with LC50 values of 10.07, 12.07, and 6.56 mg/L, respectively. Moreover, compounds 5, 18, and 24 exhibited moderate insecticidal activities against N. lugens (Stal) with LC50 values of 37.91, 53.44, and 31.21 mg/L, respectively. Furthermore, compounds 9 and 10 exhibited moderate insecticidal activities against T. urticae.

Three New Pregnanes Isolated from the Cynanchum auriculatum.

Three new compounds named cynansteroid A (1), cynansteroid B (2) and cynansteroid C (3), together with nine known C21 -steroidal pregnane sapogenins (4-12) were isolated from the hydrolytic extract of the roots of Cynanchum auriculatum. The structures of cynansteroid A-C (1-3) were ascertained via the detailed analysis of the HR-ESI-MS, 1D and 2D NMR, and the calculated and experimental ECD data of cynansteroid B (2). Compound 11 displayed moderate inhibitory activity toward Verticillium dahliae Kleb (IC50 =37.15 µM), furthermore, compounds 11 and 12 showed significant inhibitory activity against Phomopsis sp. (IC50 =16.49 µM and 17.62 µM, respectively).